Vance G Nielsen

Interests

Research

Heme oxygenase mediated hypercoagulability; snake venom effects on coagulation; effects of iron and carbon monoxide on coagulation.

Teaching

Cardiothoracic Anesthesiology; Coagulation; Fibrinolysis

Courses

No activities entered.

Scholarly Contributions

Journals/Publications

• Nielsen, V. G., Goff, T., Hunsaker, B. D., & Neves, C. D. (2023). The Gilded Clot: Review of Metal-Modulated Platelet Activation, Coagulation, and Fibrinolysis. International journal of molecular sciences, 24(4).
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  The processes of blood coagulation and fibrinolysis that in part maintain the physical integrity of the circulatory system and fluidity of its contents are complex as they are critical for life. While the roles played by cellular components and circulating proteins in coagulation and fibrinolysis are widely acknowledged, the impact of metals on these processes is at best underappreciated. In this narrative review we identify twenty-five metals that can modulate the activity of platelets, plasmatic coagulation, and fibrinolysis as determined by in vitro and in vivo investigations involving several species besides human beings. When possible, the molecular interactions of the various metals with key cells and proteins of the hemostatic system were identified and displayed in detail. It is our intention that this work serve not as an ending point, but rather as a fair evaluation of what mechanisms concerning metal interactions with the hemostatic system have been elucidated, and as a beacon to guide future investigation.
• Kazui, T., Nielsen, V. G., Audie, S. D., Venkataramani, R. M., Bryant, J. T., Swenson, K., & Ford, P. M. (2022). A Case Report of Severe Factor XI Deficiency during Cardiac Surgery: Less Can Be More. Journal of cardiovascular development and disease, 9(4).
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  Severe congenital Factor XI (FXI) deficiency (
• Nielsen, V. G., & Wagner, M. T. (2022). Review of the Mechanisms of Snake Venom Induced Pain: It's All about Location, Location, Location. International journal of molecular sciences, 23(4).
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  Pain-acute, chronic and debilitating-is the most feared neurotoxicity resulting from a survivable venomous snake bite. The purpose of this review is to present in a novel paradigm what we know about the molecular mechanisms responsible for pain after envenomation. Progressing from known pain modulating peptides and enzymes, to tissue level interactions with venom resulting in pain, to organ system level pain syndromes, to geographical level distribution of pain syndromes, the present work demonstrates that understanding the mechanisms responsible for pain is dependent on "location, location, location". It is our hope that this work can serve to inspire the molecular and epidemiologic investigations needed to better understand the neurotoxic mechanisms responsible for these snake venom mediated diverse pain syndromes and ultimately lead to agent specific treatments beyond anti-venom alone.
• Sniecinski, R. M., Nielsen, V. G., & Tanaka, K. (2022). Searching for an Alternate Anticoagulant for Cardiopulmonary Bypass: Does Two Plus Two Equal Two?. Anesthesia & Analgesia. doi:10.1213/ane.0000000000006059
• Nielsen, V. G. (2021). Modulation of Diverse Procoagulant Venom Activities by Combinations of Platinoid Compounds.. International journal of molecular sciences, 22(9). doi:10.3390/ijms22094612
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  Procoagulant snake venoms have been inhibited by the ruthenium containing compounds CORM-2 and RuCl3 separately, presumably by interacting with critical histidine or other sulfur-containing amino acids on key venom enzymes. However, combinations of these and other platinoid containing compounds could potentially increase, decrease or not affect the procoagulant enzyme function of venom. Thus, the purpose of this investigation was to determine if formulations of platinoid compounds could inhibit venom procoagulant activity and if the formulated compounds interacted to enhance inhibition. Using a human plasma coagulation kinetic model to assess venom activity, six diverse venoms were exposed to various combinations and concentrations of CORM-2, CORM-3, RuCl3 and carboplatin (a platinum containing compound), with changes in venom activity determined with thrombelastography. The combinations of CORM-2 or CORM-3 with RuCl3 were found to enhance inhibition significantly, but not in all venoms nor to the same extent. In sharp contrast, carboplatin-antagonized CORM-2 mediated the inhibition of venom activity. These preliminary results support the concept that platinoid compounds may inhibit venom enzymatic activity at the same or different molecular sites and may antagonize inhibition at the same or different sites. Further investigation is warranted to determine if platinoid formulations may serve as potential antivenoms.
• Nielsen, V. G. (2021). Ruthenium chloride inhibits the anticoagulant activity of the phospholipase A-dependent neurotoxin of Mojave rattlesnake Type A venom. Journal of thrombosis and thrombolysis, 52(4), 1020-1022.
• Nielsen, V. G., Kazui, T., Horn, E. A., & Dotson, V. E. (2021). Thrombocytosis and neutrophilia associated with oxygenator failure and protamine reaction after cardiopulmonary bypass: a case report and literature review. Journal of thrombosis and thrombolysis, 52(4), 1220-1226.
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  Thrombocytosis has been feared as a source of thrombotic complications during the conduct of cardiopulmonary bypass (CPB) for patients undergoing cardiac procedures. We present a patient urgently requiring repair/replacement of three heart valves that had preexisting myelofibrosis with thrombocytosis (platelet count of 800,000 per µl) and neutrophilia (40,000 per µl). Despite achieving an activated clotting time > 500 s with heparin and antithrombin concentrate administration prior to CPB, the pump oxygenator and reservoir demonstrated significant clot just prior to restoration of the patient's circulation. The patient subsequently suffered a severe protamine reaction that was successfully managed. A review of the literature of similar patients and the relevant cellular and biochemical mechanisms in this setting are presented, with potential therapeutic approaches to prevent such complications noted.
• Nielsen, V. G. (2020). Ruthenium, Not Carbon Monoxide, Inhibits the Procoagulant Activity of , , and Venoms. International journal of molecular sciences, 21(8).
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  The demonstration that carbon monoxide releasing molecules (CORMs) affect experimental systems by the release of carbon monoxide, and not via the interaction of the inactivated CORM, has been an accepted paradigm for decades. However, it has recently been documented that a radical intermediate formed during carbon monoxide release from ruthenium (Ru)-based CORM (CORM-2) interacts with histidine and can inactivate bee phospholipase A activity. Using a thrombelastographic based paradigm to assess procoagulant activity in human plasma, this study tested the hypothesis that a Ru-based radical and not carbon monoxide was responsible for CORM-2 mediated inhibition of , and species snake venoms. Assessment of the inhibitory effects of ruthenium chloride (RuCl) on snake venom activity was also determined. CORM-2 mediated inhibition of the three venoms was found to be independent of carbon monoxide release, as the presence of histidine-rich albumin abrogated CORM-2 inhibition. Exposure to RuCl had little effect on venom activity, but and venom had procoagulant activity significantly reduced. In conclusion, a Ru-based radical and ion inhibited procoagulant snake venoms, not carbon monoxide. These data continue to add to our mechanistic understanding of how Ru-based molecules can modulate hemotoxic venoms, and these results can serve as a rationale to focus on perhaps other, complementary compounds containing Ru as antivenom agents in vitro and, ultimately, in vivo.
• Nielsen, V. G. (2020). The anticoagulant effect of Apis mellifera phospholipase A2 is inhibited by CORM-2 via a carbon monoxide-independent mechanism.. Journal of thrombosis and thrombolysis, 49(1), 100-107. doi:10.1007/s11239-019-01980-0
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  Bee venom phospholipase A2 (PLA2) has potential for significant morbidity. Ruthenium (Ru)-based carbon monoxide releasing molecules (CORM) inhibit snake venoms that are anticoagulant and contain PLA2. In addition to modulating heme-bearing proteins with carbon monoxide, these CORM generate reactive Ru species that form adducts with histamine residues resulting in changes in protein function. This study sought to identify anticoagulant properties of bee venom PLA2 via catalysis of plasma phospholipids required for thrombin generation. Another goal was to determine if Ru-based CORM inhibit bee venom PLA2 via carbon monoxide release or via potential binding of reactive Ru species to a key histidine residue in the catalytic site of the enzyme. Anticoagulant activity of bee venom PLA2 was assessed via thrombelastography with normal plasma. Bee venom PLA2 was then exposed to different CORM and a metheme forming agent and anticoagulant activity was reassessed. Using Ru, boron and manganese-based CORM and a metheme forming agent, it was demonstrated that it was unlikely that carbon monoxide interaction with a heme group attached to PLA2 was responsible for inhibition of anticoagulant activity by Ru-based CORM. Exposure of PLA2 to a Ru-based CORM in the presence of histidine-rich human albumin resulted in loss of inhibition of PLA2. Ru-based CORM likely inhibit bee venom PLA2 anticoagulant activity via formation of reactive Ru species that bind to histidine residues of the enzyme.
• Nielsen, V. G. (2021). Platinoid effects on human plasmatic coagulation kinetics: a viscoelastic analysis. Journal of thrombosis and thrombolysis.
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  In recent years a variety of metals (cadmium, chromium, copper, iron) have been demonstrated to modulate coagulation in vitro and in vivo. One group of metals, the platinoids, have not been assessed, and such investigation is justified given the thromboembolic phenomena associated with platinum-based chemotherapy. Thus, the goal of the present investigation was to assess the effects of carboplatin, cisplatin (platinum compounds), NAMI-A, and ruthenium chloride (ruthenium compounds) on human plasmatic coagulation. Human plasma was exposed to clinically relevant, equimolar concentrations of the aforementioned platinum and ruthenium compounds, with changes in plasmatic coagulation assessed via thrombelastography. The first series of experiments demonstrated no significant modulation of coagulation by the platinum compounds, while NAMI-A demonstrated mild hypercoagulability and ruthenium chloride exerted marked hypercoagulability. A second series of experiments utilizing a variety of specialized modifications of thrombelastography focused on ruthenium chloride revealed that this compound enhances prothrombin activation. While the hypercoagulability associated with platinum compounds in vivo do not appear to have a basis in plasmatic biochemistry, it appears that ruthenium compounds can exert procoagulant properties by enhancing the common pathway of human plasmatic coagulation. Future investigation of Ru based chemotherapeutic agents in development to assess procoagulant activity as part of evaluating their potential clinical safety is warranted.
• Nielsen, V. G., Wagner, M. T., & Frank, N. (2020). Mechanisms Responsible for the Anticoagulant Properties of Neurotoxic Venoms: A Viscoelastic Analysis. International journal of molecular sciences, 21(6).
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  Using thrombelastography to gain mechanistic insights, recent investigations have identified enzymes and compounds in and species' neurotoxic venoms that are anticoagulant in nature. The neurotoxic venoms of the four extant species of (the Black and green mambas) were noted to be anticoagulant in nature in human blood, but the mechanisms underlying these observations have never been explored. The venom proteomes of these venoms are unique, primarily composed of three finger toxins (3-FTx), Kunitz-type serine protease inhibitors (Kunitz-type SPI) and
• Johnson, T. E., Wells, R. J., Bell, A., Nielsen, V. G., & Olver, C. S. (2019). Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo. Basic & clinical pharmacology & toxicology, 125(4), 328-336.
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  Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis. We hypothesized that CORM-2 enhances coagulation and attenuates fibrinolysis in canine plasma exposed to C viridis venom. We measured the effects of C viridis venom on clot strength, rates of coagulation and fibrinolysis in both pooled canine plasma and plasma from individual naturally envenomed dogs, with and without CORM-2, using thromboelastography (TEG). We tested venom effects on coagulation using tissue factor (TF) activated TEG and on both coagulation and fibrinolysis using TF-activated TEG with added tPA. We found that 17.9 µg/mL of venom causes a mean 26.4% decrease in clot strength, a 61.8% decrease in maximum rate of thrombus generation, 75% faster clot lysis, a 226% increase in maximum rate of lysis and a 92% decrease in total clot life span (CLS). CORM-2 ameliorated these effects, increasing CLS in the presence of venom by 603%. Additionally, we showed that CORM-2 has similar effects in vitro on plasma from naturally envenomed dogs, showing promise as an adjunct therapy for snake envenomation.
• Nielsen, V. G. (2019). Carbon monoxide inhibits the anticoagulant activity of Mojave rattlesnake venoms type A and B. Journal of thrombosis and thrombolysis, 48(2), 256-262.
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  The Mojave rattlesnake is a unique species of pit viper that expresses either a highly potent phospholipase A (PLA)-dependent neurotoxin containing venom nearly devoid of fibrinogenolytic metalloproteinases (venom type A) or a hemotoxic venom with a high percentage of metalloproteinases and PLA without any neurotoxin present (venom type B) depending on its geographical location in the Southwestern United States and Mexico. Given that PLA have been demonstrated to affect coagulation, it was hypothesized that the anticoagulant effects of both type A and B venoms could be assessed by thrombelastography, and determination made if these venoms were heme modulated. Both venom types were exposed to carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100 µM) in isolation and then placed in human plasma with consequent coagulation kinetics assessed by thrombelastography. It was determined that type A venom was twice as potent as an anticoagulant compared to type B venom, and that both venoms were inhibited by carbon monoxide releasing molecule-2 but not its inactivated molecule. Given the lack of proteolytic activity of type A venom and the dependence of neurotoxicity on PLA activity, it may be possible that carbon monoxide could inhibit neurotoxicity based on inhibition of PLA anticoagulant activity. These data may serve as the rationale for extension of these observations into animal models to determine if CO may inhibit not just hemotoxic venom, but also PLA-dependent neurotoxic venom.
• Nielsen, V. G. (2019). Characterization of L-amino Acid Oxidase Derived from Venom: Procoagulant and Anticoagulant Activities. International journal of molecular sciences, 20(19).
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  Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.
• Nielsen, V. G. (2019). Lethal concentrations of mercury or lead do not affect coagulation kinetics in human plasma. Journal of thrombosis and thrombolysis, 48(4), 697-698.
• Nielsen, V. G. (2019). The anticoagulant effect of Apis mellifera phospholipase A is inhibited by CORM-2 via a carbon monoxide-independent mechanism. Journal of thrombosis and thrombolysis.
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  Bee venom phospholipase A (PLA) has potential for significant morbidity. Ruthenium (Ru)-based carbon monoxide releasing molecules (CORM) inhibit snake venoms that are anticoagulant and contain PLA. In addition to modulating heme-bearing proteins with carbon monoxide, these CORM generate reactive Ru species that form adducts with histamine residues resulting in changes in protein function. This study sought to identify anticoagulant properties of bee venom PLA via catalysis of plasma phospholipids required for thrombin generation. Another goal was to determine if Ru-based CORM inhibit bee venom PLA via carbon monoxide release or via potential binding of reactive Ru species to a key histidine residue in the catalytic site of the enzyme. Anticoagulant activity of bee venom PLA was assessed via thrombelastography with normal plasma. Bee venom PLA was then exposed to different CORM and a metheme forming agent and anticoagulant activity was reassessed. Using Ru, boron and manganese-based CORM and a metheme forming agent, it was demonstrated that it was unlikely that carbon monoxide interaction with a heme group attached to PLA was responsible for inhibition of anticoagulant activity by Ru-based CORM. Exposure of PLA to a Ru-based CORM in the presence of histidine-rich human albumin resulted in loss of inhibition of PLA. Ru-based CORM likely inhibit bee venom PLA anticoagulant activity via formation of reactive Ru species that bind to histidine residues of the enzyme.
• Nielsen, V. G., & Frank, N. (2019). Role of heme modulation in inhibition of Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera hemotoxic venom activity.. Human & experimental toxicology, 38(2), 216-226. doi:10.1177/0960327118793186
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  Venomous snake bite and subsequent coagulopathy is a significant source of morbidity and mortality worldwide. The gold standard to treat coagulopathy caused by these venoms is the administration of antivenom; however, despite this therapy, coagulopathy still occurs and recurs. Of interest, our laboratory has demonstrated in vitro and in vivo that coagulopathy-inducing venom exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the African genera Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera that have no or limited antivenoms available could be inhibited with CO or with the metheme-inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms were exposed in isolation to CO or PHA. Eight species were found to have procoagulant activity consistent with the generation of human thrombin, while one was likely fibrinogenolytic. All venoms were significantly inhibited by CO/PHA with species-specific variation noted. These data demonstrate indirectly that the heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, future investigation is warranted to determine if heme could serve as a potential therapeutic target to be modulated during treatment of envenomation by hemotoxic enzymes.
• Nielsen, V. G., & Frank, N. (2019). The kallikrein-like activity of Heloderma venom is inhibited by carbon monoxide. Journal of thrombosis and thrombolysis, 47(4), 533-539.
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  Lizards in the genus Heloderma are the most ancient venomous reptiles, with a traceable lineage nearly 100 million years old. The proteome of the venom of three of the remaining species (Heloderma suspectum, H. exasperatum, H. horridum) are very conserved, with kallikrein-like activity present to cause critical hypotension to immobilize and outright kill prey. Kallikrein-like activity would be expected to activate the contact protein pathway of coagulation, which would be detectable with thrombelastography in human plasma. Thus, it was proposed to determine if kallikrein-like activity could be detected with thrombelastography, and if this activity could be inhibited by carbon monoxide (CO) via a putative heme-based mechanism. Procoagulant activity of each venom was assessed via thrombelastography with normal plasma, and kallikrein-like activity confirmed with FX-depleted plasma. Venom was then exposed to carbon monoxide releasing molecule-2 (CORM-2) or its inactive releasing molecule to assess CO inhibition. All three venoms demonstrated kallikrein-like activity with the same potency and inhibition of activity by CO. In conclusion, the present work documented that procoagulant, kallikrein-like activity containing venoms of the oldest species of venomous reptiles was inhibited by CO, potentially via heme modulation. This is also the first identification and characterization of a kallikrein-like enzyme utilizing coagulation factor-depleted plasma to assess venom that inflicts hypotension. Future investigations will continue to define the vulnerability of venom enzymatic activities to CO.
• Nielsen, V. G., Frank, N., & Afshar, S. (2019). De Novo Assessment and Review of Pan-American Pit Viper Anticoagulant and Procoagulant Venom Activities via Kinetomic Analyses. Toxins, 11(2).
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  Snakebite with hemotoxic venom continues to be a major source of morbidity and mortality worldwide. Our laboratory has characterized the coagulopathy that occurs in vitro in human plasma via specialized thrombelastographic methods to determine if venoms are predominantly anticoagulant or procoagulant in nature. Further, the exposure of venoms to carbon monoxide (CO) or -phenylhydroxylamine (PHA) modulate putative heme groups attached to key enzymes has also provided mechanistic insight into the multiple different activities contained in one venom. The present investigation used these techniques to characterize fourteen different venoms obtained from snakes from North, Central, and South America. Further, we review and present previous thrombelastographic-based analyses of eighteen other species from the Americas. Venoms were found to be anticoagulant and procoagulant (thrombin-like activity, thrombin-generating activity). All prospectively assessed venom activities were determined to be heme-modulated except two, wherein both CO and its carrier molecule were found to inhibit activity, while PHA did not affect activity ( and ). When divided by continent, North and Central America contained venoms with mostly anticoagulant activities, several thrombin-like activities, with only two thrombin-generating activity containing venoms. In contrast, most venoms with thrombin-generating activity were located in South America, derived from species. In conclusion, the kinetomic profiles of venoms obtained from thirty-two Pan-American Pit Viper species are presented. It is anticipated that this approach will be utilized to identify clinically relevant hemotoxic venom enzymatic activity and assess the efficacy of locally delivered CO or systemically administered antivenoms.
• Nielsen, V. G., Frank, N., & Turchioe, B. J. (2019). Anticoagulant activity of krait, coral snake, and cobra neurotoxic venoms with diverse proteomes are inhibited by carbon monoxide. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 30(8), 379-384.
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  A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide.
• Nielsen, V. G., Johnson, T. E., Wells, R. J., Bell, A., & Olver, C. S. (2019). Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo. Basic & Clinical Pharmacology & Toxicology, 125(4), 328-336. doi:10.1111/bcpt.13242
• Donaghy, D., Yoo, S., Johnson, T., Nielsen, V., & Olver, C. (2018). Carbon Monoxide-Releasing Molecule Enhances Coagulation and Decreases Fibrinolysis in Normal Canine Plasma. Basic & clinical pharmacology & toxicology, 123(3), 257-262.
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  The dog is an important companion animal and also purpose-bred for research studies. Coagulopathies in dogs are common, although the availability of blood products for therapy is inconsistent throughout the profession. A pro-coagulant therapeutic that is readily available and easily stored would be useful for the treatment of coagulopathies. Tricarbonyldichlororuthenium (II) dimer [Carbon monoxide-releasing molecule-2 (CORM-2)] acts as a prothrombotic agent in plasma by increasing the velocity of clot formation and clot strength, and by decreasing the clot's vulnerability to fibrinolysis. We sought to test CORM-2's effect on coagulation and fibrinolysis in vitro in canine plasma using thromboelastography. Measures of the rate of clot formation and clot strength in plasma without CORM-2 were highly correlated with fibrinogen concentration. We found that CORM-2 significantly enhanced the rate of clot formation and clot strength and significantly reduced the rate of fibrinolysis and the clot lysis time. The per cent change in rate of clot formation and clot strength was not significantly correlated with fibrinogen concentration, indicating that CORM-2's pro-coagulant effect is not dependent on fibrinogen concentration. This study corroborates studies in other species that show that CORM-2 is pro-coagulant in plasma, and lays the groundwork for developing CORM-2 as a therapeutic agent for canine coagulopathies. Future studies will evaluate the effect of CORM-2 on whole blood both in vitro and in vivo.
• Nielsen, V. G. (2018). Carbon monoxide inhibits the anticoagulant activity of phospholipase A purified from Crotalus adamanteus venom. Journal of thrombosis and thrombolysis.
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  Snake venom contains a myriad of classes of enzyme which have been investigated for medicinal and toxinological purposes, including phospholipase A (PLA), which is responsible for anticoagulant, myotoxic and neurotoxic effects. Given the importance of PLA, the purposes of the present investigation were to characterize the coagulation kinetic behavior of a PLA purified from Crotalus adamanteus venom (Ca-PLA) in human plasma with thrombelastography and determine if carbon monoxide could inhibit its activity. Coagulation kinetics were determined in human plasma with a range of Ca-PLA activity (0-2 U/ml) via thrombelastography. Then, using carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100 µM), the vulnerability of Ca-PLA activity to carbon monoxide mediated inhibition was assessed. Lastly, the inhibitory response of Ca-PLA activity to exposure to carbon monoxide releasing molecule-2 (0-100 µM) was determined. Ca-PLA activity degraded the velocity of clot growth and clot strength in an activity dependent, exponential manner. Carbon monoxide inhibited Ca-PLA activity in a concentration dependent fashion, with loss of detectable activity at 100 µM of carbon monoxide releasing molecule-2. These findings, while preliminary, open the possibility that other PLA contained in snake venom with multiple toxicities (e.g., myotoxin, neurotoxin) may be heme bearing and CO-inhibitable, which have profound potential basic and clinical science implications.
• Nielsen, V. G. (2018). Crotalus atrox Venom Exposed to Carbon Monoxide Has Decreased Fibrinogenolytic Activity In Vivo in Rabbits. Basic & clinical pharmacology & toxicology, 122(1), 82-86.
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  Envenomation by haemotoxic enzymes remains a significant source of human morbidity and mortality worldwide, with administration of long-acting or multiple doses of antivenom first-line therapy. However, coagulopathy can still occur and recur. Of interest, it has been recently demonstrated that direct, isolated exposure of snake venom enzymes with fibrinogenolytic activity to carbon monoxide (CO) abrogates venom-mediated loss of coagulation in human plasma. These observations of CO inhibition of venom fibrinogenolytic activity were subsequently repeated in rabbit whole blood. This study sought to translate these findings in an in vivo rabbit model of envenomation with fibrinogenolytic Crotalus atrox venom. Sedated rabbits were intravenously administered C. atrox venom (400 μg/kg) pre-exposed to 0 or 1000 μM carbon monoxide-releasing molecule-2 (CORM-2) in vitro. Arterial whole-blood samples demonstrated that compared to pre-envenomation values, the CORM-2-naïve venom significantly prolonged the onset of coagulation, decreased the velocity of clot growth and decreased clot strength as determined by thromboelastography an hour after venom injection. In contrast, CORM-2 pre-exposure prevented or attenuated C. atrox venom effects on coagulation kinetics. Future studies to determine whether rabbits injected with such venom subcutaneously/intramuscularly can have consequent coagulopathy abrogated by injection of carbon monoxide-releasing molecules into the 'bite site' are justified.
• Nielsen, V. G., & Ford, P. M. (2018). The ratio of concentrations of aminocaproic acid and tranexamic acid that prevent plasmin activation of platelets does not provide equivalent inhibition of plasmatic fibrinolysis. Journal of thrombosis and thrombolysis.
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  Aminocaproic acid (EACA) availability has recently been decreased whereas tranexamic acid (TXA) is still available as an antifibrinolytic agent to decrease blood loss associated with procedures involving cardiopulmonary bypass (CPB) by inhibiting plasmin mediated platelet activation. Given that the clinical inclination is to substitute TXA for EACA, we sought to compare the antifibrinolytic efficacy of the two agents using the clinically accepted molar ratio of EACA:TXA (7.9:1) that prevents platelet activation in a viscoelastic based system under a variety of conditions in human plasma; 25-50% therapeutic concentration (EACA 32.5-65 µg/ml, TXA 5-10 µg/ml) in the presence of 1500-3000 IU tissue-type plasminogen activator, with 0-50% dilution of plasma with buffer. In all equipotent concentrations, TXA provided superior antifibrinolytic action compared to EACA. It is hoped that this work will serve as a rationale to further investigate these and other similar agents, especially now in a time of unpredictable unavailability of key medications needed to optimize patient care. It is also our wish that these data assist perfusionists, anesthesiologists and cardiothoracic surgeons with their consideration of using an antifibrinolytic agent when managing complex patients with hypercoagulable states (e.g., ventricular assist device explant, infective endocarditis) undergoing CPB.
• Nielsen, V. G., & Frank, N. (2018). Differential heme-mediated modulation of Deinagkistrodon, Dispholidus, Protobothrops and Pseudonaja hemotoxic venom activity in human plasma. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 31(6), 951-959.
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  Envenomation by vipers with hemotoxic enzymes continues to be a worldwide source of morbidity and mortality. The present work examined the effects of exposure of venom enzymes to carbon monoxide and O-phenylhydroxylamine, agents that modulate the biometal heme, by forming carboxyheme and metheme, respectively. Four venoms obtained from medically important, diverse snake venom found in Africa, Asia and Australia were analyzed. The species that had venom tested in human plasma with thrombelastography and heme modulating agents were Deinagkistrodon acutus, Protobothrops mucrosquamatus, Dispholidus typus and Pseudonaja textilis. These venoms varied four hundred-fold in potency (ng-µg/ml) to exert procoagulant effects on human plasma; further, there was species specific variability in venom inhibition after exposure to carboxyheme or metheme agents. Lastly, using a wide range of carbon monoxide concentrations, it was determined that the factor V component of P. textilis venom was likely inhibited before the factor X component. Further investigation using this thrombelastograph-based, venom "kinetomic" methodology involving heme modulation will demonstrate in time its laboratory and clinical utility.
• Nielsen, V. G., & Frank, N. (2018). Role of heme modulation in inhibition of Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera hemotoxic venom activity. Human & experimental toxicology, 960327118793186.
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  Venomous snake bite and subsequent coagulopathy is a significant source of morbidity and mortality worldwide. The gold standard to treat coagulopathy caused by these venoms is the administration of antivenom; however, despite this therapy, coagulopathy still occurs and recurs. Of interest, our laboratory has demonstrated in vitro and in vivo that coagulopathy-inducing venom exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the African genera Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera that have no or limited antivenoms available could be inhibited with CO or with the metheme-inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms were exposed in isolation to CO or PHA. Eight species were found to have procoagulant activity consistent with the generation of human thrombin, while one was likely fibrinogenolytic. All venoms were significantly inhibited by CO/PHA with species-specific variation noted. These data demonstrate indirectly that the heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, future investigation is warranted to determine if heme could serve as a potential therapeutic target to be modulated during treatment of envenomation by hemotoxic enzymes.
• Nielsen, V. G., Frank, N., & Matika, R. W. (2018). Carbon monoxide inhibits hemotoxic activity of Elapidae venoms: potential role of heme. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 31(1), 51-59.
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  Envenomation by hemotoxic enzymes continues to be a major cause of morbidity and mortality throughout the world. With regard to treatment, the gold standard to abrogate coagulopathy caused by these venoms is still the administration of antivenom; however, despite antivenom therapy, coagulopathy still occurs and recurs. Of interest, this laboratory has demonstrated in vitro and in vivo that coagulopathy inducing venom derived from snakes of the family Viperidae exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the Elapidae family (taipans and cobras) could also be inhibited with CO or with the metheme inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms from Elapidae snakes were exposed in isolation to CO (five species) or PHA (one specie) and placed in human plasma to assess changes in procoagulant or anticoagulant activity. The procoagulant activity of two taipan venoms and anticoagulant activity of three cobra venoms were significantly inhibited by CO. The venom of the inland taipan was also inhibited by PHA. In sum, these data demonstrate indirectly that the biometal heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, CO may not just be a potential therapeutic agent to treat envenomation but also may be a potential modulator of heme as a protective mechanism for venomous snakes against injury from their own proteolytic venoms.
• Nielsen, V. G., Frank, N., & Matika, R. W. (2018). Effects of Heme Modulation on and Venom Activity in Human Plasma. Toxins, 10(8).
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  Geographic isolation and other factors result in evolution-driven diversity of the enzymatic composition of venom of pit vipers in the same genus. The present investigation sought to characterize venoms obtained from such genetically diverse and pit vipers utilizing thrombelastographic coagulation kinetic analyses. The coagulation kinetics of human plasma were assessed after exposure to venom obtained from two and three species. The potency of each venom was defined (µg/mL required to equivalently change coagulation); additionally, venoms were exposed to carbon monoxide (CO) or a metheme-inducing agent to modulate any enzyme-associated heme. All venoms had fibrinogenolytic activity, with four being CO-inhibitable. While venoms had similar potency, one demonstrated the presence of a thrombin-like activity, whereas the other demonstrated a thrombin-generating activity. There was a 10-fold difference in potency and 10-fold different vulnerability to CO inhibition between the species. Metheme formation enhanced fibrinogenolytic-like activity in both species venoms, whereas the three species venoms had fibrinogenolytic-like activity enhanced, inhibited, or not changed. This novel "venom kinetomic" approach has potential to identify clinically relevant enzymatic activity and assess efficacy of antivenoms between genetically and geographically diverse species.
• Nielsen, V. G., Kim, E. J., Chyatte, D. A., & Acevedo, F. A. (2018). Rare cause of delirium and hypoxemia after coronary bypass surgery: transdermal lidocaine patch-associated methemoglobinemia.. International journal of legal medicine, 132(3), 767-769. doi:10.1007/s00414-017-1682-7
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  We present a case of a patient administered parasternal transdermal lidocaine patch therapy as part of a multimodal analgesic regime designed to diminish opioid-associated delirium after coronary bypass surgery. The patient presented with delirium and severe methemoglobinemia (41%) that responded to discontinuation of lidocaine therapy, oxygen administration, and methylene blue administration. The clinical contributors and medicolegal implications of this degree of lidocaine-associated methemoglobin-mediated delirium are presented in the hope of avoiding similar complications in the postoperative setting after coronary bypass surgery.
• Nielsen, V. G., Sanchez, E. E., Langlais, P. R., & Suntravat, M. (2018). CatroxMP-II: a heme-modulated fibrinogenolytic metalloproteinase isolated from Crotalus atrox venom.. Biometals.
• Nielsen, V. G., Sánchez, E. E., & Redford, D. T. (2018). Characterization of the Rabbit as an In Vitro and In Vivo Model to Assess the Effects of Fibrinogenolytic Activity of Snake Venom on Coagulation. Basic & clinical pharmacology & toxicology, 122(1), 157-164.
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  Several in vitro investigations have demonstrated that anticoagulant effects of fibrinogenolytic snake venom metalloproteinases have been abrogated in human plasma by modifying fibrinogen with iron (Fe) and carbon monoxide (CO) to prevent catalysis or by directly inhibiting these enzymes with CO. To translate these findings, we chose to assess the rabbit as a model of envenomation with Crotalus atrox venom. It was determined with thrombelastography that 15 times the concentration of venom noted to compromise coagulation in plasma in vitro was required to cause coagulopathy in vivo, likely secondary to venom binding to blood cells and being cleared from the circulation rapidly. Unlike human plasma, rabbit plasma pre-treated with Fe/CO was not protected from fibrinogenolysis by venom. Consequently, the administration of purified human fibrinogen (with or without Fe/CO) would be required before venom administration to rabbits. Of greater interest, venom exposed to CO had complete loss of fibrinogenolytic effect in rabbit plasma and partial loss of activity in whole blood, indicative of unbinding of CO from venom and binding to haemoglobin. Thus, venom exposed to CO could remain partially or completely inhibited in whole blood long enough for clearance from the circulation, allowing rabbits to be a useful model to test the efficacy of regional CO administration to the bite site. Future investigations are planned to test these novel approaches to attenuate venom-mediated coagulopathy in the rabbit.
• Nielsen, V. G., Ward, T. D., & Ford, P. M. (2018). Effects of cupric chloride on coagulation in human plasma: role of fibrinogen. Journal of thrombosis and thrombolysis.
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  Copper poisoning is associated with severe multiorgan injury and potentially death if chelation therapy is not administered. Of interest, while important gastrointestinal and urinary tract hemorrhage is associated with copper poisoning, very little is known concerning the nature of copper induced coagulopathy.
• Nielsen, V., Donaghy, D., Yoo, S., Johnson, T., & Olver, C. (2018). Carbon Monoxide-Releasing Molecule Enhances Coagulation and Decreases Fibrinolysis in Normal Canine Plasma. Basic & Clinical Pharmacology & Toxicology, 123(3), 257-262. doi:10.1111/bcpt.13015
• Suntravat, M., Langlais, P. R., Sánchez, E. E., & Nielsen, V. G. (2018). CatroxMP-II: a heme-modulated fibrinogenolytic metalloproteinase isolated from Crotalus atrox venom. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine.
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  It has been recently demonstrated that the hemotoxic venom activity of several species of snakes can be inhibited by carbon monoxide (CO) or a metheme forming agent. These and other data suggest that the biometal, heme, may be attached to venom enzymes and may be modulated by CO. A novel fibrinogenolytic metalloproteinase, named CatroxMP-II, was isolated and purified from the venom of a Crotalus atrox viper, and subjected to proteolysis and mass spectroscopy. An ion similar to the predicted singly charged m/z of heme at 617.18 was identified. Lastly, CORM-2 (tricarbonyldichlororuthenium (II) dimer, a CO releasing molecule) inhibited the fibrinogenolytic effects of CatroxMP-II on coagulation kinetics in human plasma. In conclusion, we present the first example of a snake venom metalloproteinase that is heme-bound and CO-inhibited.
• Yu, S., Khalpey, Z. I., Wong, R. K., Huynh, T., & Nielsen, V. G. (2018). Complete antithrombin replacement for anticoagulation for cardiopulmonary bypass to repair severe infective mitral valve endocarditis. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 29(1), 123-125.
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  : We present a case of a 26-year-old patient with severe infective endocarditis complicated with cerebral septic emboli that required essentially complete replacement of his circulating antithrombin activity to achieve an activated coagulation time near 480 s. The need for this degree of antithrombin administration may have been secondary to ongoing systemic inflammation and consequent thrombin generation despite blood culture results demonstrating no bacteremia. In sum, ongoing loss of endogenous antithrombin activity secondary to inflammation and the need for more than 80% normal activity to conduct safe cardiopulmonary bypass may require extraordinary administration of exogenous antithrombin in similar settings.
• Acevedo, F. A., Kim, E. J., Chyatte, D. A., & Nielsen, V. G. (2017). Rare cause of delirium and hypoxemia after coronary bypass surgery: transdermal lidocaine patch-associated methemoglobinemia. International journal of legal medicine.
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  We present a case of a patient administered parasternal transdermal lidocaine patch therapy as part of a multimodal analgesic regime designed to diminish opioid-associated delirium after coronary bypass surgery. The patient presented with delirium and severe methemoglobinemia (41%) that responded to discontinuation of lidocaine therapy, oxygen administration, and methylene blue administration. The clinical contributors and medicolegal implications of this degree of lidocaine-associated methemoglobin-mediated delirium are presented in the hope of avoiding similar complications in the postoperative setting after coronary bypass surgery.
• Boyer, L. V., Redford, D. T., Ford, P. M., Redford, D. T., Nielsen, V. G., Ford, P. M., & Boyer, L. V. (2017). Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 28(3), 211-217. doi:10.1097/mbc.0000000000000577
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  : Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 μg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).
• Nielsen, V. G. (2017). Crotalus atroxVenom Exposed to Carbon Monoxide Has Decreased Fibrinogenolytic ActivityIn Vivoin Rabbits. Basic & Clinical Pharmacology & Toxicology, 122(1), 82-86. doi:10.1111/bcpt.12846
• Nielsen, V. G. (2017). Effects of purified human fibrinogen modified with carbon monoxide and iron on coagulation in rabbits injected with Crotalus atrox venom. Journal of thrombosis and thrombolysis, 44(4), 481-488.
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  While snake venom derived enzymes, such as the thrombin-like activity possessing ancrod, have been used to treat thrombotic disease by defibrinogenating patients, the therapeutic potential of fibrinogenolytic snake venom enzymes, such as those derived from Crotalus atrox, have not been fully explored. However, one of the potential risks of administering fibrinogenolytic enzymes to effect defibrinogenation is hemorrhage secondary to hypofibrinogenemia. The present investigation sought to determine if human fibrinogen modified with carbon monoxide (CO) and iron (Fe) could resist degradation by C. atrox venom as has been seen in vitro in a recently developed rabbit model of envenomation. Compared with unmodified human fibrinogen, CO/Fe modified fibrinogen administered prior to envenomation had significantly shorter onset of coagulation and greater strength; however, when administered after envenomation, there was no differences between the two types of fibrinogen. Of interest, when administered after envenomation, both types of fibrinogen delayed the onset of coagulation while increasing plasma clot strength, a mixed effect likely secondary to formation of fibrinogen degradation products. Further preclinical investigations are needed to further define the benefits and risks of the use of fibrinogenolytic enzymes as defibrinogenating agents, as well as the risks of the "biochemical brakes" used to modulate the activity or substrate of the fibrinogenolytic enzyme.
• Nielsen, V. G., & Bazzell, C. M. (2017). Carbon monoxide releasing molecule-2 inhibition of snake venom thrombin-like activity: novel biochemical "brake"?. Journal of thrombosis and thrombolysis, 43(2), 203-208.
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  A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels. We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity. In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.
• Nielsen, V. G., & Losada, P. A. (2017). Direct Inhibitory Effects of Carbon Monoxide on Six Venoms Containing Fibrinogenolytic Metalloproteinases. Basic & clinical pharmacology & toxicology, 120(2), 207-212.
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  Since the introduction of antivenom administration over a century ago to treat venomous snake bite, it has been the most effective therapy for saving life and limb. However, this treatment is not always effective and not without potential life-threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases (SVMP) by inhibiting these Zn+2 -dependent enzymes directly with carbon monoxide (CO) exposure. Assessment of the fibrinogenolytic effects of venoms collected from the Arizona black rattlesnake, Northern Pacific rattlesnake, Western cottonmouth, Eastern cottonmouth, Broad-banded copperhead and Southern copperhead on human plasmatic coagulation kinetics was performed with thrombelastography in vitro. Isolated exposure of all but one venom (Southern copperhead) to CO significantly decreased the ability of the venoms to compromise coagulation. These results demonstrated that direct inhibition of transition metal-containing venom enzymes by yet to be elucidated mechanisms (e.g. CO, binding to Zn+2 or displacing Zn+2 from the catalytic site, CO binding to histidine residues) can in many instances significantly decrease fibrinogenolytic activity. This new paradigm of CO-based inhibition of the anticoagulant effects of SVMP could potentially diminish haemostatic compromise in envenomed patients until antivenom can be administered.
• Nielsen, V. G., & Matika, R. W. (2017). Effects of iron and carbon monoxide on Lachesis muta muta venom-mediated degradation of plasmatic coagulation. Human & experimental toxicology, 36(7), 727-733.
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  Hypofibrinogenemia is an important clinical consequence following envenomation by Lachesis muta muta, usually attenuated or prevented by administration of antivenom. The venom of L. m. muta contains both a metalloproteinase fibrinogenase and a serine protease thrombin-like enzyme, and exposure of fibrinogen to iron (Fe) and carbon monoxide (CO) has been demonstrated to decrease its catalysis by such enzymes. Using thrombelastographic analytical techniques, it was determined that this venom displayed weak procoagulant effects combined with fibrinogenolytic effects, and pretreatment of plasma with Fe and CO markedly attenuated venom-mediated effects. Additional experiments involving heparin exposure and varying calcium concentrations demonstrated that modification of fibrinogen with Fe and CO in human plasma rendered fibrinogen not recognizable to the fibrinogenolytic metalloproteinase but did not prevent polymerization by the thrombin-like serine protease. Lastly, when venom was exposed to CO in isolation and then placed in plasma, the fibrinogenase was inhibited but the thrombin-like enzyme was not inhibited. In sum, utilizing relatively facile modifications, we demonstrated with thrombelastography that Fe and/or CO addition can protect human plasmatic coagulation from fibrinogenase activity but not the effects of the thrombin-like activity of L. m. muta venom.
• Nielsen, V. G., Paidy, S. R., McLeod, W., Fox, A., & Nfonsam, V. N. (2017). Treatment of accidental perianal injection of topical thrombin with intravenous antithrombin. Journal of thrombosis and thrombolysis, 43(3), 423-425.
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  While topical thrombin application can markedly improve surgical hemostasis, rapid absorption of thrombin can result in pulmonary embolism and death. We report a case of accidental interstitial infiltration of topical thrombin after hemorrhoidectomy that was treated with administration of human antithrombin and heparin anticoagulation. Except for a marked decrease in antithrombin activity from super normal to normal values, the patient exhibited no laboratory or clinical signs of pulmonary embolism, thrombin mediated consumptive loss of procoagulants, or regional thrombosis. The patient had an uncomplicated recovery without sign of thrombotic morbidity. While it is hoped that such a medical misadventure should not occur, our case may serve as a reference to guide anticoagulant therapy if such a clinical scenario arises.
• Nielsen, V. G., Paidy, S. R., Meek, C. A., Thornton, T. K., & Lick, S. D. (2017). Survival after intravenous thrombin prior to cardiopulmonary bypass. International journal of legal medicine, 131(2), 485-487.
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  We present a case of a patient undergoing aortic valve replacement being inadvertently administered 5000 U of bovine thrombin instead of heparin for anticoagulation for cardiopulmonary bypass. The labeling error was made within the operating room pharmacy. The key to survival of this patient was a rapid diagnosis, administration of antithrombin and heparin, and removal of cardiac and great vessel thrombi. It is recommended that point of care anesthesia providers `prepare heparin for cardiopulmonary bypass anticoagulation, as thrombin is not used in anesthetic practice and is not contained within anesthesia cabinet medication drawers.
• Nielsen, V. G., Swanepoel, A. C., Naidoo, P., & Pretorius, E. (2017). Clinical relevance of hypercoagulability and possible hypofibrinolysis associated with estrone and estriol: SWANEPOEL et al.. Microscopy Research and Technique, 80(7), 697-703. doi:10.1002/jemt.22854
• Olver, C. S., & Nielsen, V. G. (2017). Iron protects porcine plasma coagulation kinetics from degradation by Crotalus atrox venom. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 30(5), 677-683.
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  While the administration of antivenom to treat hemotoxic snake bite injury remains the gold standard of therapy, we have demonstrated that modifying human fibrinogen with iron and carbon monoxide renders it resistant to fibrinogenolytic snake venom enzymes. In order to translate these findings into a possible biometal-based therapy complementary to antivenom administration, a preclinical model that possesses fibrinogen that closely mimics the human molecule in response to iron and carbon monoxide needed to be identified. The goal of this investigation was to determine if a swine model could serve in this capacity by assessing the thrombelastographic response of porcine plasma to iron and carbon monoxide exposure, without or with further exposure to the fibrinogenolytic venom of the viper Crotalus atrox. Using plasma obtained from eight swine, it was determined that their plasma responded to iron and carbon monoxide in a manner similar to that of human plasma by displaying enhanced coagulation kinetics. However, in sharp contrast to the response seen with human plasma, only iron significantly protected porcine plasma coagulation kinetics from C. atrox venom degradation. Therefore the pig is an animal beyond humans that could derive benefit from the biometal-focused therapy of iron infusion to protect against venom mediated compromise of coagulation. Thus, future investigation to assess the effects of iron administration to attenuate the effects of fibrinogenolytic envenomation with a pig model is justified.
• Redford, D. T., Boyle, P. K., Redford, D. T., Nielsen, V. G., & Boyle, P. K. (2017). Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of four Crotalus species.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 28(1), 34-39. doi:10.1097/mbc.0000000000000529
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  Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. Iron and carbon monoxide (CO) improve coagulation kinetics by modulation of fibrinogen as demonstrated in various Agkistrodon species and Crotalus atrox. Thus, we sought to determine whether pretreatment of plasma with iron and CO could attenuate venom-mediated catalysis of fibrinogen obtained from four common Crotalus species with known fibrinogenase activity. Human plasma was pretreated with ferric chloride (0-10 μmol/l) and CO-releasing molecule-2 (0-100 μmol/l) prior to exposure to venom from a Northern Pacific rattlesnake, Arizona black rattlesnake, prairie rattlesnake, or red diamond rattlesnake. The concentration of venom used decreased coagulation function of one or more kinetic parameters by at least 50% of normal values. Coagulation kinetics were determined with thrombelastography.Three snake venoms significantly degraded plasmatic coagulation kinetics, prolonging the onset to clot formation, diminishing velocity of clot growth and decreasing clot strength. However, red diamond rattlesnake venom exposure resulted in mixed coagulation kinetics, significantly decreasing the time to onset of coagulation without decreasing the velocity of clot growth. Iron and CO attenuated these coagulation kinetic changes in a species-specific manner. Further in vitro investigation of other fibrinogenolytic venoms is indicated to determine if iron and CO can attenuate venom compromised coagulation.
• Swanepoel, A. C., Naidoo, P., Nielsen, V. G., & Pretorius, E. (2017). Clinical relevance of hypercoagulability and possible hypofibrinolysis associated with estrone and estriol. Microscopy research and technique, 80(7), 697-703.
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  Estrone (E1 ) and Estriol (E3 ) are endogenous female hormones, present in increased concentrations during female specific physiological processes (menopause and pregnancy respectively) that are associated with increased venous thrombotic risk. These hormones are also used as hormone therapies that are also associated with increased thromboembolism risk. Viscoelastic analysis revealed no significant difference to clot formation after hormone addition, however morphological analysis showed that the addition of both E1 and E3 result in fibrin clots composed of thinner fibrin fibers arranged in dense matted networks. These changes to the fibrin network ultrastructure are indicative of a prothrombotic state but may also indicate hypofibrinolysis. We therefore conclude that the increased risk of venous thrombosis during pregnancy and menopause may originate from a combination of hypercoagulation and a possible hypofibrinolytic mechanism of these hormones. Therefore females with a hypercoagulable tendency that fall pregnant or enter menopause need to be monitored to prevent venous thrombotic events. The decision to use hormone therapies during and after menopause should not be taken lightly and the risk-reward scale should be closely examined to ensure it does not tip towards thrombosis and subsequent thrombotic events that ultimately could have been prevented.
• Bazzell, C. M., Nielsen, V. G., & Bazzell, C. M. (2016). Carbon monoxide attenuates the effects of snake venoms containing metalloproteinases with fibrinogenase or thrombin-like activity on plasmatic coagulation. MedChemComm, 7(10), 1973-1979. doi:10.1039/c6md00336b
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  Exposure of plasma to iron and carbon monoxide (CO) renders fibrinogen resistant to fibrinogenolytic or thrombin-like activity contained in pit viper venom. However, the direct effects of iron/CO on venom activity are unknown. Thus, we assessed if four different, metalloproteinase containing snake venoms exposed to iron/CO or CO alone could attenuate their fibrinogenolytic or thrombin-like activity. Venom (0–500 μg ml−1) was exposed to 0–10 μM FeCl3 and/or 0–100 μM carbon monoxide releasing molecule-2 (CORM-2), or inactivated CORM-2 (iCORM-2) for 3 min at room temperature. Venom solution (0–8 μg ml−1 final concentration) was then placed in citrated human plasma containing tissue factor, followed by CaCl2 addition for commencement of coagulation. Data were determined with thrombelastography for 10–15 min at 37 °C. Iron had no effect on the first venom tested, so only CO was investigated subsequently. Exposure of venom to CO attenuated fibrinogenolytic or thrombin-like activity, and iCORM-2 did not affect the venom activities. Further investigation of the effect of CO exposure on similar venoms is justified.
• Matika, R., & Nielsen, V. (2016). Effects of iron and carbon monoxide on Lachesis muta muta venom-mediated degradation of plasmatic coagulation. Human & Experimental Toxicology, 36(7), 727-733. doi:10.1177/0960327116661401
• Nielsen, V. (2016). The Contribution of Pin End-Cup Interactions to Clot Strength Assessed with Thrombelastography. Anesthesia and analgesia, 122(1), 43-5.
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  Viscoelastic methods have been developed to assess the contribution of plasma proteins and platelets to coagulation in vitro to guide clinical transfusion therapy. One of the cardinal precepts of determining clot strength is making sure that the viscoelastic technique includes complete exposure of the plastic pin in the testing chamber with the fluid analyzed so as to assure maximal interaction of the cup wall with the pin surface. However, the various contributions of the pin surface area to final clot strength have not been investigated. That is, it is not clear what is more important in the in vitro determination of clot strength, the surface area shared between the cup and pin filled with fluid or the final viscoelastic resistance of the gel matrix formed. Thus, the purpose of this investigation was to determine the clot strength when only the tip of the pin was engaged with plasma thrombus and to compare these values with clot strength values obtained when the pin was completely in plasma. After determining the minimal amount of plasma required to cover a pin tip in a thrombelastographic system (30 μL), clot strength (elastic modulus, G) was determined in plasma samples of 30 or 360 μL final volume (n = 12 per condition) after tissue factor activation. The G value with 30 μL volume was 1057 ± 601 dynes/cm (mean ± SD; 95% confidence interval, 675-1439 dynes/cm), which was (P = 0.0015) smaller than the G value associated with 360-μL sample volumes, that was 1712 ± 48 dynes/cm (confidence interval, 1681-1742 dynes/cm). In conclusion, these data demonstrate that clot strength is not determined by a simple ratio of surface area of pin and cup to volume of sample, but rather strength is importantly influenced by the viscoelastic resistance of the fluid assessed.
• Nielsen, V. G. (2016). Ancrod revisited: viscoelastic analyses of the effects of Calloselasma rhodostoma venom on plasma coagulation and fibrinolysis.. Journal of thrombosis and thrombolysis, 42(2), 288-93. doi:10.1007/s11239-016-1343-6
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  Fibrinogen depletion via catalysis by snake venom enzymes as a therapeutic strategy to prevent or treat thrombotic disorders was utilized for over four decades, with ancrod being the quintessential agent. However, ancrod eventually was found to not be of clinical utility in large scale stroke trial, resulting in the eventual discontinuation of the administration of the drug for any indication. It was hypothesized that ancrod, possessing thrombin-like activity, may have unappreciated robust coagulation kinetics. Using thrombelastographic methods, a comparison of equivalent tissue factor initiated thrombin generation and Calloselasma rhodostoma venom (rich in ancrod activity) on plasmatic coagulation kinetics was performed. The venom resulted in thrombi that formed nearly twice as fast compared to thrombin formed clots, and there was no difference in fibrinolytic kinetics initiated by tissue-type plasminogen activator. In plasma containing iron and carbon monoxide modified fibrinogen, which may be found in patients at risk of stroke, the coagulation kinetic differences observed with venom was still more vigorous than that seen with thrombin. These phenomena may provide insight into the clinical failure of ancrod, and may serve as an impetus to revisit the concept of fibrinogen depletion via fibrinogenolytic enzymes, not those with thrombin-like activity.
• Nielsen, V. G. (2016). Iron and carbon monoxide prevent degradation of plasmatic coagulation by thrombin-like activity in rattlesnake venom.. Human & experimental toxicology, 35(10), 1116-22. doi:10.1177/0960327115621366
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  Thousands suffer poisonous snake bite, often from defibrinogenating species annually. Three rattlesnake species in particular, the timber rattlesnake, Eastern diamondback rattlesnake, and Southern Pacific rattlesnake, cause clinically relevant hypofibrinogenemia via thrombin-like activity in their venom. It has been demonstrated that iron (Fe) and carbon monoxide (CO) change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, the present investigation sought to determine if pretreatment of plasma with Fe and CO could attenuate venom-mediated catalysis of fibrinogen via thrombin-like activity. Human plasma was pretreated with ferric chloride (0-10 μM) and CO-releasing molecule-2 (0-100 μM) prior to exposure to 2.5-10 μg/ml of venom obtained from the aforementioned three species of rattlesnake. Coagulation kinetics were determined with thrombelastography. All three snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially diminishing the speed of clot growth and strength. Pretreatment of plasma with Fe and CO completely abrogated the effects of all three venoms on coagulation kinetics. Further in vitro investigation of other pit viper venoms that possess thrombin-like activity is indicated to see if there is significant conservation of venom enzymatic target recognition of specific amino acid sequences such that Fe and CO can reliably attenuate venom-mediated catalysis of fibrinogen. These data also serve as a rationale for future preclinical investigation.
• Nielsen, V. G. (2016). Southern copperhead venom enhances tissue-type plasminogen activator induced fibrinolysis but does not directly lyse human plasma thrombi.. Journal of thrombosis and thrombolysis, 42(1), 33-7. doi:10.1007/s11239-015-1287-2
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  In addition to degrading fibrinogen as a source of consumptive coagulopathy, purified fractions of southern copperhead (Agkistrodon contortrix contortrix; A. c. contortrix) venom has been demonstrated to enhance fibrinolysis. The goal of this investigation was to characterize the kinetic fibrinolytic profile of A. c. contortrix venom in the absence and presence of tissue-type plasminogen activator (tPA) to determine if intact venom had tPA independent fibrinolytic properties. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to A. c. contortrix venom (0-6 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, plasma was exposed to 0-6 μg/ml of venom without tPA added and coagulation observed for 3 h. Venom significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth but did not significantly decrease clot strength. In the presence of tPA, venom significantly decreased clot strength, shortened the time of onset of fibrinolysis, decreased clot lysis time but did not significantly affect the maximum rate of lysis. Lastly, while venom exposure in the absence of tPA significantly prolonged the onset of coagulation and decreased the velocity of clot growth, venom exposure did not result in detectable fibrinolysis over the 3 h observation period. A. c. contortrix venom enhances tPA mediated fibrinolysis by degrading plasma coagulation kinetics. Intact A. c. contortrix venom does not possess sufficient fibrinolytic activity to cause fibrinolysis in human plasma at the concentration tested.
• Nielsen, V. G., & Henderson, J. H. (2016). Sonoclot(®)-based method to detect iron enhanced coagulation.. Journal of thrombosis and thrombolysis, 42(1), 1-5. doi:10.1007/s11239-015-1293-4
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  Thrombelastographic methods have been recently introduced to detect iron mediated hypercoagulability in settings such as sickle cell disease, hemodialysis, mechanical circulatory support, and neuroinflammation. However, these inflammatory situations may have heme oxygenase-derived, coexistent carbon monoxide present, which also enhances coagulation as assessed by the same thrombelastographic variables that are affected by iron. This brief report presents a novel, Sonoclot-based method to detect iron enhanced coagulation that is independent of carbon monoxide influence. Future investigation will be required to assess the sensitivity of this new method to detect iron mediated hypercoagulability in clinical settings compared to results obtained with thrombelastographic techniques.
• Nielsen, V. G., & Losada, P. A. (2016). Direct Inhibitory Effects of Carbon Monoxide on Six Venoms Containing Fibrinogenolytic Metalloproteinases. Basic & Clinical Pharmacology & Toxicology, 120(2), 207-212. doi:10.1111/bcpt.12654
• Nielsen, V. G., Boyer, L. V., Redford, D. T., & Ford, P. (2016). Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.
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  Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 μg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).
• Nielsen, V., & Jacobsen, W. (2016). Iron modulates the alpha chain of fibrinogen. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine.
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  Iron-bound fibrinogen has been noted to accelerate plasmatic coagulation in patients with divergent conditions involving upregulation of heme oxygenase activity, including hemodialysis, Alzheimer's disease, sickle cell anemia, and chronic migraine. Our goal was to determine if a site of iron-fibrinogen interaction was on the alpha chain. Using thrombelastography, we compared the coagulation kinetic profiles of plasma exposed to 0-10 µM ferric chloride after activation of coagulation with thrombin generated by contact activation of plasma with the plastic sample cup or by exposure to 1 µg/ml of Calloselasma rhodostoma venom (rich in ancrod activity), which causes coagulation via polymerization of alpha chain monomers. Venom mediated coagulation always occurred before thrombin activated thrombus formation, and ferric chloride always diminished the time of onset of coagulation and increased the velocity of clot growth. Iron enhances plasmatic coagulation kinetics by modulating the alpha chain of fibrinogen.
• Nielsen, V., Redford, D., & Boyle, P. (2016). Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of four Crotalus species. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.
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  Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. Iron and carbon monoxide (CO) improve coagulation kinetics by modulation of fibrinogen as demonstrated in various Agkistrodon species and Crotalus atrox. Thus, we sought to determine whether pretreatment of plasma with iron and CO could attenuate venom-mediated catalysis of fibrinogen obtained from four common Crotalus species with known fibrinogenase activity. Human plasma was pretreated with ferric chloride (0-10 μmol/l) and CO-releasing molecule-2 (0-100 μmol/l) prior to exposure to venom from a Northern Pacific rattlesnake, Arizona black rattlesnake, prairie rattlesnake, or red diamond rattlesnake. The concentration of venom used decreased coagulation function of one or more kinetic parameters by at least 50% of normal values. Coagulation kinetics were determined with thrombelastography.Three snake venoms significantly degraded plasmatic coagulation kinetics, prolonging the onset to clot formation, diminishing velocity of clot growth and decreasing clot strength. However, red diamond rattlesnake venom exposure resulted in mixed coagulation kinetics, significantly decreasing the time to onset of coagulation without decreasing the velocity of clot growth. Iron and CO attenuated these coagulation kinetic changes in a species-specific manner. Further in vitro investigation of other fibrinogenolytic venoms is indicated to determine if iron and CO can attenuate venom compromised coagulation.
• Visagie, A., Swanepoel, A. C., Pretorius, E., Nielsen, V. G., Lange, Z. D., & Emmerson, O. (2016). The clinical relevance of altered fibrinogen packaging in the presence of 17β-estradiol and progesterone.. Thrombosis research, 146, 23-34. doi:10.1016/j.thromres.2016.08.022
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  The effect of endogenous hormone concentrations, specifically 17β-estradiol and progesterone, on fibrin network formation has not been established..It is essential to understand natural hormone mechanisms since these hormones are still present in circulation while hormonal contraceptives, which are associated with increased risk of venous thromboembolism, are used..Due to the fact that these hormones are known to increase hypercoagulability and the prothrombotic state scanning electron microscopy (SEM), atomic force microscopy (AFM), thromboelastography (TEG) and turbidimetry were employed to investigate the morphology, surface roughness, viscoelastic properties and formation and lysis of fibrin..17β-estradiol and progesterone showed hypercoagulable viscoelastic properties and decreased the diameter and surface roughness of fibrin while increasing dense matted deposit occurrence. Our results suggest that the additional burden of hormonal load, together with the presence of endogenous estrogen and progesterone, may result in a prothrombotic and hypercoagulable state in females with an inflammatory predisposition..Our results are of clinical importance when considering hormones as either pathological agent or therapeutic intervention as will be assessed in future investigation.
• Boyle, P. K., Nielsen, V. G., & Redford, D. T. (2015). Effect of Iron and Carbon Monoxide on Fibrinogenase-like Degradation of Plasmatic Coagulation by Venoms of SixAgkistrodonSpecies. Basic & Clinical Pharmacology & Toxicology, 118(5), 390-395. doi:10.1111/bcpt.12504
• Faraoni, D., Van der Linden, P., Ducloy-Bouthors, A. S., Goobie, S. M., DiNardo, J. A., & Nielsen, V. G. (2015). Quantification of Fibrinolysis Using Velocity Curves Measured with Thromboelastometry in Children with Congenital Heart Disease. Anesthesia and analgesia, 121(2), 486-91.
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  In this pilot study, we hypothesized that velocity parameters obtained from changes in clot amplitude (A) and clot elasticity (E) measured with thromboelastometry (ROTEM, Tem International GmbH, Munich, Germany) could improve detection of fibrinolysis in whole blood obtained from children undergoing surgery for congenital heart disease.
• N, S., Welsby, I. J., Fielder, M. A., Jacobsen, W. K., & Nielsen, V. G. (2015). Sickle cell disease is associated with iron mediated hypercoagulability. Journal of thrombosis and thrombolysis, 40(2), 182-5.
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  Sickle cell disease (SCD) is associated with a significant hypercoagulable state and several hemostatic anomalies have been identified in this disease state. Of interest, SCD patients can become iron overloaded after transfusion, and iron can enhance fibrinogen as a substrate for thrombin, resulting in thrombi that commence coagulation quickly and form rapidly. We hypothesized that SCD patients would display hypercoagulable plasma coagulation kinetics and an iron enhancement of coagulation. After obtaining IRB approval, we assessed coagulation kinetics and iron enhancement with viscoelastic methods in archived, citrated plasma obtained from ambulatory or hospitalized SCD patients (n = 20). All SCD patients had plasmatic hypercoagulability, and 65 % were positive for iron enhancement of coagulation. In conclusion, continuing investigation correlating such viscoelastic data with clinical symptoms may provide insight into the role played by iron in the setting of SCD, including complications such as vaso-occlusive crisis.
• Nielsen, V. G. (2015). Iron and carbon monoxide prevent degradation of plasmatic coagulation by thrombin-like activity in rattlesnake venom. Human & experimental toxicology.
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  Thousands suffer poisonous snake bite, often from defibrinogenating species annually. Three rattlesnake species in particular, the timber rattlesnake, Eastern diamondback rattlesnake, and Southern Pacific rattlesnake, cause clinically relevant hypofibrinogenemia via thrombin-like activity in their venom. It has been demonstrated that iron (Fe) and carbon monoxide (CO) change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, the present investigation sought to determine if pretreatment of plasma with Fe and CO could attenuate venom-mediated catalysis of fibrinogen via thrombin-like activity. Human plasma was pretreated with ferric chloride (0-10 μM) and CO-releasing molecule-2 (0-100 μM) prior to exposure to 2.5-10 μg/ml of venom obtained from the aforementioned three species of rattlesnake. Coagulation kinetics were determined with thrombelastography. All three snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially diminishing the speed of clot growth and strength. Pretreatment of plasma with Fe and CO completely abrogated the effects of all three venoms on coagulation kinetics. Further in vitro investigation of other pit viper venoms that possess thrombin-like activity is indicated to see if there is significant conservation of venom enzymatic target recognition of specific amino acid sequences such that Fe and CO can reliably attenuate venom-mediated catalysis of fibrinogen. These data also serve as a rationale for future preclinical investigation.
• Nielsen, V. G. (2015). Old mineshaft, new canary: can circulating osteopontin concentrations predict septic shock?. Minerva anestesiologica, 81(2), 116-8.
• Nielsen, V. G. (2015). Southern copperhead venom enhances tissue-type plasminogen activator induced fibrinolysis but does not directly lyse human plasma thrombi. Journal of thrombosis and thrombolysis.
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  In addition to degrading fibrinogen as a source of consumptive coagulopathy, purified fractions of southern copperhead (Agkistrodon contortrix contortrix; A. c. contortrix) venom has been demonstrated to enhance fibrinolysis. The goal of this investigation was to characterize the kinetic fibrinolytic profile of A. c. contortrix venom in the absence and presence of tissue-type plasminogen activator (tPA) to determine if intact venom had tPA independent fibrinolytic properties. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to A. c. contortrix venom (0-6 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, plasma was exposed to 0-6 μg/ml of venom without tPA added and coagulation observed for 3 h. Venom significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth but did not significantly decrease clot strength. In the presence of tPA, venom significantly decreased clot strength, shortened the time of onset of fibrinolysis, decreased clot lysis time but did not significantly affect the maximum rate of lysis. Lastly, while venom exposure in the absence of tPA significantly prolonged the onset of coagulation and decreased the velocity of clot growth, venom exposure did not result in detectable fibrinolysis over the 3 h observation period. A. c. contortrix venom enhances tPA mediated fibrinolysis by degrading plasma coagulation kinetics. Intact A. c. contortrix venom does not possess sufficient fibrinolytic activity to cause fibrinolysis in human plasma at the concentration tested.
• Nielsen, V. G., & Boyer, L. V. (2015). Iron and carbon monoxide attenuate degradation of plasmatic coagulation by Crotalus atrox venom. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.
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  Hypofibrinogenemia is an important clinical consequence following envenomation by Crotalus species, usually attenuated or prevented by administration of antivenom. It has been determined that iron and carbon monoxide (CO) enhance fibrinogen as a thrombin substrate, likely secondary to conformational changes in molecular structure. We tested the hypothesis that pretreatment of plasma with iron and CO could attenuate the effects of exposure to Crotalus atrox venom. Human plasma was exposed to 0 to 10 μmol/l ferric chloride (iron source) and 0 to 100 μmol/l CO-releasing molecule-2 (CO source) followed by exposure to 0 to 0.5 μg/ml venom for 5 to 20 min. Changes in coagulation kinetics were determined with thrombelastography. Iron and CO significantly attenuated venom-mediated degradation of plasmatic coagulation in terms of onset time, velocity of clot growth and final clot strength. Further preclinical investigation of iron and CO administration as a 'bridge-to-antivenom' to preserve plasmatic coagulation is justified.
• Nielsen, V. G., & Henderson, J. (2015). Sonoclot(®)-based method to detect iron enhanced coagulation. Journal of thrombosis and thrombolysis.
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  Thrombelastographic methods have been recently introduced to detect iron mediated hypercoagulability in settings such as sickle cell disease, hemodialysis, mechanical circulatory support, and neuroinflammation. However, these inflammatory situations may have heme oxygenase-derived, coexistent carbon monoxide present, which also enhances coagulation as assessed by the same thrombelastographic variables that are affected by iron. This brief report presents a novel, Sonoclot-based method to detect iron enhanced coagulation that is independent of carbon monoxide influence. Future investigation will be required to assess the sensitivity of this new method to detect iron mediated hypercoagulability in clinical settings compared to results obtained with thrombelastographic techniques.
• Nielsen, V. G., Boyer, L. V., Matika, R. W., Amos, Q., & Redford, D. T. (2015). Iron and carbon monoxide attenuate Crotalus atrox venom-enhanced tissue-type plasminogen activator-initiated fibrinolysis. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.
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  In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10 μmol/l) or CO (carbon monoxide-releasing molecule-2, 100 μmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.
• Nielsen, V. G., Galvani, C. A., Boyle, P. K., Steinbrenner, E. B., & Matika, R. W. (2015). Bariatric patients have plasmatic hypercoagulability and systemic upregulation of heme oxygenase activity. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 26(2), 200-4.
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  Morbid obesity is associated with significant thrombophilia. Of interest, adipocytes obtained from obese patients have increased heme oxygenase (Hmox) activity, the endogenous enzyme responsible for carbon monoxide (CO) production. Given that CO enhances plasmatic coagulation, we determined whether morbidly obese patients undergoing bariatric surgery had an increase in endogenous CO and plasmatic hypercoagulability. CO was determined by noninvasive pulse oximetry measurement of carboxyhemoglobin (COHb). A thrombelastographic method to assess plasma coagulation kinetics and formation of carboxyhemefibrinogen (COHF) was utilized. Nonsmoking bariatric patients (n = 20, BMI 47 ± 8 kg/m, mean ± SD) had abnormally increased COHb concentrations of 2.7 ± 1.9%, indicative of Hmox upregulation. When coagulation kinetics of these bariatric patients were compared with values obtained from normal individuals' (n = 30) plasma, 70% (95% confidence interval 45.7-88.1%) had abnormally great velocity of clot formation, abnormally large clot strength, and COHF formation. Future investigation of Hmox-derived CO in the pathogenesis of obesity-related thrombophilia is warranted.
• Nielsen, V. G., Kulin, W., LaWall, J. S., MacFarland, F. N., Chen, A., Hadley, H. A., DaDeppo, A. J., Steinbrenner, E. B., & Matika, R. W. (2015). Chronic Migraineurs Form Carboxyhemefibrinogen and Iron-Bound Fibrinogen. CNS & neurological disorders drug targets, 14(8), 1079-85.
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  Chronic migraine (CM) is a disabling painful condition that is associated with dementia and thrombotic disease. It has been proposed that carbon monoxide (CO) and iron may play a role in CM, and CO and iron are products of the heme oxygenase system which is widespread within the brain. Further, CO and iron enhance plasmatic coagulation in part via a fibrinogen-dependent mechanism. Thus, our goal was to determine whether patients with CM had experienced carboxyhemefibrinogen formation, iron bound fibrinogen formation and plasmatic hypercoagulability. Nonsmokers with CM were recruited after informed, written consent. Blood was collected, anticoagulated with sodium citrate, and then centrifuged with plasma stored at -80ºC. Carboxyhemefibrinogen formation, iron bound fibrinogen formation and coagulation kinetics were determined via thrombelastographic methods. Patient results were compared with laboratory values generated from normal control plasmas. Incidence (95% confidence intervals) of the various parameters was determined using the Clopper-Pearson method. Twenty-six CM patients (24 female) were recruited; they were 46±12 years old. With regard to fibrinogen modification, 88.5% (69.8%-97.6%) of CM patients had formation of carboxyhemefibrinogen, iron bound fibrinogen, or both. With regard to coagulation, 42.3% (23.4%-63.1%) of patients had abnormally decreased time to clot initiation, 80.8% (60.6%-93.4%) had abnormally large velocity of clot formation, and 46.2% (26.6%-66.7%) had abnormally strong clot strength. Patients with CM have a large incidence of carboxyhemefibrinogen and iron bound fibrinogen formation and hypercoagulability. Confirmatory and potential therapeutic clinical trials targeting CO and iron modified hypercoagulation as a source of pain and vascular disease in CM patients are indicated.
• Nielsen, V. G., Pretorius, E., Bester, J., Jacobsen, W. K., Boyle, P. K., & Reinhard, J. P. (2015). Carbon monoxide and iron modulate plasmatic coagulation in Alzheimer's disease. Current neurovascular research, 12(1), 31-9.
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  Alzheimer's disease (AD) is a significant source of morbidity and mortality for millions of people worldwide, and multiple potential etiologies have been postulated to contribute to AD. Among these, spontaneous cerebral emboli and increased cerebral and circulating heme oxygenase (Hmox) activity in AD patients are of particular interest, as two of the products of Hmox activity, carbon monoxide (CO) and iron enhance plasmatic coagulation and modify the ultrastructure of thrombi. We hypothesized that patients afflicted with AD would have coagulation kinetics modulated by CO and iron. Using viscoelastic assessments of coagulation, it was determined with a small cohort (n=11) of AD patients that all had enhancement of coagulation by CO, iron, or both. In a complementary fashion, it was determined that a separate cohort (n=12) of AD patients had thrombi with ultrastructural features consistent with iron and CO exposure as assessed with scanning electron microscopy. Further, when stratified by normal or abnormally increased serum ferritin concentrations (which can be increased by Hmox), the AD patients with abnormal ferritin concentrations had significantly thinner fibrin fiber diameters, not unlike that noted when normal plasma is mixed with iron or CO. In sum, AD patients were noted to have plasmatic coagulation kinetic and thrombus ultrastructural changes consistent with exposure to CO and iron. Future investigation of CO and iron in the pathogenesis of Alzheimer's disease is warranted.
• Nielsen, V. G., Redford, D. T., & Boyle, P. K. (2015). Effect of Iron and Carbon Monoxide on Fibrinogenase-like Degradation of Plasmatic Coagulation by Venoms of Six Agkistrodon Species. Basic & clinical pharmacology & toxicology.
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  Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. It has been demonstrated that iron and carbon monoxide change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, the present investigation sought to determine if pre-treatment of plasma with iron and carbon monoxide could attenuate venom-mediated catalysis of fibrinogen obtained from Agkistrodon species with fibrinogenase activity. Human plasma was pre-treated with ferric chloride (0-10 μM) and carbon monoxide releasing molecule-2 (CORM-2, 0-100 μM) prior to exposure to 0.5-11 μg/ml of six different Agkistrodon species' venom. The amount of venom used for experimentation needed to decrease coagulation function of one or more kinetic parameters by at least 50% of normal values for (e.g., half the normal speed of clot formation). Coagulation kinetics were determined with thrombelastography. All six snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially prolonging the onset to clot formation and diminishing the speed of clot growth. Pre-treatment of plasma with iron and carbon monoxide attenuated these venom-mediated coagulation kinetic changes in a species-specific manner, with some venom effects markedly abrogated while others were only mildly decreased. Further in vitro investigation of other pit viper venoms that possess fibrinogenolytic activity is indicated to identify species amenable to or resistant to iron and carbon monoxide-mediated attenuation of venom-mediated catalysis of fibrinogen. Lastly, future preclinical investigation with animal models (e.g., rabbit ear bleed model) is planned to determine if iron and carbon monoxide can be used therapeutically after envenomation. This article is protected by copyright. All rights reserved.
• Nielsen, V. G., Sobieski II, M. A., & Slaughter, M. S. (2015). Left Ventricular Assist Device-Associated Carbon Monoxide and Iron-Enhanced Hypercoagulation: Impact of Concurrent Disease. ASAIO journal (American Society for Artificial Internal Organs : 1992), 61(4), 417-23.
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  Left ventricular assist device (LVAD) therapy is associated with thrombophilia despite anticoagulation. Of interest, LVAD patients have increased carboxyhemoglobin, a measure of upregulated heme oxygenase (Hmox) activity that releases carbon monoxide (CO) and iron. Given that CO and iron enhance plasmatic coagulation, we determined if LVAD patients had hypercoagulability and decreased fibrinolytic vulnerability with measurable CO and iron-mediated effects. Blood samples were obtained a month or more after implantation of the LVAD. Thrombelastographic methods to assess coagulation kinetics, fibrinolytic kinetics, formation of carboxyhemefibrinogen, and iron-mediated enhancement of clot growth were utilized. Coagulation and fibrinolytic parameter normal individual (n = 30) plasma values were determined. Sixteen LVAD patients were studied. CO and iron enhancement of coagulation were observed in the majority of LVAD patients, contributing to hypercoagulation. However, most patients demonstrated abnormally increased rates of clot lysis. Critically, hemolysis as assessed by circulating lactate dehydrogenase activity was small in this cohort, and only four patients without comorbid states (e.g., obesity, diabetes, sleep apnea) were hypercoagulable with evidence of Hmox upregulation. However, seven patients with comorbidities were hypercoagulable with Hmox upregulation. Future investigation of CO and iron-related thrombophilia and comorbid disease is warranted to define its role in LVAD-related thrombosis.
• Redford, D. T., Paidy, S. R., Steinbrenner, E. B., & Nielsen, V. G. (2015). Effects of profound hypoxemia on coagulation & fibrinolysis in normal individuals. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.
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  Hypoxia has been proposed to enhance, diminish, or have no effect on laboratory measures of coagulation or clinical thrombosis. Further, there usually are significant pathological or environmental factors concurrently present with hypoxia. Thus, the goal of the present investigation was to determine whether whole blood or plasmatic coagulation and fibrinolytic kinetics would change in response to progressive hypoxia to a systemic oxygenation (SpO2) of 70%. Healthy, conscious volunteers (n = 9) breathing a hypoxic mixture of gases during an in-vivo validation of noninvasive cerebral oximetry had blood samples collected and assessed with thrombelastography at normoxia and after SpO2 of 70%. A mild release of endogenous heparin-like activity occurred that diminished plasmatic coagulation, and a mild increase in clot lysis time also was noted. Further investigation to determine whether these phenomena occur in more chronic, less hypoxic states as sources of hypocoagulation or thrombophilia is needed.
• Swanepoel, A. C., Nielsen, V. G., & Pretorius, E. (2015). Viscoelasticity and Ultrastructure in Coagulation and Inflammation: Two Diverse Techniques, One Conclusion. Inflammation, 38(4), 1707-26.
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  The process of blood clotting has been studied for centuries. A synopsis of current knowledge pertaining to haemostasis and the blood components, including platelets and fibrin networks which are closely involved in coagulation, are discussed. Special emphasis is placed on tissue factor (TF), calcium and thrombin since these components have been implicated in both the coagulation process and inflammation. Analysis of platelets and fibrin morphology indicate that calcium, tissue factor and thrombin at concentrations used during viscoelastic analysis (with thromboelastography or TEG) bring about alterations in platelet and fibrin network ultrastructure, which is similar to that seen in inflammation. Scanning electron microscopy indicated that, when investigating platelet structure in disease, addition of TF, calcium or thrombin will mask disease-induced alterations associated with platelet activation. Therefore, washed platelets without any additives is preferred for morphological analysis. Furthermore, morphological and viscoelastic analysis confirmed that thrombin activation is the preferred method of fibrin activation when investigating fibrin network ultrastructure.
• Thompson III, J. L., Nielsen, V. G., Castro, A., & Chen, A. (2015). Heme oxygenase derived carbon monoxide and iron mediated plasmatic hypercoagulability in a patient with calcific mitral valve disease. Journal of thrombosis and thrombolysis, 39(4), 532-5.
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  We present a case of a patient with calcific mitral valve stenosis and plasmatic hypercoagulability. Using thrombelastography, the patient was determined to have an abnormally large velocity of plasma thrombus growth and strength with reduced vulnerability to lysis. Critically, increased carboxyhemoglobin concentration (2.4 %) was present, likely secondary to hemolysis from mitral stenosis and engagement of systemic heme oxygenase. It was determined that the patient's plasmatic hypercoagulability was in part due to carboxyhemefibrinogen formation and iron-enhancement of coagulation via two thrombelastographic methods. In conclusion, future investigation of the involvement of both carbon monoxide and iron mediated hypercoagulability in the setting of stenotic valve disease is warranted.
• Matika, R. W., Kim, S. S., Zelman, E. A., Steinbrenner, E. B., Nielsen, V. G., Matika, R. W., Kim, S. S., & Gharagozloo, F. (2014). Thoracic tumor effects on plasmatic coagulation: role of hemeoxygenase-1.. Lung cancer (Amsterdam, Netherlands), 83(2), 288-91. doi:10.1016/j.lungcan.2013.11.012
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  Lung cancer is an important health threat worldwide, and is associated with a 3.8-13.9% incidence of thrombophilia. Of interest, patients with lung tumors have been noted to have an increase in endogenous carbon monoxide production via upregulation of hemeoxygenase-1 activity. Given that it has been demonstrated that carbon monoxide enhances plasmatic coagulation in vitro and in vivo via formation of carboxyhemefibrinogen, we sought to determine if patients with thoracic tumors undergoing lung resection/pneumonectomy had an increase in endogenous carbon monoxide and concurrent plasmatic hypercoagulability..Nonsmoking patients with thoracic tumors (n=19) had preoperative carboxyhemoglobin (a measure of carbon monoxide production) determined, and a thromboelastometric method to assess citrated plasma coagulation kinetics and the formation of carboxyhemefibrinogen was utilized. Thoracic tumor patient coagulation kinetics was compared with normal subject (n=30) plasma samples..Patients with thoracic tumors were determined to have an abnormally increased carboxyhemoglobin concentration of 2.1±0.6%, indicative of hemeoxygenase-1 upregulation. It was found that 84% of thoracic tumor patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal subjects, and 44% of this hypercoagulable subgroup had carboxyhemefibrinogen formation. Future investigation of the role played by plasmatic hypercoagulability and hemeoxygenase-1 derived carboxyhemefibrinogen in the pathogenesis of thoracic tumor related thrombophilia is warranted.
• Matika, R. W., Sussman, A. N., Steinbrenner, E. B., Nielsen, V. G., Matika, R. W., & Madhrira, M. (2014). Hemodialysis patients have plasmatic hypercoagulability and decreased fibrinolytic vulnerability: role of carbon monoxide.. ASAIO journal (American Society for Artificial Internal Organs : 1992), 60(6), 716-21. doi:10.1097/mat.0000000000000144
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  Chronic hemodialysis is associated with significant thrombophilia. Of interest, hemodialysis patients have increased carboxyhemoglobin (COHb) and exhaled carbon monoxide (CO), signs of upregulated heme oxygenase (Hmox) activity. Given that CO enhances plasmatic coagulation, we determined whether patients requiring chronic hemodialysis had an increase in endogenous CO, plasmatic hypercoagulability and decreased fibrinolytic vulnerability. Carbon monoxide was determined by noninvasive pulse oximetry measurement of COHb. Blood samples were obtained just before hemodialysis. Thrombelastographic methods to assess plasma coagulation kinetics, fibrinolytic kinetics, and formation of carboxyhemefibrinogen (COHF) were used. Hemodialysis patients (n = 45) had abnormally increased COHb concentrations of 2.2 ± 1.9%, indicative of Hmox upregulation. Coagulation and fibrinolytic parameter normal values were determined with normal individual (n = 30) plasma. Thirty-seven patients of the hemodialysis cohort had COHF formation (82.2%, [67.9%-92.0%]; mean, [95% confidence interval]), and many of this group of patients had abnormally great velocity of clot growth (73.3%, [58.1%-85.4%]) and strength (75.6%, [60.5%-87.1%]). Furthermore, over half of COHF positive patients had a hypofibrinolytic state, evidenced by an abnormally prolonged time to maximum rate of lysis (53.3%, [37.9%-68.6%]) and clot lysis time (64.4%, [48.8%-78.1%]). Carbon monoxide enhanced coagulation and diminished fibrinolytic vulnerability in hemodialysis patients. Future investigation of hemodialysis, CO-related thrombophilia is warranted.
• Matika, R. W., Waer, A. L., Warneke, J. A., Warneke, J. A., Waer, A. L., Steinbrenner, E. B., Ong, E. S., Nielsen, V. G., Nfonsam, V. N., Matika, R. W., Ley, M. L., Kim, S., Jie, T., & Gharagozloo, F. (2014). Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 25(3), 248-53. doi:10.1097/mbc.0000000000000040
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  Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.
• Matika, R. W., Warneke, J. A., Warneke, J. A., Steinbrenner, E. B., Ong, E. S., Nielsen, V. G., Nfonsam, V. N., Matika, R. W., & Jie, T. (2014). Colon and pancreas tumors enhance coagulation: role of hemeoxygenase-1.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 25(5), 435-8. doi:10.1097/mbc.0000000000000075
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  Colon and pancreatic cancer are associated with significant thrombophilia. Colon and pancreas tumor cells have an increase in hemeoxygenase-1 (HO-1) activity, the endogenous enzyme responsible for carbon monoxide production. Given that carbon monoxide enhances plasmatic coagulation, we determined if patients undergoing resection of colon and pancreatic tumors had an increase in endogenous carbon monoxide and plasmatic hypercoagulability. Patients with colon (n = 17) and pancreatic (n = 10) tumors were studied. Carbon monoxide was determined by the measurement of carboxyhemoglobin (COHb). A thrombelastographic method to assess plasma coagulation kinetics and formation of carboxyhemefibrinogen (COHF) was utilized. Nonsmoking patients with colon and pancreatic tumors had abnormally increased COHb concentrations of 1.4 ± 0.9 and 1.9 ± 0.7%, respectively, indicative of HO-1 upregulation. Coagulation analyses comparing both tumor groups demonstrated no significant differences in any parameter; thus the data were combined for the tumor groups for comparison with 95% confidence interval values obtained from normal individuals (n = 30) plasma. Seventy percent of tumor patients had a velocity of clot formation greater than the 95% confidence interval value of normal individuals, with 53% of this hypercoagulable group also having COHF formation. Further, 67% of tumor patients had clot strength that exceeded the normal 95% confidence interval value, and 56% of this subgroup had COHF formation. Finally, 63% of all tumor patients had COHF formation. Future investigation of HO-1-derived carbon monoxide in the pathogenesis of colon and pancreatic tumor-related thrombophilia is warranted.
• Matika, R. W., Weinand, M. E., Steinbrenner, E. B., Nielsen, V. G., Matika, R. W., Lemole, G. M., Hussaini, S., & Baaj, A. A. (2014). Brain tumors enhance plasmatic coagulation: the role of hemeoxygenase-1.. Anesthesia and analgesia, 118(5), 919-24. doi:10.1213/ane.0000000000000048
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  Patients with brain tumors suffer significant thrombotic morbidity and mortality. In addition to increased thrombin generation via tumor release of tissue factor-bearing microparticles and hyperfibrinogenemia, brain tumors and surrounding normal brain likely generate endogenous carbon monoxide (CO) via the hemeoxygenase-1 (HO-1) system. CO has been shown to enhance plasmatic coagulation via formation of carboxyhemefibrinogen (COHF). Thus, our goals in this study were to determine whether patients with brain tumors had increased HO-1 upregulation/CO production, plasmatic hypercoagulability, and formation of COHF..Patients with brain tumors (N = 20) undergoing craniotomy had blood collected for determination of carboxyhemoglobin as a marker of HO-1 activity, plasmatic hypercoagulability (defined as clot strength > 95% confidence interval value of normal subject plasma), and COHF formation (determined with a thrombelastograph-based assay). Plasma obtained from commercially available normal subjects (N = 30) was used for comparison with brain tumor patient samples..Brain tumor patients had carboxyhemoglobin concentrations of 1.5% ± 0.5% (mean ± SD), indicative of HO-1 upregulation. Compared with normal subject plasma, brain tumor patient plasma had significantly (P < 0.0001) greater clot formation velocity (5.2 ± 1.5 vs 9.5 ± 2.3 dynes/cm/s, respectively) and significantly (P = 0.00016) stronger final clot strength (166 ± 28 vs 230 ± 78 dynes/cm, respectively). Ten of the brain tumor patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal subjects, and 12 of the brain tumor patients had COHF formation. Five of the brain tumor patients in the hypercoagulable subgroup had COHF formation. Last, 5 of the hypercoagulable patients had primary brain tumors, whereas the other 5 patients had metastatic tumors or an inflammatory mass lesion..A subset of patients with brain tumors has increased endogenous CO production, plasmatic hypercoagulability, and COHF formation. Future investigation of the role played by HO-1 derived CO in the pathogenesis of brain tumor-associated thrombophilia is warranted.
• Nielsen, V. G., & Garza, J. I. (2014). Comparison of the effects of CORM-2, CORM-3 and CORM-A1 on coagulation in human plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 25(8), 801-5. doi:10.1097/mbc.0000000000000146
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  Carbon monoxide derived from the catalytic action of heme oxygenase-1 or carbon monoxide-releasing molecules (CORMs) has been found to potentially be an anticoagulant or procoagulant agent. Of interest, two water-soluble CORMs, CORM-3 and CORM-A1, recently became commercially available. Thus, the purpose of the present study was to assess and compare the effects of the previously well studied CORM-2 to the effects of CORM-3 and CORM-A1 on coagulation in citrated human plasma with thrombelastography. Plasma exposed to CORMs was incubated at 37°C for at least one carbon monoxide release half-time, and then tissue factor-activated coagulation was commenced with calcium addition. CORM-2 and CORM-3 enhanced the velocity of clot formation and thrombus strength in a similar manner, whereas CORM-A1 did not affect coagulation. However, CORM-A1 did diminish tissue-type plasminogen activator initiated fibrinolysis. The similarity in effect on coagulation by CORM-2 and CORM-3 was likely secondary to the relatively inert effect of their ruthenium-containing carrier molecule, whereas the boron-containing CORM-A1 may have had no effect secondary to boron binding to fibrinogen, preventing carbon monoxide-mediated changes in fibrinogen protein structure via attached heme group(s). Future investigations with CORMs should have special attention to confounding effects of the carrier molecule.
• Pretorius, E., & Nielsen, V. G. (2014). Carbon monoxide: Anticoagulant or procoagulant?. Thrombosis research, 133(3), 315-21. doi:10.1016/j.thromres.2013.12.004
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  Within the past decade there have been several investigations attempting to define the impact of exogenous and endogenous carbon monoxide exposure on hemostasis. Critically, two bodies of literature have emerged, with carbon monoxide mediated platelet inhibition cited as a cause of in vitro human and in vitro/in vivo rodent anticoagulation. In contrast, interaction with heme groups associated with fibrinogen, α₂-antiplasmin and plasmin by carbon monoxide has resulted in enhanced coagulation and decreased fibrinolysis in vitro in human and other species, and in vivo in rabbits. Of interest, the ultrastructure of platelet rich plasma thrombi demonstrates an abnormal increase in fine fiber formation and matting that are obtained from humans exposed to carbon monoxide. Further, thrombi obtained from humans and rabbits have very similar ultrastructures, whereas mice and rats have more fine fibers and matting present. In sum, there may be species specific differences with regard to hemostatic response to carbon monoxide. Carbon monoxide may be a Janus-faced molecule, with potential to attenuate or exacerbate thrombophilic disease.
• Pretorius, E., & Nielsen, V. G. (2014). Iron and carbon monoxide enhance coagulation and attenuate fibrinolysis by different mechanisms.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 25(7), 695-702. doi:10.1097/mbc.0000000000000128
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  Two parallel lines of investigation elucidating novel mechanisms by which iron (scanning electron microscopy-based) and carbon monoxide (viscoelastic-based) enhance coagulation and diminish fibrinolysis have emerged over the past few years. However, a multimodal approach to ascertain the effects of iron and carbon monoxide remained to be performed. Such investigation could be important, as iron and carbon monoxide are two of the products of heme catabolism via heme oxygenase-1, an enzyme upregulated in a variety of disease states associated with thrombophilia. Human plasma was exposed to ferric chloride, carbon monoxide derived from carbon monoxide-releasing molecule-2, or their combination. Viscoelastic studies demonstrated ferric chloride and carbon monoxide mediated enhancement of velocity of growth, and final clot strength, with the combination of the two molecules noted to have all the prothrombotic kinetic effects of either separately. Parallel ultrastructural studies demonstrated separate types of fibrin polymer cross-linking and matting in plasma exposed to ferric chloride and carbon monoxide, with the combination sharing features of each molecule. In conclusion, we present the first evidence that iron and carbon monoxide interact with key coagulation and fibrinolytic processes, resulting in thrombi that begin to form more quickly, grow faster, become stronger, and are more resistant to lysis.
• Pretorius, E., & Nielsen, V. G. (2014). Iron-enhanced coagulation is attenuated by chelation: thrombelastographic and ultrastructural analysis.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 25(8), 845-50. doi:10.1097/mbc.0000000000000160
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  Increased circulating ferritin and free iron have been found in a variety of disease states associated with thrombophilia. When blood or plasma is exposed to iron addition, characteristic changes in thrombus formation are observed by scanning electron microscopy, which include fusion of fibrin polymers, matting, and even sheeting of fibrin. A primary mechanism posited to explain iron-mediated hypercoagulability is hydroxyl radical formation and modification of fibrinogen; however, iron has also been demonstrated to bind to fibrinogen. We have recently demonstrated that iron enhances coagulation, manifested as a decrease in the time of onset of coagulation. Using clinically encountered concentrations of iron created by addition of FeCl3 to human plasma, we demonstrated that iron-mediated changes in reaction time determined by thrombelastography or changes in thrombus ultrastructure were significantly, but not completely, reversed by iron chelation with deferoxamine. Thus, reversible iron binding to fibrinogen mechanistically explains a significant portion of coagulation kinetic and ultrastructural hypercoagulability. Further investigation is needed to determine whether residual iron binding or other iron-mediated effects is responsible for hypercoagulability observed after chelation.
• Redford, D. T., Thompson, J. L., Thompson, J. L., Redford, D. T., Nielsen, V. G., & Mcculloch, J. C. (2014). Left atrial myxoma presenting as pulmonary embolism: potential role of heme oxygenase-1.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 25(6), 621-4. doi:10.1097/mbc.0000000000000097
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  We present the case of a patient with left atrial myxoma that presented with pulmonary embolism. The patient did not have any intracardiac communication between right and left sides of the heart. Using thrombelastography, the patient was determined to have an abnormally large velocity of plasma thrombus growth and strength with reduced vulnerability to lysis. Critically, increased carboxyhemoglobin concentrations were present, likely secondary to hemolysis from the tumor and engagement of systemic heme oxygenase-1. It was determined that the patient's plasmatic hypercoagulability was in part due to carboxyhemefibrinogen formation via a thrombelastographic method. In addition to circulating hypercoagulability, the patient also had an area of chronic venous stasis in his left ankle that had not changed for over a decade prior to this thrombophilic episode. In conclusion, we present the first case of paradoxical pulmonary embolism in the presence of a left atrial myxoma, potentially secondary to a combination of hemolysis, heme oxygenase-1 up-regulation, systemic hypercoagulability/hypofibrinolysis, and regional venous stasis.
• Matika, R. W., Waer, A. L., Steinbrenner, E. B., Nielsen, V. G., Matika, R. W., Ley, M. L., & Alger, P. W. (2013). Plasmatic hypercoagulation in patients with breast cancer: role of heme oxygenase-1.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 24(8), 809-13. doi:10.1097/mbc.0b013e3283658b00
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  Breast cancer is an important health threat to women worldwide, and is associated with a 9-14% incidence of thrombophilia. Of interest, patients with breast cancer have been noted to have an increase in endogenous carbon monoxide production via upregulation of heme oxygenase-1 activity. Given that it has been demonstrated that carbon monoxide enhances plasmatic coagulation in vitro and in vivo, we sought to determine whether patients with breast cancer had an increase in endogenous carbon monoxide and concurrent plasmatic hypercoagulability. Breast cancer patients who were not smokers scheduled to undergo partial or complete mastectomy (n = 18) had 15 ml of whole blood collected via an indwelling intravenous catheter and anticoagulated with sodium citrate. Whole blood was centrifuged and citrated plasma assessed with a thromboelastometric method to measure coagulation kinetics and the formation of carboxyhemefibrinogen. Breast cancer patients were determined to have an abnormally increased carboxyhemoglobin concentration of 2.5 ± 1.3%, indicative of heme oxygenase-1 upregulation. Breast cancer patient plasma on average clotted 73% more quickly and had 32% stronger thrombus strength than normal individual (n = 30) plasma. Further, 44% of breast cancer patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal individuals, and 75% of this hypercoagulable subgroup had carboxyhemefibrinogen formation. Future investigation of the role played by heme oxygenase-1-derived carbon monoxide in the pathogenesis of breast cancer-related thrombophilia is warranted.
• Olver, C. S., & Nielsen, V. G. (2013). Thrombelastographic characterization of coagulation/fibrinolysis in horses: role of carboxyheme and metheme states.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 24(3), 273-8. doi:10.1097/mbc.0b013e32835bfd6e
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  Carboxyheme and metheme states modulate hemostasis in humans and other species. Further, carbon monoxide and/or nitric oxide production increase in inflammatory disorders involving the gastrointestinal tract, with associated hypercoagulability or hypocoagulability. In particular, the horse suffers both thrombotic or coagulopathic complications during acute gastrointestinal disease. This investigation characterized the thrombelastographic response to carboxyheme (via CORM-2) or metheme (via phenylhydroxylamine, PHA) states without/with addition of tissue type plasminogen activator. Citrated plasma was obtained from 14 normal mares and three horses with enteritis. In normal horses, a carboxyheme state did not enhance the velocity of clot growth and minimally enhanced clot strength (9%). In contrast, a metheme state was associated with a decrease in the velocity of clot formation (54%) and clot strength (47%). During fibrinolysis, a carboxyheme state significantly decreased the onset (113%) and velocity (27%) of fibrinolysis; however, in contrast, a metheme state more markedly increased the onset (84%) and velocity (133%) of fibrinolysis. These data support a carbon monoxide-dominant modulation of hemostasis in normal horses. In contrast, an increase in the severity of acute gastrointestinal disease was associated with a likely nitric oxide-mediated, metheme state-induced hypocoagulable/hyperfibrinolytic state. Additional investigation is warranted to determine the role played by carbon monoxide and nitric oxide in equine thrombotic and coagulopathic disease.
• Smith, M. C., & Nielsen, V. G. (2013). Detection of carboxyhemefibrinogen and methemefibrinogen in a patient with thrombosis of a HeartMate II ventricular assist device.. ASAIO journal (American Society for Artificial Internal Organs : 1992), 59(1), 93-5. doi:10.1097/mat.0b013e31827986e6
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  Ventricular assist device (VAD) thrombosis is a devastating, potentially fatal complication suffered by patients requiring mechanical circulatory support. We present a patient with thrombosis of a HeartMate II VAD with concurrent hemolysis and increased carbon monoxide formation. Using a specialized thrombelastographic assay, we detected marked plasmatic hypercoagulability mediated in part by the formation of carboxyhemefibrinogen.
• Smith, M. C., Pearson, E. C., & Nielsen, V. G. (2013). Increased carbon monoxide production by hemeoxygenase-1 caused by device-mediated hemolysis: thrombotic phantom menace?. Artificial organs, 37(11), 1008-14. doi:10.1111/aor.12122
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  Replacement of key components of the circulatory system with artificial devices has become the mainstay of therapy for conditions such as end-stage valvular disease or congestive heart failure. Unfortunately, device thrombosis and thromboembolic morbidity persist despite optimized anticoagulation. This work reviews the commonly known causes of device-associated thrombophilia, introduces recent literature concerning the effect of carbon monoxide on coagulation, and presents new patient data linking endogenously produced carbon monoxide with device-associated thrombosis. A new paradigm involving the interaction of red blood cell lysis-induced upregulation of hemoxygenase-1, increased endogenous carbon monoxide, hyperfibrinogenemia, and contact protein/microparticle-induced thrombin generation is presented.
• Steinbrenner, E. B., Nielsen, V. G., & Hafner, D. T. (2013). Can divergent plasmin-antiplasmin-carbon monoxide interactions in young, healthy tobacco smokers explain the 'smoker's paradox'?. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 24(4), 381-5. doi:10.1097/mbc.0b013e32835d53ec
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  In the setting of acute myocardial infarction, decreases in early/late mortality, reocclusion after thrombolysis, and restenosis rate after percutaneous intervention are lower in smokers - this phenomenon has been designated as the 'smoker's paradox'. These benefits of smoking, however, are abrogated by stent placement. We hypothesized that fibrinolytic vulnerability would change in response to smoking, and that inhaled carbon monoxide may play a role. Smoking patients (n = 20, two cigarettes consumed within 90 min, average carboxyhemoglobin concentration of 5%) had plasma collected and normal individual (n = 20) plasma was also obtained. Thrombelastographic analyses conducted with addition of tissue-type plasminogen activator revealed that with the exception of the rate of thrombus generation, there was little difference in fibrinolytic kinetics between normal and smoking individuals. Addition of exogenous carbon monoxide resulted in diminished fibrinolytic response to the same extent in both groups. Subanalyses demonstrated that the smoking cohort had both hyperfibrinolytic and hypofibrinolytic patients as defined by confidence interval (5-95%) values generated from normal individuals. Addition of carbon monoxide reduced hyperfibrinolytic parameter values by 80% in smokers, whereas only a 17% decrease in hypofibrinolytic values changed. Our investigation suggests that 'smoker's paradox' involves a marked change in the character of the plasmin-antiplasmin-carbon monoxide interaction. Further investigation will be required to further define the molecular mechanism responsible for the 'smoker's paradox'.
• Steinbrenner, E. B., Nielsen, V. G., & Hafner, D. T. (2013). Tobacco smoke-induced hypercoagulation in human plasma: role of carbon monoxide.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 24(4), 405-10. doi:10.1097/mbc.0b013e32835d5458
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  Virtually every disease state associated with chronic or acute thrombosis has had smoking identified as a risk factor. Further, smoking enhances clot strength as assessed by thrombelastography. Critically, carbon monoxide, a product of cigarette smoking, has been demonstrated to enhance plasmatic coagulation in vitro via modulation of a heme associated with fibrinogen. We hypothesized that plasmatic hypercoagulability and formation of carboxyhemefibrinogen (COHF) detected with thrombelastographic methods would be observed after cigarette smoking. Smoking participants (n = 20, two cigarettes consumed within 90 min, average carboxyhemoglobin concentration of 5%) had plasma collected and normal participant (n = 20) plasma was also obtained. Thrombelastographic analyses revealed that plasma obtained from smokers had an 86% greater velocity of clot growth and 65% larger clot strength than normal participant plasma. Forty-five percent of smokers had plasma clot strength that exceeded the 95th percentile of normal participant plasma values; 45% of smoking participants had detectable COHF; and 20% of smoking participants were both hypercoagulable with COHF present. We conclude that smoking induced a hypercoagulable state and COHF formation in an important portion of participants tested. Future investigations of the effects of smoking, plasmatic hypercoagulation and COHF formation are planned in populations with established atherosclerotic/thrombotic disease.
• Zelman, E. A., Nielsen, V. G., Guerrero, M. A., & Garol, B. D. (2013). Hemeoxygenase-1 mediated hypercoagulability in a patient with thyroid cancer.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 24(6), 663-5. doi:10.1097/mbc.0b013e328363ab86
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  Thyroid cancers can cause significant regional thrombotic morbidity and mortality. Of interest, thyroid cancer cell lines can have upregulation of the carbon monoxide-producing enzyme, hemeoxygenase-1. Carbon monoxide has been demonstrated to markedly enhance plasmatic coagulation in vitro and in vivo via enhancement of fibrinogen's substrate properties by binding to a fibrinogen-associated heme group(s). We present a patient undergoing removal of a malignant thyroid tumour who was serendipitously found to have abnormally increased carboxyhaemoglobin concentration (2.4%) and plasmatic hypercoagulability with a carbon monoxide-mediated clot strength as determined by a thrombelastographic method. This initial observation serves as a rationale to further investigate the role played by hemeoxygenase-1 upregulation in the setting of cancers associated with increased endogenous carbon monoxide production.
• Nielsen, V. G. (2012). Coagulation crystal ball: why can't we predict bleeding after cardiac surgery?. Anesthesia and analgesia, 115(3), 490-2. doi:10.1213/ane.0b013e318261f425
• Nielsen, V. G., & Hafner, D. T. (2012). Freezing does not decrease carbon monoxide-mediated hypercoagulation and hypofibrinolysis in human plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 23(8), 784-6. doi:10.1097/mbc.0b013e328358e8d5
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  Carbon monoxide (CO) has been demonstrated to enhance coagulation and attenuate fibrinolysis in vitro and in vivo. Hemostasis is affected by CO interactions with key heme-modulated molecules. We wished to determine whether freezing would affect CO-mediated changes in coagulation/fibrinolysis in plasma in anticipation of collecting samples both within our institution and from collaborating centers. Plasma was exposed to CO by addition of 0-100 μmol/l tricarbonyldichlororuthenium (II) dimer, with a portion of plasma immediately frozen at -80 °C. Unfrozen plasma was subjected to thrombelastographic analysis following tissue factor activation, with some samples exposed to tissue type plasminogen activator. Frozen plasma was subsequently thawed and similarly analyzed. Freezing did not significantly change CO-mediated enhancement of coagulation or attenuation of fibrinolysis. Hemostatic changes in plasma exposed to CO are not affected by a freeze-thaw cycle, which will permit local batch processing of samples and transport of samples on dry ice from collaborating centers.
• Persaud, J. M., Nielsen, V. G., Malayaman, S. N., & Cohen, J. B. (2012). Carbon monoxide releasing molecule-2 improves protamine-mediated hypocoagulation/hyperfibrinolysis in human plasma in vitro.. The Journal of surgical research, 173(2), 232-9. doi:10.1016/j.jss.2010.09.007
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  Protamine sulfate has been implicated as a possible cause of coagulopathy for over 20 y. Protamine has been demonstrated to decrease thrombin activity and to prolong bleeding. We tested the hypothesis that a new hemostatic agent, carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2), could attenuate protamine-mediated hypocoagulation/hyperfibrinolysis in plasma..Normal plasma was exposed to 0, 12.5, 25, or 50 μg/mL of protamine, with or without addition of 100 μM CORM-2. Tissue factor was used to initiate coagulation, and tissue type plasminogen activator was added in some experiments. Additional experiments were performed wherein plasma was exposed to protamine combined with 0% or 30% dilution with normal saline, with or without CORM-2 addition. Thrombelastography was performed until either stable clot strength or clot lysis occurred..Protamine, in a concentration-dependent fashion, significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth, and decreased clot strength in the absence or presence of tissue type plasminogen activator. Further, protamine significantly decreased the time to onset of fibrinolysis and decreased clot lysis time. CORM-2 exposure significantly diminished all aforementioned protamine-mediated effects on coagulation and fibrinolysis. Lastly, CORM-2 addition significantly increased the velocity of clot growth and strength in diluted, protamine-exposed plasma..CORM-2 attenuated protamine-mediated hypocoagulation/hyperfibrinolysis at clinically encountered concentrations. Additional preclinical investigation is warranted to determine if CORM-2 administration will be efficacious in diminishing coagulopathy caused by protamine.
• Wasko, K. A., Vosseller, K., Nielsen, V. G., Malayaman, S. N., & Arkebauer, M. R. (2012). Carbon monoxide-releasing molecule-2 decreases fibrinolysis in vitro and in vivo in the rabbit.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 23(1), 104-7. doi:10.1097/mbc.0b013e32834ea012
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  Administration of carbon monoxide derived from carbon monoxide-releasing molecules (CORMs) have been demonstrated to enhance coagulation and diminish fibrinolysis in vitro at small concentrations (100-200 μmol/l) in human and rabbit plasma, whereas in vivo administration of large concentrations (>1400 μmol/l) of carbon monoxide has mildly increased bleeding time in vivo in rats. We sought to determine whether CORM-2 [tricarbonyldichlororuthenium (II) dimer] would improve coagulation and attenuate tissue-type plasminogen activator (tPA)-mediated fibrinolysis in rabbit whole blood as determined in vitro by thrombelastography and in an in vivo preclinical rabbit model of ear bleeding time administered intravenous tPA (1 mg/kg). Addition of 200, 400 and 600 μmol/l CORM-2 to whole blood significantly improved coagulation and attenuated fibrinolysis compared with blood without CORM-2. Rabbits administered CORM-2 (10 mg/kg, 279 μmol/l) had a small but significant decrease in bleeding time before tPA administration. Administration of tPA resulted in bleeding times more than six-fold greater than baseline in animals not exposed to CORM-2, whereas rabbits administered CORM-2 had significantly smaller (more than five-fold less) bleeding time values after tPA administration. CORM-2 administration significantly decreases fibrinolytic bleeding in the rabbit in vivo. Additional preclinical investigation of the effects of CORM-2 on coagulopathy (e.g. heparin-mediated or clopidogrel-mediated) utilizing this rabbit model are planned.
• Welsby, I. J., Ushakumari, D. S., Nielsen, V. G., & Machovec, K. A. (2012). The procoagulant properties of purified fibrinogen concentrate are enhanced by carbon monoxide releasing molecule-2.. Thrombosis research, 129(6), 793-6. doi:10.1016/j.thromres.2011.08.005
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  Fibrinogen concentrate has been demonstrated to enhance coagulation in vitro and in several clinical settings of coagulopathy. We have recently demonstrated that carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2) enhances fibrinogen as a substrate for thrombin via an attached heme. The objective of this study was to determine if CORM-2 modified fibrinogen concentrate would enhance coagulation more effectively than CORM-2 naïve fibrinogen concentrate..In the first series of experiments, fibrinogen concentrate (final concentration 300mg/dl) was exposed to 0, 50 or 100μM CORM-2 for 5min at 37°C prior to being added to citrated, fibrinogen depleted plasma. In another series of experiments, citrated plasma obtained from 12 normal subjects was 50% diluted with crystalloid to which was added fibrinogen concentrate (final concentration 300mg/dl) exposed to 0 or 100μM CORM-2. Coagulation was activated with tissue factor (n=8 per condition). Thrombus growth was monitored with thrombelastography for 15min..CORM-2 modification of fibrinogen concentrate significantly enhanced the velocity of clot formation (30-50%) and strength (15-31%) in fibrinogen deficient plasma. Similarly, while diluted plasma-derived thrombi demonstrated a marked decrease in velocity of formation (54%) and strength (61%), fibrinogen concentrate significantly enhanced velocity (217%) and strength (171%); however, CORM-2 modified fibrinogen concentrate significantly increased velocity (303%) and strength (205%) to a greater extent. Additional in vitro investigation and in vivo preclinical assessments of the hemostatic efficacy of CORM-2 modified fibrinogen concentrate are warranted.
• Nielsen, V. G. (2011). Epsilon-aminocaproic acid, neonates, and cardiopulmonary bypass.. Anesthesia and analgesia, 112(3), 735. doi:10.1213/ane.0b013e31820547e6
• Nielsen, V. G., & George, S. J. (2011). Carbon monoxide releasing molecule-2 attenuates the anticoagulant and amplifies the hypofibrinolytic effects of hypothermia in human plasma in vitro.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(1), 67-72. doi:10.1097/mbc.0b013e3283423534
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  Hypothermia is known to contribute to coagulopathy in trauma and other major surgical procedures. Although the effects of hypothermia on coagulation have been characterized, the effects on fibrinolysis remained to be elucidated. Thus, our goals were to discern the effects of hypothermia on fibrinolysis in human plasma, and secondarily determine if a new procoagulant/antifibrinolytic molecule, carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) would modify thrombus growth/disintegration under hypothermic conditions. Normal plasma was exposed to 0 or 100 μmol/l CORM-2, with coagulation activated by tissue factor and fibrinolysis initiated with 100 U/ml tissue-type plasminogen activator. Plasma samples were exposed to 37°, 35°, 33°, 31°, 29°, or 27°C (n = 6 per temperature/CORM-2 concentration). Thrombus growth/disintegration kinetics were monitored with thrombelastography until clot lysis time occurred. Hypothermia significantly prolonged the onset and decreased the velocity of clot growth in plasma without decreasing clot strength. Although hypothermia did not affect the time to onset of fibrinolysis, it did significantly decrease the velocity of lysis. The addition of CORM-2 significantly increased the velocity of clot growth and clot strength at all temperatures tested compared with unexposed plasma. Further, CORM-2 addition significantly prolonged the onset of fibrinolysis and diminished the velocity of lysis. Hypothermia resulted in slower growing, slower lysing thrombi in normal plasma. CORM-2 enhanced coagulation and markedly attenuated fibrinolysis at all temperatures tested. Further investigation is warranted to determine if CORM-2 administration can improve hemostasis in preclinical models of hypothermia and trauma.
• Nielsen, V. G., Machovec, K. A., Chambers, B. P., & Brodsky, M. A. (2011). Platelet-mediated thrombolysis in patients with δ-storage pool deficiency: a thrombelastographic analysis.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(7), 610-2. doi:10.1097/mbc.0b013e328349a2a4
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  We present the first thrombelastographic descriptions of three patients with δ-storage pool deficiency, a platelet disorder that involves a deficiency of dense granules and moderate bleeding. The patients demonstrated a 49-54% loss of platelet-mediated clot strength over a 1-2-h period after normal thrombus formation. This pattern persisted, with some attenuation of loss of strength following administration of epsilon aminocaproic acid, desmopressin and platelets for tonsillectomy. Assessment of platelet function in patients with platelet granule disorders can be accomplished with thrombelastographic methods in ambulatory and perioperative settings; however, the effects of therapy for this disorder cannot be monitored with thrombelastography without obtaining a blood sample prior to prophylactic hemostatic intervention.
• Nielsen, V. G., Malayaman, S. N., Machovec, K. A., Cohen, J. B., Bernhardt, B. E., & Arkebauer, M. R. (2011). Carbon monoxide releasing molecule-2 enhances α2-antiplasmin activity.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(4), 345-8. doi:10.1097/mbc.0b013e328344c657
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  The objective of this study was to determine whether carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer, CORM-2) directly affects α2-antiplasmin activity. For this purpose, purified α2-antiplasmin was exposed to 0 or 100 μmol/l CORM-2 for 5 min at 37°C and then placed in α2-antiplasmin-deficient plasma (25 μg/ml α2-antiplasmin and 3.3 μmol/l CORM-2 final concentrations). In a second series of experiments, α2-antiplasmin and deficient plasma were combined and then exposed to 0 or 100 μmol/l CORM-2. Coagulation was activated with tissue factor and fibrinolysis initiated with tissue-type plasminogen activator (n = 8 per condition). Thrombus growth/disintegration kinetics were monitored with thrombelastography until clot lysis time occurred. Samples containing α2-antiplasmin preexposed to 100 μmol/l CORM-2 demonstrated no changes in the velocity of clot growth, but had a significant prolongation of the time to maximum rate of lysis, clot lysis time, and a significant decrease in the maximum rate of clot lysis compared with samples preexposed to 0 μmol/l CORM-2. In sharp contrast, addition of 100 μmol/l CORM-2 to premixed α2-antiplasmin in its deficient plasma resulted in significant, marked increases in the velocity of clot growth and the strength with concurrent antifibrinolytic effects as in the first series. In conclusion, CORM-2 exerts its antifibrinolytic effects by direct enhancement of α2-antiplasmin activity. It appears that combined modification of both fibrinogen and α2-antiplasmin are responsible for the robust procoagulant/antifibrinolytic effects of CORM-2 in the fibrinolytic environment.
• Nielsen, V. G., Martin-ross, A., Kirklin, J. K., Khan, E. S., Green, P., Green, M. S., & George, J. F. (2011). Carbon monoxide-releasing molecule-2 enhances coagulation and diminishes fibrinolytic vulnerability in diluted plasma in vitro.. The Journal of trauma, 70(4), 939-47. doi:10.1097/ta.0b013e3181e50a3b
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  A carbon monoxide-releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) enhances coagulation and attenuates vulnerability to fibrinolysis in normal and hemophiliac human plasma. We tested the hypothesis that plasma diluted with resuscitative fluids would demonstrate improved coagulation and decreased fibrinolytic vulnerability after exposure to CORM-2..Normal, platelet-poor plasma was diluted 0%, 20%, 30%, 40%, or 50% with 0.9% NaCl (NS) or low-molecular-weight hydroxyethyl starch (VOL) and, subsequently, exposed to 0 μmol/L or 100 μmol/L CORM-2 before activation with tissue factor (n = 4 per condition). Additional plasma samples diluted with NS or VOL (0% or 30%) were exposed to 0 μmol/L or 100 μmol/L CORM-2 and 0 U/mL or 100 U/mL tissue-type plasminogen activator to assess fibrinolytic vulnerability (n = 8 per condition). Thrombelastographic data were collected until either clot strength stabilized or clot lysis occurred, as appropriate..CORM-2 exposure maintained normal to supranormal velocity of clot formation and strength in plasma diluted up to 40% with NS. In contrast, although CORM-2 exposure improved coagulation kinetics, dilution with VOL markedly degraded thrombus formation kinetics. Similarly, fibrinolytic vulnerability to tissue-type plasminogen activator was markedly improved by CORM-2 exposure in samples diluted with NS, whereas VOL-diluted thrombi were still abnormally weak and easily lysed compared with undiluted samples despite CORM-2 exposure..CORM-2 exposure attenuated the decrease in coagulation kinetics and enhancement of fibrinolytic vulnerability associated with hemodilution. Extensive preclinical investigation remains to be performed to determine the route of administration, safety, and efficacy of CORM-2 and other CORMs to treat trauma-associated bleeding.
• Parekh, J., Nielsen, V. G., Heyer, A., Green, P., & Green, M. S. (2011). Endotracheal cardiac output monitor in a patient with severe tricuspid regurgitation.. Journal of cardiothoracic and vascular anesthesia, 25(5), 830-2. doi:10.1053/j.jvca.2010.05.007
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  DURING CARDIAC SURGERY, the accurate measurements: of cardiac output (CO) and intracardiac pressures an assist in guiding anesthetic management. The balloonipped pulmonary artery catheter (PAC) was introduced in 9701 and became the gold standard for measuring CO via thermodilution. Many hemodynamic values are obtained from this type of monitoring, including intracardiac pressures, pulmonary artery pressures, pulmonary artery occlusion pressures, CO, and mixed venous oxygen saturation. This technique is invasive, and many factors can lessen its accuracy, including disease processes that increase pulmonary vascular resistance, changes in intrathoracic and intrapleural pressures, variability in pulmonary blood flow, cardiac valvular abnormalities, and intracardiac shunting. Doppler echocardiography, which was developed in the 1970s, allows a more accurate estimation of cardiac values and has the advantage of allowing direct visualization of cardiac function. Impedance cardiography, which was first described in 1991, provides further advances over earlier modalities in that it provides continuous calculation of CO and cardiac index (CI). Impedance cardiography offers a less invasive alternative to the thermodilution technique for the determination of continuous CO in comparison with PAC, which often is criticized because of its uncertain risk-to-benefit ratio.2-4 The endotracheal cardiac output monitor (ECOM) is an endotracheal tube with 6 electrodes distributed evenly on the surface of the balloon positioned in proximity to the ascending aorta in order to measure CO (Fig 1).5,6 The electrodes produce mA of alternating current at 100 kHz and measure the hanges in electrical impedance caused by pulsatile blood flow n the aorta.6 The field current is produced between the balloon lectrodes and a ground electrode located on the shaft of the ndotracheal tube.6 The electrodes are used to measure impednce signals in 3 planes.6 The 3-dimensional impedance fields
• Persaud, J. M., Nielsen, V. G., Malayaman, S. N., & Cohen, J. B. (2011). Carbon monoxide releasing molecule-2 enhances coagulation and attenuates fibrinolysis by two mechanisms: insights gained with colloid dilution.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(1), 60-6. doi:10.1097/mbc.0b013e3283423516
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  Carbon monoxide releasing molecule-2 (CORM-2, tricarbonyldichlororuthenium (II) dimer) enhances coagulation and attenuates vulnerability to fibrinolysis. Our goal was to further define the CORM-2-mediated mechanisms using colloids with known effects on coagulation/fibrinolytic proteins. Plasma diluted 0 or 30% with 0.9% NaCl, 5% human albumin, low molecular weight hydroxyethyl starch (130 kDa) or high molecular weight hydroxyethyl starch (450 kDa) was exposed to 0 or 100 μmol/l CORM-2 before activation with tissue factor (n = 8 per condition). A second identically diluted series of experiments were performed with the addition of tissue-type plasminogen activator. Thrombelastographic data were collected until clot strength stabilized or clot lysis occurred. Dilution resulted in fluid-specific decreases in velocity of clot growth, strength and clot growth time with progressive increases in macromolecule size. CORM-2 exposure significantly increased the velocity of clot formation and strength (thin fibrin fiber promoting), but not clot growth time, under all conditions. Fibrinolysis was enhanced to the greatest extent by hydroxyethyl starch (antiα2-antiplasmin effect), and CORM-2 addition attenuated fibrinolysis in all conditions (α2-antiplasmin enhancement). CORM-2 exposure attenuated the decrease in coagulation kinetics mediated by hemodilution by two different mechanisms based on kinetic profile differences between the diluents tested. Further laboratory-based investigation is warranted to further define the molecular mechanisms responsible for CORM-2-mediated effects on coagulation and fibrinolysis.
• Vosseller, K., Nielsen, V. G., & Arkebauer, M. R. (2011). Redox-based thrombelastographic method to detect carboxyhemefibrinogen-mediated hypercoagulability.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(8), 657-61. doi:10.1097/mbc.0b013e32834aa7b0
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  Cigarette smoking is associated with plasmatic hypercoagulability, and carbon monoxide has been demonstrated to enhance coagulation by binding to a fibrinogen-bound heme. Our objective was to design and test a redox-based method to detect carboxyhemefibrinogen. Normal, pooled, citrated plasma was exposed to 0-100 μmol/l carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2) before or after exposure to the organic reductant phenylhydroxylamine (PHA, 0-30 mmol/l), a compound that rapidly converts Fe(+2) to Fe(+3) in heme. Addition of calcium and tissue factor activation in disposable thrombelastographic cups was performed, followed by data collection at 37°C for 15 min. Elastic modulus (G, dynes/cm(2)) was the primary endpoint. CORM-2 significantly increased G values by 67.8% compared to unexposed plasma; pretreatment with 10 mmol/l PHA significantly decreased G values in CORM-2-exposed plasma by 77.1%, whereas 30 mmol/l PHA was required to significantly decrease G values by 64.0% in plasma following CORM-2 pre-exposure. G values were not significantly different between unexposed plasma and plasma exposed to CORM-2 followed by 30 mmol/l PHA addition. Conversion of fibrinogen-bound to the metheme state alone decreased G by 34.3-38.9% following exposure to 10-30 mmol/l PHA. Conversion of fibrinogen-bound heme Fe(+2) to Fe(+3) with PHA abrogated carbon monoxide-mediated increases in clot strength. Clinical trials are planned to investigate smoking individuals to mechanistically link carboxyhemefibrinogen formation with in-vitro hypercoagulability.
• Vosseller, K., Nielsen, V. G., Malayaman, S. N., Kanaparthy, S. S., & Arkebauer, M. R. (2011). Carbon monoxide and nitric oxide modulate α₂-antiplasmin and plasmin activity: role of heme.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(8), 712-9. doi:10.1097/mbc.0b013e32834c73f9
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  Cigarette smoke and carbon monoxide (CO) released from tricarbonyldichlororuthenium (II) dimer (CORM-2) attenuate fibrinolysis. The purpose of the present study was to determine whether CO diminished fibrinolysis by enhancement of α₂-antiplasmin via a putative heme group. Plasma, isolated α₂-antiplasmin and isolated plasmin were exposed to CO released from CORM-2 and nitric oxide (NO) via a NO donor to induce carboxyheme and metheme states, respectively. Exposed, isolated enzymes were placed in either α₂-antiplasmin-deficient or normal plasma. Effects of CO and NO on tissue-type plasminogen activator initiated fibrinolysis were determined by thrombelastography. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify heme released from α₂-antiplasmin and plasmin. CO significantly enhanced α₂-antiplasmin activity, but decreased plasmin activity. NO decreased both α₂-antiplasmin and plasmin activity. Although inadequate LC-MS/MS data were obtained with α₂-antiplasmin (secondary to glycosylation), a putative plasmin-associated heme was identified. CO elicits hypofibrinolysis by enhancing α₂-antiplasmin activity and decreasing plasmin activity. On the basis of the responses to NO and LC-MS/MS data, it is highly likely that both enzymes are modulated by attached heme groups. Efforts to develop methods to detect CO-mediated hypercoagulability are ongoing, with the goal of identifying populations at risk of thrombotic morbidity secondary to cigarette smoking.
• Vosseller, K., Nowak, M., Nielsen, V. G., Malayaman, S. N., & Cohen, J. B. (2011). Fibrinogen is a heme-associated, carbon monoxide sensing molecule: a preliminary report.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(5), 443-7. doi:10.1097/mbc.0b013e328345c069
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  The objective of this study was to determine how carbon monoxide directly modifies fibrinogen utilizing liquid chromatography-mass spectrometry (LC-MS/MS) by examining fibrinogen exposed to carbon monoxide releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer; CORM-2]. Purified fibrinogen was exposed to 0, 25, 50 or 100 μmol/l CORM-2 for 5 min at 37°C and then stored at -80°C before analyses with LC-MS/MS. In a second series of experiments, normal plasma was exposed to 0 or 100 μmol/l CORM-2 in the absence or presence of the nitric oxide donor sodium nitroprusside and hydroquinone (an organic reductant) to compete with carbon monoxide binding to a putative heme group found on fibrinogen. Coagulation was activated with tissue factor (n=8 per condition). Thrombus growth was monitored with thrombelastography for 15 min. LC-MS/MS did not detect any direct modifications of amino acids in fibrinogen, but detection of small regions of both the alpha and gamma chains was lost following exposure to CORM-2 and endoproteinase digestion with trypsin and Glu-C. An ion with the same m/z and expected retention time as heme was found in the purified fibrinogen. Exposure of plasma to nitric oxide/hydroquinone significantly decreased CORM-2-mediated enhancement of coagulation without affecting the coagulation kinetics of plasma not exposed to CORM-2. Carbon monoxide derived from CORM-2 likely modifies fibrinogen via modulation of a fibrinogen-associated heme group(s). Whereas the precise molecular location of heme attachment and three-dimensional conformational change secondary to carbon monoxide exposure remain to be determined, fibrinogen appears to be a carbon monoxide sensing molecule.
• Wasko, K. A., Vosseller, K., Sadacharam, K., Nielsen, V. G., Mangla, D., Gomes, S. B., Chawla, N., & Arkebauer, M. R. (2011). Carbon monoxide-releasing molecule-2 enhances coagulation in rabbit plasma and decreases bleeding time in clopidogrel/aspirin-treated rabbits.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(8), 756-9. doi:10.1097/mbc.0b013e32834c7412
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  Administration of carbon monoxide derived from carbon monoxide-releasing molecules has been demonstrated to enhance coagulation in vitro at small concentrations (100-200 μmol/l) in human and rabbit plasma. We sought to determine if carbon monoxide-releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer, CORM-2] would improve coagulation in rabbit plasma in vitro via thrombelastography and in an in vivo preclinical rabbit model of ear bleeding time following administration of clopidogrel (20 mg/kg) with aspirin (10 mg/kg) via gavage. Addition of 100 μmol/l CORM-2 to rabbit plasma significantly improved coagulation. This procoagulant effect was blocked by pre-exposure of plasma to an agent that converts hemefibrinogen to methemefibrinogen in human plasma, preventing carbon monoxide binding and enhancement of coagulation. Rabbit ear bleeding time was 5.8 ± 1.1 min 2-3 h after clopidogrel/aspirin administration. Bleeding time significantly decreased to 2.6 ± 0.6 min, 5 min after administration of CORM-2 (10 mg/kg; 279 μmol/l 'best-case' instantaneous concentration) intravenously. CORM-2 enhances plasmatic coagulation in a manner similar to that of human plasma in vitro, and plasmatic coagulation is enhanced in vivo by CORM-2 as well. Additional preclinical investigation of the effects of CORM-2 on coagulopathy (e.g. heparin or hemodilution mediated) utilizing this rabbit model is planned.
• Wechsler, A. S., Persaud, J. M., Nielsen, V. G., Malayaman, S. N., Kirklin, J. K., Entwistle, J. W., Cohen, J. B., & Boateng, P. (2011). Carbon monoxide releasing molecule-2 improves coagulation in patient plasma in vitro following cardiopulmonary bypass.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 22(5), 362-8. doi:10.1097/mbc.0b013e328344c66c
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  The objective of the present study was to determine if a new procoagulant molecule, carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) would improve coagulation following cardiopulmonary bypass (CPB). Plasma was obtained from patients undergoing elective cardiac surgery requiring CPB. Whole blood was collected and anticoagulated with sodium citrate after induction of anesthesia and again after CPB and heparin neutralization with protamine. Blood samples were centrifuged for 15 min, with plasma collected and stored at -80°C prior to analysis. Samples were subsequently exposed to 0 or 100 μmol/l CORM-2, with coagulation activated with tissue factor. Data were collected with thrombelastography until clot strength stabilized. Patients underwent CPB for 133 ± 61 min (mean ± SD). The velocity of thrombus formation was significantly decreased (52%) by CPB, as was clot strength (53%). Addition of CORM-2 to plasma samples obtained after CPB significantly increased the velocity of clot formation (75%) and strength (52%) compared to matched unexposed samples. The lesion of plasmatic coagulation associated with CPB was significantly improved in vitro by addition of CORM-2. If preclinical assessments of efficacy and safety of CORM-2 are favorable, future clinical trials involving CORM-2 or other CORMs as a hemostatic intervention in the setting of CPB are justified.
• Nielsen, V. G. (2010). The antifibrinolytic effects of carbon monoxide-releasing molecule-2 are fibrin and alpha2-antiplasmin dependent.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 21(6), 584-7. doi:10.1097/mbc.0b013e32833cea13
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  Carbon monoxide derived from a carbon monoxide-releasing molecule [tricarbonyldichlororuthenium (II) dimer; CORM-2] has been recently demonstrated to diminish tissue-type plasminogen activator (tPA)-mediated fibrinolysis of plasma thrombi via enhancement of alpha2-antiplasmin-plasmin interactions. The goal of this study was to confirm this mechanism by comparing tPA-mediated fibrinolysis with fibrin-independent, alpha2-antiplasmin-resistant streptokinase-mediated fibrinolysis in CORM-2 exposed plasma. Normal plasma was exposed to 0 or 100 micromol/l CORM-2, with coagulation activated with tissue factor and fibrinolysis initiated with 100 U/ml tPA or 50 U/ml streptokinase. Thrombus growth/disintegration kinetics were monitored with thrombelastography until clot lysis time occurred. Unlike tPA-lysed clots, streptokinase-exposed thrombi demonstrated no significant CORM-2-mediated prolongation of clot growth time or clot lysis time. In contrast, streptokinase-mediated lysis did not significantly change the CORM-2-mediated percentage increase in the maximum rate of clot growth or maximum rate of clot lysis compared with tPA-exposed thrombi. CORM-2 likely attenuates fibrinolysis by a fibrin-dependent/alpha2-antiplasmin-dependent mechanism. Additional molecular investigation (e.g., mass spectroscopy) is planned to further elucidate how CORM-2 modifies fibrinogen/fibrin.
• Nielsen, V. G., & Asmis, L. M. (2010). Hypercoagulability in the perioperative period.. Best practice & research. Clinical anaesthesiology, 24(1), 133-44. doi:10.1016/j.bpa.2009.09.012
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  One of the greatest disappointments associated with a successful surgical procedure is a thrombotic or thrombo-embolic complication in the postoperative period. Morbidity and mortality of the perioperative period are related, to a relevant degree, to perioperative thrombo-embolic events. Ranging from simple deep venous thrombosis to pulmonary embolism or arterial thrombosis, this class of complication invariably increases length of hospital stay or may result in mortality. The purpose of this review is to identify the procedures and patient populations noted to have thrombophilia in the postoperative period, link the changes in circulating and in situ haematological/biochemical substrates most likely responsible for morbidity, identify the clinical diagnostic modalities that detect recent/impending thrombosis and, lastly, consider the rational therapeutic approaches recommended for minimising postoperative thrombotic complications.
• Nielsen, V. G., & Malayaman, S. N. (2010). Protamine sulfate: crouching clot or hidden hemorrhage?. Anesthesia and analgesia, 111(3), 593-4. doi:10.1213/ane.0b013e3181eb6388
• Nielsen, V. G., Khan, E. S., & Huneke, R. B. (2010). Carbon monoxide releasing molecule-2 enhances coagulation in rat and rabbit plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 21(3), 298-9. doi:10.1097/mbc.0b013e3283380c88
• Nielsen, V. G., Kirklin, J. K., & George, J. F. (2010). Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in hemophilia A, hemophilia B and factor VII-deficient plasmas.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 21(1), 41-5. doi:10.1097/mbc.0b013e328331fd00
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  Carbon monoxide derived from carbon monoxide releasing molecules (CORMs) has been demonstrated to enhance normal plasma thrombus speed of growth and strength in vitro. We tested the hypothesis that tricarbonyldichlororuthenium (II) dimer (CORM-2) improves the velocity of formation and strength of hemophiliac plasma thrombi as determined by thrombelastography. Plasma deficient (
• Nielsen, V. G., Kirklin, J. K., Khan, E. S., & George, J. F. (2010). Carbon monoxide releasing molecule-2 enhances coagulation and diminishes fibrinolytic vulnerability in plasma exposed to heparin or argatroban.. Anesthesia and analgesia, 111(6), 1347-52. doi:10.1213/ane.0b013e3181fbc120
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  It has been recently demonstrated that a carbon monoxide releasing molecule (tricarbonyldichlororuthenium [II] dimer; CORM-2) enhances coagulation and attenuates vulnerability to fibrinolysis in normal and hemophiliac human plasma. We tested the hypothesis that plasma anticoagulated with heparin or argatroban would demonstrate improved coagulation and decreased fibrinolytic vulnerability after exposure to CORM-2..Normal plasma was anticoagulated with 0 to 0.1 U/mL unfractionated heparin or 0 to 1 μg/mL argatroban. Samples were subsequently exposed to 0 or 100 μM CORM-2 and activated with tissue factor. Additional samples with the same anticoagulant and CORM-2 exposure schema were incubated with 100 U/mL tissue-type plasminogen activator (tPA) to assess fibrinolytic vulnerability. Thrombelastographic data were collected until either clot strength stabilized or clot lysis occurred as appropriate..In the absence of tPA, CORM-2 significantly increased the velocity of clot growth in heparin (75%) and argatroban-exposed (40%) samples. Clot strength was also significantly increased in heparin (69%) and argatroban-exposed (72%) samples. In the presence of tPA, CORM-2-treated samples had even greater (94%-731%) increases in velocity of growth and strength after exposure to either anticoagulant and significantly increased clot lysis time (103%-200%)..CORM-2 exposure resulted in faster-growing, stronger, longer-lived thrombi after anticoagulation with heparin or argatroban. Additional preclinical investigation is warranted to determine whether CORM-2 administration will be useful in attenuating bleeding complications associated with thromboprophylaxis.
• Nielsen, V. G., Kirklin, J. K., Khan, E. S., & George, J. F. (2010). Carbon monoxide releasing molecule-2 enhances coagulation and diminishes fibrinolytic vulnerability in subjects exposed to warfarin.. Thrombosis research, 126(1), 68-73. doi:10.1016/j.thromres.2010.03.011
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  It has been recently demonstrated that a carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) enhances coagulation and attenuates vulnerability to fibrinolysis in normal and hemophiliac human plasma. We tested the hypothesis that plasma obtained from warfarin-treated subjects would demonstrate improved coagulation and decreased fibrinolytic vulnerability following exposure to CORM-2..Anonymous donor plasma samples with international normalized ratios (INR) values ranging from 1.5-5.4 were exposed to 0 or 100 microM CORM-2 and activated with tissue factor (12 samples). Additional samples within the same INR range were exposed to 0 or 100 microM CORM-2 and 0 or 100 U/ml tissue-type plasminogen activator (tPA) to assess fibrinolytic vulnerability (8 samples). Thrombelastographic data were collected until either clot strength stabilized or clot lysis occurred as appropriate..In the absence of tPA, all but one sample (INR=1.5) demonstrated a marked increase in the velocity of clot formation (40-577%) and strength (42-180%) following CORM-2 exposure. Of interest, in the presence of tPA, all samples (including the previously unresponsive sample) were noted to have an increase in the velocity of clot formation and strength, coupled with a prolonged onset to maximal rate of clot lysis (60-242%) and increased clot lysis time (74-149%). As with normal and hemophilic plasma, both enhancement of coagulation and attenuation of fibrinolysis occur following CORM-2 exposure in plasma from warfarin-treated subjects. Future investigation must determine if carbon monoxide releasing molecules could be used therapeutically to control bleeding in warfarin-treated patients.
• Nielsen, V. G., Malayaman, S. N., Kirklin, J. K., Khan, E. S., & George, J. F. (2010). Carbon monoxide releasing molecule-2 increases fibrinogen-dependent coagulation kinetics but does not enhance prothrombin activity.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 21(4), 349-53. doi:10.1097/mbc.0b013e328338948f
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  We have previously determined that tricarbonyldichlororuthenium (II) dimer (CORM-2) increases plasma clot velocity of formation and strength by enhancing thrombin-fibrinogen interactions as determined by thrombelastography. The purpose of the present investigation was to further define the nature of CORM-2 interaction with prothrombin and fibrinogen by exposing purified proteins to CORM-2 or generating protein concentration-response curves in the absence or presence of CORM-2. Purified prothrombin was exposed to 0 or 100 micromol/l CORM-2 prior to being added to prothrombin-deficient plasma (n=7-8 per condition). Fibrinogen-deficient plasma had fibrinogen added for a final concentration of 100-800 mg/dl and was exposed to 0 or 100 micromol/l CORM-2 (n=4 per condition). Following tissue factor activation, thrombelastographic data were collected until clot strength stabilized. Exposure of prothrombin to CORM-2 did not significantly enhance coagulation kinetics. In sharp contrast, CORM-2 exposure enhanced fibrinogen coagulation kinetics in a concentration-dependent fashion, with peak effect seen at a fibrinogen concentration of 300 mg/dl that then progressively decreased throughout the range tested. Our data demonstrate that CORM-2 does not enhance plasma coagulation kinetics by modifying prothrombin; instead, the concept that CORM-2 modifies fibrinogen is the most likely explanation for the enhanced thrombin-fibrinogen interactions observed.
• Nielsen, V. G., Messinger, J. D., Kirklin, J. K., & George, J. F. (2010). Carbon monoxide releasing molecule-2 decreases thick diameter fibrin fibre formation in normal and Factor XIII deficient plasmas.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 21(1), 101-5. doi:10.1097/mbc.0b013e3283333c5d
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  Carbon monoxide derived from carbon monoxide releasing molecules (CORMs) has been demonstrated to enhance normal plasma thrombus speed of growth and strength as well as diminish vulnerability to fibrinolysis in vitro. We tested the hypothesis that tricarbonyldichlororuthenium (II) dimer (CORM-2) would modify plasma thrombi ultrastructure as determined by electron microscopy. Normal and FXIII-deficient (
• Nielsen, V. G. (2009). Corn trypsin inhibitor decreases tissue-type plasminogen activator-mediated fibrinolysis of human plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 20(3), 191-6. doi:10.1097/mbc.0b013e3283258011
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  Corn trypsin inhibitor (CTI) has been considered the molecule of choice to inhibit activated factor XII (FXIIa) during the conduct of experimentation focusing on tissue factor-initiated coagulation. However, CTI-mediated attenuation of fibrinolysis following celite activation of coagulation was observed in pilot studies with the clot lifespan model. The goal of the present study was thus to characterize the mechanism(s) responsible for CTI-mediated hypofibrinolysis. Normal plasma was exposed to 0 or 49.6 microg/ml CTI, with coagulation initiated with celite or tissue factor. Fibrinolysis was initiated with tissue-type plasminogen activator (tPA). Additional experiments utilized plasminogen activator inhibitor 1 deficient or alpha2-antiplasmin-deficient plasma. Coagulation/fibrinolysis kinetics were monitored with the thrombelastography-based clot lifespan model. In addition to delaying the initiation of coagulation, CTI prolonged clot growth time, delayed the onset of lysis, and prolonged clot lysis time in normal plasma after celite activation. Conversely, CTI increased the speed of clot growth, clot strength, and prolonged clot lysis time after tissue factor activation. Experiments with plasma deficient in antifibrinolytic proteins supported a primary inhibition of tPA by CTI. In addition to anti-FXIIa effects following celite activation, CTI likely exerts an anti-tPA effect, which contributed to hypofibrinolysis in this model.
• Nielsen, V. G., Kirklin, J. K., & George, J. F. (2009). Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in human plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 20(5), 377-80. doi:10.1097/mbc.0b013e32832ca3a3
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  Carbon monoxide derived from degradation of heme by heme oxygenase or carbon monoxide releasing molecules (CORMs) has been demonstrated to decrease thrombosis in vivo and to weakly inhibit platelet aggregation. We tested the hypothesis that carbon monoxide released from tricarbonyldichlororuthenium (II) dimer (CORM-2) would diminish the velocity of formation and strength of plasma thrombi as determined by thrombelastography. Normal plasma was exposed to 0 or 100 micromol/l CORM-2 or inactivated CORM-2 (iCORM-2), with coagulation initiated with tissue factor or celite (n = 8 per condition). Additional experiments utilized factor XIII (FXIII) deficient plasma activated with celite. Coagulation kinetics was monitored with thrombelastography for 15 min. CORM-2, and to a lesser extent, iCORM-2, significantly (P < 0.05) increased the velocity of formation (122 and 56%, respectively) and strength (66 and 57%, respectively) of plasma clots initiated with either tissue factor or celite compared with thrombi not exposed to CORM-2 or iCORM. In FXIII deficient plasma CORM-2 significantly increased the velocity of clot formation (264%) and strength (240%). Carbon monoxide and iCORM-2 derived from CORM-2 markedly enhance the velocity of clot growth and strength. These findings serve as the rationale for further investigations to determine if CORMs could be utilized as hemostatic agents.
• Nielsen, V. G., Kirklin, J. K., & George, J. F. (2009). Carbon monoxide-releasing molecule-2 decreases fibrinolysis in human plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 20(6), 448-55. doi:10.1097/mbc.0b013e32832f4335
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  Carbon monoxide, derived from carbon monoxide-releasing molecules, has been recently demonstrated to enhance the velocity of formation and strength of plasma thrombi. We tested the hypothesis that carbon monoxide-releasing molecule-2 would modulate fibrinolysis of plasma thrombi. Normal plasma was exposed to 0, 25, 50, 100 or 200 micromol/l carbon monoxide-releasing molecule-2, with coagulation activated with tissue factor and fibrinolysis initiated with tissue-type plasminogen activator. Additional experiments utilized factor XIII, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor or alpha2-antiplasmin-deficient plasmas. Thrombus growth/disintegration kinetics was monitored with thrombelastography. Carbon monoxide-releasing molecule-2, in a concentration-dependent fashion, increased the velocity of thrombus formation and strength, and markedly attenuated fibrinolysis in normal plasma. In factor XIII-deficient plasma, carbon monoxide-releasing molecule-2 mediated effects on thrombus growth/disintegration kinetics were similar to that seen with normal plasma; however, carbon monoxide-releasing molecule-2 had a less marked effect on thrombus growth/disintegration in both plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor-deficient plasma, with even less carbon monoxide-releasing molecule-2-mediated effects noted in alpha2-antiplasmin-deficient plasma. Carbon monoxide-releasing molecule-2 attenuated fibrinolysis by enhancing the velocity of clot growth and strength while augmenting the effects of plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and alpha2-antiplasmin. These findings serve as the rationale for further investigations to determine if carbon monoxide-releasing molecules could be utilized as hemostatic agents.
• Steenwyk, B. L., Nielsen, V. G., Kirklin, J. K., & Holman, W. L. (2009). Clot lifespan model analysis of the effects of warfarin on thrombus growth and fibrinolysis: role of contact protein and tissue factor initiation.. ASAIO journal (American Society for Artificial Internal Organs : 1992), 55(1), 33-40. doi:10.1097/mat.0b013e318190c1a9
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  Warfarin therapy has served as the backbone of chronic anticoagulation therapy for decades to prevent thrombotic morbidity secondary to blood-biomaterial interfaces. Unfortunately, thrombotic and bleeding complications are observed despite maintenance of therapeutic international normalized ratio (INR) values. We proposed to define the effects of warfarin therapy on thrombus growth and disintegration following contact pathway protein or tissue factor (TF) initiation. Normal subject or patient plasma with INR values between 1.8 and 9.6 were exposed to TF or celite and tissue-type plasminogen activator (tPA). Thrombus growth/disintegration kinetics were determined by changes in resistance over time with the clot lifespan model (CSLM), a thrombelastographic-based methodology. Data were collected until clot lysis time was observed. Linear relationships of the difference between the CLSM parameter values obtained from paired celite and TF-activated samples and corresponding INR value were determined and reported as r. The time to clot initiation was progressively prolonged with increasing INR values in all samples, and speed of clot formation, clot strength, and time to onset of fibrinolysis decreased as INR increased. Throughout the range of INR values tested, contact activation resulted in faster growing, stronger, and longer lived thrombi when compared with matched TF-activated plasma samples. Only the time to maximum rate of lysis was correlated with INR (r=0.36, p=0.005). INR values have little correlation with the difference between contact protein and TF-activated coagulation/fibrinolysis.
• Nielsen, V. G. (2008). Beyond cell based models of coagulation: analyses of coagulation with clot "lifespan" resistance-time relationships.. Thrombosis research, 122(2), 145-52. doi:10.1016/j.thromres.2007.09.003
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  Cell based models of coagulation (CBM) have provided mechanistic insight into numerous hematological issues for nearly two decades. This review discusses another coagulation model system--the clot lifespan model (CLSM)--that has been designed to compliment the CBM-based approach to elucidating the mechanisms responsible for a variety of hemostatic disorders/phenomena. The CLSM is a thrombelastograph-based approach that utilizes a standardized clotting stimulus (e.g., celite, tissue factor) and a fibrinolytic stimulus (e.g., tissue type plasminogen activator) to assess clot growth and disintegration via changes in clot resistance. The CLSM utilizes parametric, elastic modulus-based parameters to document these phenomena. The CLSM has recently been employed to discern the effects of protamine and hydroxyethyl starch on key fibrinolytic-antifibrinolytic protein interactions, as well as demonstrating differences in fibrinolytic kinetics dependent on whether contact pathway proteins or tissue factor is used to initiate coagulation. The CLSM is presently being utilized to investigate the effects of ventricular assist device placement on fibrinolysis, and it is anticipated that this model system will be employed in both basic science and clinical investigations in the future.
• Nielsen, V. G. (2008). Clot life span model analysis of clot growth and fibrinolysis in normal subjects: role of thrombin activatable fibrinolysis inhibitor.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 19(4), 283-7. doi:10.1097/mbc.0b013e3282ff76c3
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  Hypofibrinolysis plays a role in thrombophilic states, and a thrombelastography-based method incorporating tissue-type plasminogen activator to determine vulnerability to fibrinolytic stress has recently been developed. This study proposed to kinetically define fibrinolytic vulnerability and the contribution of thrombin activatable fibrinolysis inhibitor to fibrinolytic defenses in normal subjects. Plasma from 30 normal subjects was exposed to tissue factor/kaolin and tissue-type plasminogen activator (100 IU/ml). Prior to activation of coagulation, samples were either not exposed or exposed to potato carboxypeptidase inhibitor (25 microg/ml, a thrombin activatable fibrinolysis inhibitor). Data were collected until clot lysis time was observed. In plasma, time to onset of maximum rate of fibrinolysis was 200-1125 s (95% confidence interval), maximum rate of lysis was -2.0--0.8 dynes/cm2 per s, and clot lysis time was 555-1595 s. Thrombin activatable fibrinolysis inhibitor's inhibition decreased the time to onset of maximum fibrinolysis by 45%, increased the rate of maximum lysis by 50%, and decreased clot lysis time by 45%. The study established a range of fibrinolytic kinetic values and the contribution of thrombin activatable fibrinolysis inhibitor in normal subjects. Study of disease states involving potential hypofibrinolysis (e.g., in-situ ventricular assist device, cancer) could be conducted using this system to link fibrinolytic vulnerability and thrombophilia.
• Nielsen, V. G. (2008). Hydroxyethyl starch does not decrease prothrombin to thrombin conversion.. Acta anaesthesiologica Scandinavica, 52(1), 163-4. doi:10.1111/j.1399-6576.2007.01497.x
• Nielsen, V. G., & Kirklin, J. K. (2008). Argatroban enhances fibrinolysis by differential inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor and factor XIII.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 19(8), 793-800. doi:10.1097/mbc.0b013e328317f5aa
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  Direct thrombin inhibitors enhance fibrinolysis more efficiently than heparin. Direct thrombin inhibitors and heparin enhance fibrinolysis by inhibition of activation of thrombin activatable fibrinolysis inhibitor (TAFI); however, the role played by other thrombin-activated proteins [e.g., factor XIII (FXIII)] in fibrinolysis remained to be elucidated. Our goal was thus to define the roles of TAFI and FXIII in direct thrombin inhibitor-mediated fibrinolysis enhancement. Plasma was exposed to argatroban or heparin, with coagulation initiated with kaolin/tissue factor and fibrinolysis initiated with tissue plasminogen activator. Additional experiments utilized TAFI and FXIII-deficient plasmas. Coagulation/fibrinolysis kinetics were monitored with thrombelastography. Argatroban (1.25, 2.5 microg/ml) significantly decreased clot lysis time and increased the maximum rate of lysis compared with unexposed plasma, whereas heparin exposure only diminished clot lysis time. When changes in maximum rate of lysis were related to changes in the maximum rate of thrombus generation, argatroban was associated with a greater increase in maximum rate of lysis per decrease in maximum rate of thrombus generation compared with heparin. Experiments with TAFI-deficient and FXIII-deficient plasma demonstrated a sparing of thrombin-mediated FXIII activation with concurrent inhibition of TAFI activation. The mechanism by which argatroban more efficiently enhanced fibrinolysis was via a differential inhibition of thrombin-mediated activation of TAFI and FXIII.
• Steenwyk, B. L., Nielsen, V. G., Kirklin, J. K., Holman, W. L., George, J. F., & Ellis, T. C. (2008). Patient with hypofibrinolysis-mediated thromboembolism converted to a hypercoagulable/hyperfibrinolytic state via ventricular assist device placement.. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 27(10), 1169-71. doi:10.1016/j.healun.2008.06.009
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  Mechanical circulatory support with ventricular assist devices (VADs) for end-stage heart failure has been a focus of intense interest for nearly four decades. Despite improvements in VAD design and biomaterial composition, it is common to administer anti-coagulation (e.g., warfarin, anti-platelet agents) to prevent both device thrombosis and thromboembolism. We present a patient with pre-operative thrombophilia (pulmonary embolism, intracardiac thrombus) and hypofibrinolysis, who subsequently developed hypercoagulability with hyperfibrinolysis with normalization of clot lifespan after left VAD placement. Such complex patient-VAD-hemostatic-state interactions serve as the rationale for continuing investigation of the effects of mechanical circulation on the fibrinolytic system and thrombophilia.
• Steenwyk, B. L., Nielsen, V. G., Mcgiffin, D. C., & Cankovic, L. (2008). Practical approach to anticoagulation for cardiopulmonary bypass in the patient with congenital prolonged activated partial thromboplastin time.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 19(7), 725-6. doi:10.1097/mbc.0b013e32830891ab
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  Patients with rare, congenital deficiencies of contact proteins (e.g., factor XII, prekallikrein, high-molecular-weight kininogen) present an important challenge with regard to safe anticoagulation during cardiopulmonary bypass. Specifically, activated coagulation time values are obtained with devices that utilize contact protein activators to generate thrombin and assess the efficacy of heparin-mediated antithrombin activation, with an activated coagulation time value of 480 s considered 'safe'. Patients with contact protein deficiencies will routinely have activated coagulation time values that exceed normal baseline values to an unpredictable extent, which, when coupled with heparin administration may well exceed 480 s but still potentially not reflect adequate antithrombin activation. We present the successful management of anticoagulation of a patient with either a prekallikrein or kininogen deficiency during cardiopulmonary bypass for coronary artery bypass graft surgery with Hepcon-based heparin concentration determinations. This approach, and the other alternatives previously mentioned, can be utilized to safely care for these rare patients in the setting of cardiac surgery.
• Zhou, F., Steenwyk, B. L., Parks, D. A., Nielsen, V. G., Kirklin, J. K., Holman, W. L., George, J. F., & Ellis, T. C. (2008). Mechanical circulatory device thrombosis: a new paradigm linking hypercoagulation and hypofibrinolysis.. ASAIO journal (American Society for Artificial Internal Organs : 1992), 54(4), 351-8. doi:10.1097/mat.0b013e31817f3e03
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  This review considers the perhaps unappreciated role of contact pathway proteins in the pathogenesis of thrombotic/thromboembolic morbidity associated with mechanical circulatory support. Placement of ventricular assist devices (VADs) has been associated with consumption of circulating contact proteins and persistent generation of activated contact proteins such as Factor XII and high molecular weight kininogen. Importantly, activated contact proteins are absorbed to the surface of VADs via the Vroman effect. Further, hyperfibrinogenemia and persistent platelet activation exist in patients with VADs, likely contributing to speed of clot growth. Using thrombelastographic-based analyses, it has been determined that contact pathway protein activated coagulation results in a thrombus that develops strength at a significantly faster rate that tissue factor initiated coagulation. Further, thrombelastographic analyses that include the addition of tissue-type plasminogen activator have demonstrated that contact protein pathway activation results in thrombin activatable fibrinolysis inhibitor activation to a far greater extent than that observed with tissue factor initiated coagulation, resulting in a thrombus that takes significantly longer to lyse. These observations serve as the rational basis for clinical investigation to determine if regional suppression of thrombin generation with FXII/high molecular weight kininogen inhibition in concert with thrombin-activatable fibrinolysis inhibitor inhibition may decrease mechanical circulatory support-associated thrombotic morbidity.
• Nielsen, V. G. (2007). A ROTEM-based method of drug assessment developed with human experimentation without consent.. Acta anaesthesiologica Scandinavica, 51(10), 1403; author reply 1403-4. doi:10.1111/j.1399-6576.2007.01395.x
• Nielsen, V. G. (2007). A comparison of the Thrombelastograph and the ROTEM.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(3), 247-52. doi:10.1097/mbc.0b013e328092ee05
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  Elastic modulus-based assessment of hemostasis in clinical and research settings has been conducted for nearly 60 years. Two systems utilizing this technology include the Thrombelastograph (Haemoscope Corporation, Niles, Illinois, USA) and the ROTEM (Pentapharm GmbH, Munich, Germany). The study goal was to compare the Thrombelastograph and the ROTEM to determine whether differences in data could be detected. Plasma (n = 8 per condition) was not activated (native); was celite-activated, hypocoagulable by dilution with hydroxyethyl starch; was celite-activated, normal; and was celite-activated, hypercoagulable by addition of fibrinogen. Equivalent ratios of plasma mixture to CaCl2 were maintained between the Thrombelastograph and ROTEM systems. Data collected included the reaction time, angle, maximum amplitude and maximum elastic modulus. The ROTEM system generated significantly (P
• Nielsen, V. G. (2007). High-molecular-weight hydroxyethyl starch accelerates kallikrein-dependent clot initiation.. The Journal of trauma, 62(6), 1491-4. doi:10.1097/01.ta.0000228881.95200.26
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  A decrease in reaction time (R; seconds) has been considered a thrombelastographic hallmark of hypercoagulability. However, the cause of changes in R has been influenced by the method of activation (e.g., celite) and the clinical/laboratory setting (e.g., hemodilution). Although antithrombin deficiency has been implicated as a cause of decreased R in unactivated samples after crystalloid dilution, dilution with hydroxyethyl starch (HES) solutions such as Hextend (6% HES solution; average molecular weight 450 kDa) or Voluven (6% HES solution; average molecular weight 130 kDa) has decreased R values in celite-activated samples in vitro and in vivo, with modulation of these R values observed after aprotinin exposure. Thus, this study proposed to define whether HES affects kallikrein-dependent clot initiation..Citrated human plasma was subjected to 0% or 30% dilution with 0.9% NaCl, Hextend, or Voluven, in the absence or presence of aprotinin (200 KIU/mL final concentration). Prekallikrein-deficient (
• Nielsen, V. G. (2007). Hydroxyethyl starch enhances fibrinolysis in human plasma by diminishing alpha2-antiplasmin-plasmin interactions.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(7), 647-56. doi:10.1097/mbc.0b013e3282a167dc
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  Hydroxyethyl starch (HES) solutions are effective volume expanders but are also associated with poorly understood coagulopathy. Enhanced fibrinolysis following dilution with HES has been demonstrated. This investigation sought to identify the interactions of HES with critical fibrinolytic/antifibrinolytic enzymes. Normal plasma or plasmas deficient in factor XIII, thrombin activatable fibrinolysis inhibitor or alpha2-antiplasmin were either not diluted or were diluted 20% with 0.9% NaCl, 5% human albumin, high-molecular-weight HES (HES 450) or low-molecular-weight HES (HES 130). Plasma was activated with celite and exposed to 75 IU/ml tissue-type plasminogen activator. Coagulation growth/disintegration kinetics were determined with thrombelastography. Compared with undiluted plasma, diluted plasma had a significant decrease in the clot lysis time and the time to maximum rate of lysis in all plasma types except in alpha2-antiplasmin-deficient plasma. The hierarchy of the decrease in clot lysis time and time to maximum rate of lysis was HES 450 = HES 130 > 5% human albumin = 0.9% NaCl. In conclusion, HES dilution enhances fibrinolysis by diminishing alpha2-antiplasmin-plasmin interactions. Further laboratory and clinical investigation is warranted to better define the mechanisms by which HES enhances clot disintegration and to find new therapeutic roles for HES to either prevent or treat thrombosis.
• Nielsen, V. G. (2007). Pharmacological upregulation of heme oxygenase-1 activity: a novel approach to the treatment of sepsis?. Anesthesia and analgesia, 104(2), 258-9. doi:10.1213/01.ane.0000253908.41555.ee
• Nielsen, V. G., & Ellis, T. C. (2007). Quantification of the effects of thrombin activatable fibrinolysis inhibitor and alpha2-antiplasmin on fibrinolysis in normal human plasma.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(1), 29-33. doi:10.1097/mbc.0b013e3280129afe
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  Two major proteins that inhibit fibrinolysis include thrombin activatable fibrinolysis inhibitor (TAFI) and alpha2-antiplasmin. Our goal was to quantify the contribution of TAFI and alpha2-antiplasmin to antifibrinolytic defenses with thrombelastography. Plasma activated with tissue factor/kaolin was subjected to fibrinolysis with tissue-type plasminogen activator (100 U/ml). Prior to activation, TAFI activity was inhibited with either potato carboxypeptidase inhibitor (25 microg/ml) or an anti-TAFI antibody, and alpha2-antiplasmin activity was inhibited with an anti-alpha2-antiplasmin antibody. Data were collected for 30 min, with the time of onset and rate of fibrinolysis determined. Compared with uninhibited samples, TAFI inhibition significantly (P < 0.05) decreased the time of onset of fibrinolysis by 70% and increased the rate of lysis by 70%. There was no difference between potato carboxypeptidase inhibitor and anti-TAFI antibody inhibition. Inhibition of alpha2-antiplasmin resulted in a significantly (P < 0.05) decreased time of onset (85%) and increased the rate of lysis (557%) compared with uninhibited samples. Inhibition of alpha2-antiplasmin activity resulted in a significantly (P < 0.05) greater fibrinolytic response than TAFI inhibition. In conclusion, utilization of standard inhibitors and thrombelastography permitted quantification of the effects of TAFI and alpha2-antiplasmin on fibrinolysis in plasma. Future investigation of diseases involving hypofibrinolysis (e.g. left ventricular assist devices) could be conducted using this assay system.
• Nielsen, V. G., & Kirklin, J. K. (2007). Hydroxyethyl starch enhances argatroban-mediated decreases in clot propagation and strength by diminishing thrombin-fibrinogen interactions.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(1), 49-54. doi:10.1097/mbc.0b013e3280111aa4
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  Direct thrombin inhibitors (DTIs) have been administered for anticoagulation during cardiopulmonary bypass for patients with heparin-induced thrombocytopenia. While DTIs prolonged clot initiation and decreased clot propagation, clot strength did not change. Hydroxyethyl starches (HES), however, significantly decreased clot propagation and strength. We hypothesized that DTI with HES could significantly decrease hemostasis more than DTI alone. Plasma was exposed to 0 or 5 microg/ml argatroban with 0 or 30% dilution with 0.9% NaCl, 10% pentastarch or 6% Voluven. Additional argatroban-exposed samples diluted with HES had addition of alpha-thrombin (0.25 U/ml) and fibrinogen (150 mg/ml). Clot kinetics were determined via thrombelastography. While dilution with 0.9% NaCl significantly (P < 0.05) decreased the clot strength by 17% compared with samples only exposed to argatroban, dilution with pentastarch and Voluven significantly (P < 0.05) markedly decreased clot strength (53 and 78%, respectively). Voluven dilution significantly increased the time to clot initiation and decreased the velocity of clot propagation compared with samples only exposed to argatroban. Addition of alpha-thrombin/fibrinogen restored clot strength. DTI/HES administration diminished hemostasis to a greater extent than DTI exposure alone. Further investigation is warranted to determine whether this therapeutic approach can improve the safety of anticoagulation during cardiopulmonary bypass in patients with heparin-induced thrombocytopenia.
• Nielsen, V. G., & Levy, J. H. (2007). Fibrinogen and bleeding: old molecule--new ideas.. Anesthesia and analgesia, 105(4), 902-3. doi:10.1213/01.ane.0000286775.33975.6e
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  ydroxyethyl starches (HES) are often administered to surgical patients asa colloid intravascular volume expander. Although all volume expandersadministeredtoableedingpatientmayproducedilutionalcoagulopathy,HESsolutionscanalsoproduceplateletinhibitionanddecreasedclotstrength(1,2).However, hemodilution with HES of all molecular weights significantlydecreases whole blood and plasma clot strength in a fibrinogen-reversiblemanner (3–8). Fenger-Eriksen et al. (3) were the first to demonstrate
• Nielsen, V. G., Kirklin, J. K., Hoogendoorn, H., Holman, W. L., & Ellis, T. C. (2007). Thrombelastographic method to quantify the contribution of factor XIII to coagulation kinetics.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(2), 145-50. doi:10.1097/mbc.0b013e32802f7d91
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  Factor XIII (FXIII) plays a critical role in clot strength, and FXIII deficiency or excess is associated with hemorrhage or thrombosis, respectively. Our goal was to design a thrombelastography-based method to characterize the effects of FXIII on plasma clot strength. Normal human plasma was exposed to 0 or 200 mug/ml anti-FXIII antibodies for 20 min prior to celite activation and calcium addition. Other plasma had addition of fibrinogen (625 mg/dl)/FXIII (2 U/ml) or 30% dilution with hydroxyethyl starch before exposure to 0 or 200 mug/ml anti-FXIII antibodies. Thromboelastography was performed and data were collected until stable clot strength was observed. The exposure of normal plasma to anti-FXIII antibodies resulted in a significant (P < 0.05) decrease in clot strength (63%) compared with plasma without antibodies. Further samples exposed to anti-FXIII antibodies had clot strength no different from FXIII-deficient plasma. The FXIII-mediated clot strength varied between 44 and 50% in hypercoagulable and hypocoagulable plasma, respectively. In conclusion, the present investigation successfully demonstrated a novel method to detect the impact of FXIII activity in plasma samples. Further actuarial investigation will be required to determine the utility of this approach in the diagnosis and treatment of patients with either acquired FXIII deficiency or excess and concordant coagulopathy.
• Nielsen, V. G., Marques, M. B., Kirklin, J. K., & Ellis, T. C. (2007). Thrombelastographic measures of clot propagation: a comparison of alpha with the maximum rate of thrombus generation.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(1), 45-8. doi:10.1097/mbc.0b013e3280111a8e
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  The alpha angle alpha (degrees) is a thrombelastographic measure of clot propagation. A parametric measurement of clot propagation [maximum rate of thrombus generation (MRTG), dynes/cm2 per s], however, has recently been utilized. Thus, the relationship of changes in alpha with changes in MRTG were determined. alpha and MRTG values obtained from 859 thrombelastograms was collected from nine studies. Data were analyzed and the relationship between alpha and MRTG defined with commercially available software. Additional comparisons were made retrospectively from whole-blood and plasma data obtained from 33 normal individuals. Data from the nine studies demonstrated that MRTG increased in an exponential fashion compared with increases in alpha (R2 = 0.88, P < 0.001). Whole-blood alpha values were in the range 66.7-74.7 whereas MRTG values were 5.5-10.8, and plasma alpha values were 65.1-77.9 with corresponding MRTG values of 3.5-12.0. Assessment of clot propagation utilizing MRTG provides a more parametric evaluation than the determination of alpha. While normal alpha values may vary by only 12-20%, MRTG values vary by approximately 200-300%. The MRTG should be progressively utilized to a greater extent in both laboratory and clinical settings to parametrically quantify clot growth kinetics with thrombelastography.
• Steenwyk, B. L., Nielsen, V. G., & Cankovic, L. (2007). Epsilon-aminocaproic acid inhibition of fibrinolysis in vitro: should the 'therapeutic' concentration be reconsidered?. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(1), 35-9. doi:10.1097/mbc.0b013e328010a359
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  The therapeutic concentration of epsilon-aminocaproic acid (EACA) has been 130 microg/ml or greater for nearly 50 years. We tested the effects on clot growth/disintegration of EACA with a plasmatic model of hyperfibrinolysis in vitro. Human plasma was exposed to 1000 U/ml tissue-type plasminogen activator containing 0, 13, 65 or 130 microg/ml EACA, with clot growth/disintegration kinetics quantified via thrombelastography. Data were analyzed with one-way analysis of variance or Kruskal-Wallis analysis of variance as appropriate. Exposure of plasma to 1000 U/ml tissue-type plasminogen activator resulted in a brief-lived clot, lasting 2 min. EACA at all concentrations tested significantly increased the rate of clot growth compared with samples with 0 microg/ml EACA. Clot strength was significantly increased by EACA in a concentration-dependent fashion. Similarly, EACA significantly prolonged the time of onset of clot lysis and decreased the rate of lysis. Samples with 130 microg/ml EACA had no sign of lysis present for 30 min. Subtherapeutic to therapeutic concentrations of EACA significantly attenuated or abolished fibrinolysis in the presence of a concentration of tissue-type plasminogen activator more than 2000-fold that encountered systemically during cardiopulmonary bypass. Further clinical investigation is warranted to determine whether smaller concentrations of EACA could provide a reduction in bleeding with a concomitant decrease in thrombotic complications.
• Steenwyk, B. L., Nielsen, V. G., Kirklin, J. K., & Gurley, W. Q. (2007). The hemostatic history of a 15-month-old child implanted with a Berlin heart left ventricular assist device.. Anesthesia and analgesia, 104(3), 538-40. doi:10.1213/01.ane.0000255155.87860.94
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  We documented the hemostatic changes associated with placement of a EXCOR Berlin Heart left ventricular assist device in a 15-month-old child before heart transplantation. The development of hypercoagulability was rapid, manifested first by a plasmatic and subsequently platelet-mediated increase in coagulation kinetics and strength that persisted for weeks. The patient had no thrombotic complications for 6 wk before transplant but required extraordinary blood product administration to achieve hemostasis secondary to aggressive, multimodal anticoagulation. In summary, when proscribing anesthetic and surgical management of patients with a Berlin Heart, consideration of hypercoagulable features and anticoagulant therapy must be made to maximize patient safety.
• Steenwyk, B. L., Nielsen, V. G., Mcgiffin, D. C., King, C. K., & Burch, T. M. (2007). Hemostatic analysis of a 13 year old with antiphospholipid syndrome and restrictive pericarditis.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 18(7), 695-7. doi:10.1097/mbc.0b013e3282a14c99
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  Antiphospholipid syndrome is an autoimmune thrombophilic disorder that is uncommon in adults and remarkably rare in children. Thrombotic etiological factors are variable in antiphospholipid syndrome, including antibody-antigen complex-mediated platelet activation, inhibition of anticoagulants, or attenuation of fibrinolysis. We present the case of a child with antiphospholipid syndrome presenting with syncope, constrictive pericarditis and hepatic enlargement that was found to have platelet-mediated hypercoagulability and marked clot lysis via thrombelastography in the preoperative period. Restoration of circulation following pericardectomy and inotropic support was associated with attenuation of hypercoagulability and fibrinolysis. It is concluded that the etiological factors responsible for antiphospholipid syndrome-mediated hemostatic abnormalities and the probable effects of hepatic hypoperfusion on clot lysis in this patient were detected with thrombelastography, and similar thrombelastographic analyses are recommended to compliment standard coagulation assessments of patients with antiphospholipid syndrome.
• Steenwyk, B. L., Powell, G., Nielsen, V. G., Lyerly, R. T., Cankovic, L., Audu, P. B., & Armstead, V. E. (2007). Qualitative thrombelastographic detection of tissue factor in human plasma.. Anesthesia and analgesia, 104(1), 59-64. doi:10.1213/01.ane.0000248223.05152.a1
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  Tissue factor (TF) is the principal in vivo initiator of coagulation, with normal circulating TF concentrations reported to be approximately 23-158 pg/mL. However, patients with atherosclerosis or cancer have been reported to have TF concentrations ranging between 800 and 9000 pg/mL. Of interest, thrombelastographic (TEG)-based measures of clot initiation and propagation have demonstrated hypercoagulability in such patients at risk for thromboembolic events. Thus, our goal in the present investigation was to establish a concentration-response relationship of the effect of TF on TEG variables, and determine specificity of TF-mediated events with a monoclonal TF antibody..Thrombelastography was performed on normal human plasma exposed to 0, 500, 1000, or 2000 pg/mL TF. Additional experiments with plasma exposed to 0 or 750 pg/mL TF in the presence or absence of a monoclonal TF antibody (1:360 dilution, 10 min incubation) were also performed. Clot initiation time (R) and the speed of clot propagation (MRTG, maximum rate of thrombus generation) were determined..The addition of TF to normal plasma resulted in a significant, concentration-dependent decrease in R and increase MRTG values. The addition of TF antibody to samples with TF significantly increased R and decreased MRTG values compared to samples with TF addition..In conclusion, changes in TEG variables in conjunction with use of a TF antibody can detect pathological concentrations of TF in human plasma in vitro. Further investigation is warranted to determine if TEG(R)-based monitoring could assist in the detection and prevention of TF-initiated thromboembolic events.
• Nielsen, V. G. (2006). Effects of Hextend hemodilution on plasma coagulation kinetics in the rabbit: role of factor XIII-mediated fibrin polymer crosslinking.. The Journal of surgical research, 132(1), 17-22. doi:10.1016/j.jss.2005.07.025
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  Hydroxyethyl starch administration has been associated with decreases in hemostasis and has recently been demonstrated to decrease fibrinogen (FI)-thrombin-(FIIa)-Factor XIII (FXIII) interactions in vitro in human plasma. Thus, the purpose of the present study was to determine the effect of in vivo hemodilution with Hextend (6% hydroxyethyl starch, mean molecular weight 450 kDa) on plasma coagulation kinetics..Eight male, New Zealand White rabbits were intravenously administered with 20 ml/kg of Hextend. Citrated plasma was obtained before, 1 min after, and 1 h after hemodilution. Thrombelastographic analyses were performed, with clot initiation (R, sec), clot propagation (alpha, degrees), and clot strength (shear elastic modulus, G, dynes/cm(2)) determined over 20 min. Samples were celite-activated and had either with addition or without additions of FI, FIIa or activated FXIII (FXIIIa) to restore protein content to prediluted values..There was no significant difference in R values observed before (229 +/- 30), 1 min after (241 +/- 54), and 1 h after (214 +/- 42) hemodilution. Prediluted alpha values (75.2 +/- 1.9) were significantly decreased 1 min (53.3 +/- 5.9) and 1 h after hemodilution (56.1 +/- 10.2). Prediluted G values (1,992 +/- 434) were significantly reduced 1 min (532 +/- 195) and 1 h after (630 +/- 297) hemodilution. FI, FIIa, and FXIIIa addition significantly decreased R values after hemodilution. alpha and G values were significantly improved by FI and FXIIIa after hemodilution. FIIa addition did not significantly affect alpha or G..Hextend hemodilution in rabbits maintains clot initiation by diminishing both FIIa-FI and FXIIIa-fibrin interactions, whereas clot propagation and strength were reduced by diminished FXIIIa-fibrin interactions.
• Nielsen, V. G. (2006). Effects of aprotinin on plasma coagulation kinetics determined by thrombelastography: role of Factor XI.. Acta anaesthesiologica Scandinavica, 50(2), 168-72. doi:10.1111/j.1399-6576.2006.00935.x
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  Aprotinin is commonly administered in settings involving cardiopulmonary bypass and liver transplantation to decrease peri-operative bleeding. Thrombelastography has been utilized to monitor coagulation in these settings, and aprotinin delays clot initiation, presumably by inhibiting kallikrein; however, aprotinin also inhibits Factor XI (FXI), a contact system protein. Thus, it was hypothesized that celite-activated thrombelastography coagulation kin-etics would be decreased via aprotinin-mediated FXI inhibition..Citrated normal plasma and prekallikrein-deficient (
• Nielsen, V. G. (2006). Hemodilution modulates the time of onset and rate of fibrinolysis in human and rabbit plasma.. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 25(11), 1344-52. doi:10.1016/j.healun.2006.08.010
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  Fibrinolysis has a critical role in the development of bleeding after insertion of a ventricular assist device (VAD). However, chronically, VAD-mediated fibrinolysis may also diminish thromboembolic events. Management of VADs involves fluid administration. It was hypothesized that the fluid administered could modulate fibrinolysis..Human plasma was diluted (0% to 30%) with 0.9% NaCl, 5% albumin or Voluven (6% hydroxyethyl-starch [HES], mean molecular weight 130 kD) and exposed to tissue-type plasminogen activator (tPA). Plasma obtained from rabbits just before and after 30% dilution with PentaLyte (6% HES, 220 kD), Voluven or Hextend (6% HES, 450 kD) were exposed to tPA. Thrombelastographic clot strength and onset/rate of fibrinolysis were determined..In human plasma, 0.9% NaCl significantly decreased clot strength, but either prolonged or maintained the onset of fibrinolysis, with the rate of fibrinolysis significantly increased only at 30% dilution. Five percent albumin significantly decreased clot strength and did not affect the onset of fibrinolysis, but it did decrease the rate of fibrinolysis. Voluven significantly decreased clot strength to the greatest extent compared with the other fluids, and decreased the time of onset of fibrinolysis. Rabbit plasma after hemodilution demonstrated significantly decreased clot strength and decreased time of onset of fibrinolysis as compared with Voluven in human plasma..Compared with crystalloid or albumin, HES solutions enhance fibrinolysis by decreasing clot strength and decreasing the time of onset of fibrinolysis. Further studies are warranted to determine whether the fluid administered to patients with VADs can impact hemorrhagic and thrombotic morbidity.
• Nielsen, V. G. (2006). Protamine enhances fibrinolysis by decreasing clot strength: role of tissue factor-initiated thrombin generation.. The Annals of thoracic surgery, 81(5), 1720-7. doi:10.1016/j.athoracsur.2005.12.027
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  Excessive protamine administration to neutralize heparin after cardiopulmonary bypass has been implicated as a cause of postoperative hemorrhage. Protamine directly inhibits thrombin and tissue factor (TF)-mediated activation of factor VII. However, the half-life of protamine is only 4.5 minutes; thus the purpose of this study was to determine if protamine could enhance fibrinolysis, explaining the delayed, protamine-associated hemorrhage observed in the postoperative period..Human plasma containing 0, 6.25, 12.5, or 25 microg/mL of protamine (n = 6 per condition) was exposed to 0.01% tissue factor and tissue-type plasminogen activator (tPA, 100 U/mL) for 30 minutes, with clot growth and disintegration measured by Thromboelastograph (Haemoscope Corp, Skokie, IL). The TF was increased to 0.1% in additional experiments with plasma containing protamine (25 microg/mL) and tPA..Protamine significantly (p < 0.05) delayed the time to clot initiation, decreased the speed of clot propagation, and diminished clot strength in a concentration-dependent fashion. The onset of fibrinolysis was significantly (p < 0.05) increased only in samples with 25 microg/mL of protamine, and the rate of clot lysis was not different among the conditions. The clot duration time (from initiation to disintegration) was significantly (p < 0.05) decreased in a concentration-dependent manner by protamine. Increased TF concentration (0.1%) significantly improved clot growth kinetics and prolonged clot duration in samples with 25 microg/mL of protamine compared with samples activated with 0.01% TF..Protamine enhanced fibrinolysis by decreasing clot strength by diminishing TF-initiated thrombin generation. Additional, clinical investigation is warranted to mechanistically implicate protamine-mediated enhancement of fibrinolysis to delayed bleeding after cardiopulmonary bypass.
• Nielsen, V. G., Cohen, E., & Cohen, B. M. (2006). Elastic modulus-based thrombelastographic quantification of plasma clot fibrinolysis with progressive plasminogen activation.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 17(1), 75-81. doi:10.1097/01.mbc.0000198047.35010.77
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  Thrombelastographic detection of fibrinolysis has been critical in the identification and treatment of coagulopathy in many perioperative settings. However, the fibrinolytic assessments have been at best non-parametric, amplitude-based determinations (e.g. estimated % lysis, clot lysis time or clot lysis rate). Recognizing this limitation, a methodology was developed to measure the onset, speed and extent of clot disintegration by changes in elastic modulus derived from the amplitude. Using this approach, our goal was to characterize the clot disintegration kinetics of progressive plasminogen activation with tissue plasminogen activator (tPA) and to determine the extent of inhibition of fibrinolysis mediated by tPA with aprotinin and activated factor XIII. While the estimated % lysis and clot lysis time were significantly affected by tPA (0-300 U/ml), elastic modulus-based analyses in a more activity-specific fashion demonstrated significantly decreased onset, increased rate and increased extent of fibrinolysis. Furthermore, aprotinin was found to inhibit the onset, rate and extent of fibrinolysis in an activity-dependent fashion, whereas activated factor XIII was noted to enhance the speed of onset of clot growth and delay the onset of fibrinolysis. In summary, our results serve as the rational basis to utilize this elastic modulus-based approach to quantify the extent of fibrinolysis in clinical and laboratory settings, as well as potentially guiding antifibrinolytic therapy.
• Powell, G., Nielsen, V. G., Mehta, M., Kim, L., Kim, J., Audu, P. B., & Armstead, V. E. (2006). The impact of tissue factor pathway inhibitor on coagulation kinetics determined by thrombelastography.. Anesthesia and analgesia, 103(4), 841-5. doi:10.1213/01.ane.0000237285.40106.1e
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  Tissue factor pathway inhibitor (TFPI) is a 40-kDa, endogenous protein that inhibits tissue factor (TF)-initiated coagulation by bonding with activated factor X (FXa). The TFPI/FXa complex then subsequently binds with TF/activated factor VII (FVIIa) complex, ultimately inhibiting thrombin generation. Heparin administration causes endothelial release of TFPI concentrations up to sixfold normal values. Thrombelastography (TEG) is often used to monitor hemostasis in the perioperative period, and TFPI could potentially affect the diagnostic interpretation of TEG-based data, given its inhibition of both common and TF coagulation pathways. Thus, in this study we characterized the effect of TFPI on coagulation kinetics via TEG..Whole blood, Factor VII-deficient plasma, and normal plasma were exposed in vitro to various concentrations of TFPI, after which unmodified, celite-activated, and TF-activated TEG were performed..The addition of 87.5 ng/mL TFPI (twice normal concentration) was required to prolong clot propagation in whole blood, with propagation and strength only significantly affected by the addition of 175 ng/mL concentrations. Experiments with Factor VII-deficient plasma demonstrated that TFPI-mediated suppression of coagulation kinetics at these concentrations was secondary to FXa inhibition. Celite activation markedly attenuated TFPI-mediated effects on coagulation kinetics, whereas TF activation accentuated TFPI-mediated prolongation of clot initiation and diminution of propagation..In settings involving heparin administration (e.g., cardiopulmonary bypass), TFPI-mediated inhibition of coagulation should be considered during TEG-based hemostatic monitoring.
• Steenwyk, B. L., Nielsen, V. G., & Gurley, W. Q. (2006). Contact activation prolongs clot lysis time in human plasma: role of thrombin-activatable fibrinolysis inhibitor and Factor XIII.. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 25(10), 1247-52. doi:10.1016/j.healun.2006.06.009
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  Contact activation system proteins (e.g., Factor XII, kallikrein) have been implicated as direct or indirect activators of plasminogen. However, contact activation and Factor XI have enhanced thrombin-activatable fibrinolysis inhibitor (TAFI) activation and decreased fibrinolysis, and Factor XIII (FXIII) also delays fibrinolysis via alpha(2)-anti-plasmin deposition on fibrin polymers. Thus, the goals of this study were to define how fibrinolysis is modulated in human plasma by contact or tissue factor (TF) activation, and what role TAFI and FXIII plays in this system..Normal, TAFI-deficient and TAFI-deficient/FXIII-supplemented plasma was exposed to tissue-type plasminogen activator and activated with either celite or TF. Clot growth/disintegration kinetics were documented with thrombelastography..Normal plasma activated with celite had significantly prolonged onset and duration of clot lysis compared with samples activated with TF. TAFI-deficient plasma activated with celite was noted to have a duration of clot lysis not different from samples activated with TF, but a significant difference in time to onset of lysis persisted. Celite activation of TAFI-deficient/FXIII-supplemented plasma showed significantly prolonged onset and duration of clot lysis compared with samples activated with TF..Primarily TAFI, and to a lesser extent FXIII, contributed to contact system protein-mediated attenuation of fibrinolysis. Clinical investigation of these phenomena is warranted in clinical settings involving contact activation (e.g., intra-aortic balloon pumps and ventricular assist devices) to determine whether these devices modulate fibrinolysis and perhaps contribute to thromboembolic morbidity.
• Steenwyk, B. L., Pereira, S. J., Nielsen, V. G., Lell, W. A., Kirklin, J. K., & Gurley, W. Q. (2006). Argatroban, bivalirudin, and lepirudin do not decrease clot propagation and strength as effectively as heparin-activated antithrombin in vitro.. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 25(6), 653-63. doi:10.1016/j.healun.2006.02.010
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  Heparin-induced thrombocytopenia is a potentially limb- and life-threatening response to heparin exposure. Direct thrombin inhibitors (DTIs) have been reported to provide anti-coagulation for cardiopulmonary bypass; however, clot formation within the cardiopulmonary bypass circuit has been reported after the administration of DTIs. We present a case of thrombosis of the cardiopulmonary bypass circuit and, ultimately, death after argatroban administration. An in vitro thrombelastographic assessment of the effects of DTIs on clot kinetics was consequently performed to determine potential causes for this complication..Normal human plasma was unmodified or exposed to heparin (1, 2, 3 U/ml), argatroban (5, 10, 50 microg/ml), bivalirudin (12, 20, 120 microg/ml), or lepirudin (3, 6, 10 microg/ml) before activation with tissue factor/kaolin in a thrombelastograph. Clot initiation (R, reaction time), propagation (MTG, maximum thrombus generation), and strength (MG, maximum elastic modulus) were determined. Analysis of variance was performed, with p < 0.05 considered significant..Compared with unmodified plasma, heparin significantly prolonged R and essentially reduced MTG and MG to the limits of detection in an activity-dependent fashion. In general, the DTIs tested prolonged R in a concentration-dependent fashion but did not diminish MTG or MG nearly as well as heparin. The only exception was 10 microg/ml lepirudin, which eliminated coagulation..DTIs demonstrated a significant prolongation of clot initiation but poor attenuation of propagation and strength. Further in vitro and clinical investigations to design a heparin-equivalent regimen to provide anti-coagulation for patients with heparin-induced thrombocytopenia are indicated.
• Nielsen, V. G. (2005). Antithrombin efficiency is maintained in vitro in human plasma following dilution with hydroxyethyl starches.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 16(5), 319-22. doi:10.1097/01.mbc.0000172100.11664.cf
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  Hemodilution has been associated with changes in hemostasis secondary to modulation of procoagulant activity. However, direct effects of specific fluids on anticoagulants, such as antithrombin (AT), remained undefined. Thus, the purpose of this investigation was to determine whether hemodilution with hydroxyethyl starches (HES) directly diminishes plasma AT activity, which would be manifested by decreases in clot initiation time (reaction time, R) with thrombelastography greater than that seen with 0.9% NaCl (NS). Normal plasma and AT-deficient (
• Nielsen, V. G. (2005). Colloids decrease clot propagation and strength: role of factor XIII-fibrin polymer and thrombin-fibrinogen interactions.. Acta anaesthesiologica Scandinavica, 49(8), 1163-71. doi:10.1111/j.1399-6576.2005.00733.x
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  Colloid-mediated hypocoagulability is clinically important, but the mechanisms responsible for coagulopathy have been incompletely defined. Thus, my goal was to elucidate how colloids decrease plasma coagulation function. Plasma was diluted 0% or 40% with 0.9% NaCl, three different hydroxyethyl starches (HES, mean molecular weight 450, 220 or 130 kDa), or 5% human albumin. Samples (n=6 per condition) were activated with celite, and diluted samples had either no additions or addition of fibrinogen (FI), thrombin (FIIa) or activated Factor XIII (FXIIIa) to restore protein function to prediluted values. Thrombelastographic variables measured included clot propagation (angle, alpha), and clot strength (amplitude, A; or shear elastic modulus, G). Dilution with 0.9% NaCl significantly decreased alpha, A and G-values compared to undiluted samples. Supplementation with FI, but not FIIa or FXIIIa, resulted in 0.9% NaCl-diluted thrombelastographic variable values not different from those of undiluted samples. FI supplementation of HES 450, HES 220, HES 130 and albumin-diluted samples only partially restored alpha, A and G-values compared to undiluted samples. FIIa addition only improved clot propagation and strength in albumin-diluted samples. FXIIIa supplementation improved propagation in samples diluted with HES 450, HES 220 and albumin, and clot strength improved in HES 450 and albumin-diluted plasma. Considered as a whole, these data support compromise of FIIa-FI and FXIIIa--fibrin polymer interactions as the mechanisms by which colloids compromise plasma coagulation. Investigation to determine if clinical enhancement of FXIII activity and/or FI concentration (e.g. fresh-frozen plasma, cryoprecipitate) can attenuate colloid-mediated decreases in hemostasis is warranted.
• Nielsen, V. G. (2005). Effects of PentaLyte and Voluven hemodilution on plasma coagulation kinetics in the rabbit: role of thrombin-fibrinogen and factor XIII-fibrin polymer interactions.. Acta anaesthesiologica Scandinavica, 49(9), 1263-71. doi:10.1111/j.1399-6576.2005.00851.x
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  Hydroxyethyl starch (HES) administration has resulted in decreased hemostasis and fibrinogen (FI)-thrombin-(FIIa)-Factor XIII (FXIII) interactions. I proposed to determine the hemostatic effect of hemodilution with PentaLyte (6% HES, mean molecular weight 220 kDa) and Voluven (6% HES, 130 kDa)..Rabbits were intravenously administered 20 ml/kg PentaLyte or Voluven (n = 8 per fluid) over 10 min. Plasma was obtained prior to, 1 min and 1 h after hemodilution. Thrombelastography was performed, with clot initiation (R, sec), clot propagation (alpha, degrees), and clot strength (shear elastic modulus, G, dynes/cm2) determined over 20 min. Celite-activated samples had either no additions or addition of FI, FIIa or activated FXIII (FXIIIa) to restore protein content to pre-diluted values..While there were no significant differences between the groups, R significantly decreased 1 h after hemodilution compared with values observed before and 1 min after hemodilution, whereas alpha and G significantly decreased 1 min after hemodilution and then significantly, but only partially, increased 1 h after hemodilution compared with pre-dilution values. Addition of FI, FIIa and FXIIIa significantly decreased R in both groups. alpha and G 1 min after hemodilution were significantly enhanced by FI, FIIa, FXIIIa in both groups; however, 1 h after hemodilution, rabbits administered PentaLyte had alpha and G enhanced only by FI and FXIIIa addition, whereas animals administered Voluven had alpha and G significantly enhanced by FI addition. PentaLyte and Voluven hemodilution initially diminishes FIIa-FI and FXIIIa-fibrin, but within an hour primarily inhibit FXIIIa-fibrin interactions in the rabbit.
• Nielsen, V. G. (2005). Effects of hydroxyethyl starch and calcium on platelet activation.. Anesthesia and analgesia, 100(5), 1538; author reply 1538-9. doi:10.1213/01.ane.0000149041.17161.ff
• Nielsen, V. G., Cohen, E., & Cohen, B. M. (2005). Effects of coagulation factor deficiency on plasma coagulation kinetics determined via thrombelastography: critical roles of fibrinogen and factors II, VII, X and XII.. Acta anaesthesiologica Scandinavica, 49(2), 222-31. doi:10.1111/j.1399-6576.2005.00602.x
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  Thrombelastography (TEG) is used to assess coagulopathy. However, a comprehensive characterization of the effects of specific coagulation factor deficiencies and mode of activation on TEG data does not exist..Thrombelastography was performed for 15 min with control plasma and plasmas deficient (
• Nielsen, V. G. (2004). Hemodilution with lactated Ringer's solution causes hypocoagulability in rabbits.. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 15(1), 55-9. doi:10.1097/00001721-200401000-00009
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  Hemodilution (HD) has been associated with hypercoagulability. It was hypothesized that HD with lactated Ringer's solution (LR) would result in hypercoagulability in rabbits. Sedated rabbits (n = 12) underwent HD with LR (40% estimated blood volume replaced with five volumes of LR) via ear vessels. Key procoagulants and anticoagulant activities were assessed prior to and 3 h after HD. Hemostatic function was assessed with the activated coagulation time and platelet-inhibited thrombelastography. Circulating tissue factor activity was much more diluted (-67.2% from baseline) than tissue factor pathway inhibitor (-45.2%) or antithrombin (-9.5%) activities after HD. HD significantly decreased factor VIII complex activity (-31.5%) more than protein C activity (-5.9%), and factor X activity (-29.2%) was more diluted than antithrombin activity. The activated coagulation time and thrombelastography demonstrated a significant decrease in hemostatic function after HD. Hemodilution with LR caused hypocoagulability in the rabbit. A greater decrease in circulating procoagulant activity than anticoagulant activity appears to be the mechanism underlying HD-mediated decreases in hemostasis.
• Nielsen, V. G., & Crow, J. P. (2004). Peroxynitrite decreases rabbit tissue factor activity in vitro.. Anesthesia and analgesia, 98(3), 668-71, table of contents. doi:10.1213/01.ane.0000101988.62847.0c
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  Tissue factor (TF) is a primary initiator of physiological coagulation in vivo. Peroxynitrite (OONO(-)), a molecule formed from nitric oxide (NO) and superoxide (O(2). (-)), decreases human TF activity in vitro. Coagulopathy has been associated with hepatoenteric ischemia-reperfusion known to involve formation of OONO(-). Further, circulating TF activity decreases in rabbits after hepatoenteric ischemia-reperfusion. We hypothesized that exposure of rabbit TF to OONO(-) would result in a decrease in activity. OONO(-) generation was performed with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide. Rabbit brain TF was incubated at 37 degrees C for 90 min with 1) 0 mM SIN-1, 2) 5 mM SIN-1, 3) 5 mM SIN-1 and 2000 U/mL recombinant human superoxide dismutase (hSOD1), or 4) 2000 U/mL hSOD1 (n = 8 per condition). TF activity was assessed by addition of TF samples to human plasma and measuring clot formation kinetics with a thrombelastograph(R). TF exposure to SIN-1 resulted in a 48% decrease in activity that was significantly different from the other three conditions (P < 0.001). There were no significant differences between the other conditions. We conclude that rabbit TF is inhibited by OONO(-), and further investigation to determine the role of OONO(-) in coagulopathies associated with hepatoenteric ischemia-reperfusion is warranted..Tissue factor (TF) initiates physiological coagulation in vivo. Hepatoenteric ischemia-reperfusion injury is associated with peroxynitrite (OONO(-)) formation, coagulopathy and decreased TF activity in rabbits. We determined that OONO(-) decreased rabbit TF activity in vitro via thrombelastography(R).
• Nielsen, V. G., Gurley, W. Q., & Burch, T. M. (2004). The impact of factor XIII on coagulation kinetics and clot strength determined by thrombelastography.. Anesthesia and analgesia, 99(1), 120-3. doi:10.1213/01.ane.0000123012.24871.62
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  Fibrinogen has been shown to be responsible for most protein-mediated clot strength via thrombelastography. However, factor XIII (FXIII) activity also plays a prominent role in the development of clot strength. Thus, we hypothesized that changes in FXIII activity would significantly increase clot strength. FXIII (0%, 1%, 6.25%, 12.5%, 25%, 50%, and 100% normal activity) was placed in a fixed volume of citrated FXIII-deficient plasma with 1% tissue factor and calcium chloride and underwent thrombelastography for 10 min. We measured the variables reaction time (R; a measurement of clot initiation), alpha (a measure of the rate of clot formation), amplitude (A; a measure of clot strength), and shear elastic modulus (G; a measure of clot strength). FXIII activity significantly decreased R in a pattern of exponential decay (R2 = 0.77; P < 0.001). FXIII activity significantly increased alpha, following a sigmoidal pattern (R2 = 0.88; P < 0.001). Finally, increases in FXIII activity significantly increased A and G in a sigmoidal pattern (R = 0.89; P < 0.001). We concluded that FXHI significantly affects R, alpha, A, and G. Thus, transfusion decision making with protein-mediated thrombelastographic patterns must account for the contribution of both fibrinogen and FXIII.
• Nielsen, V. G., Lyerly, R. T., & Gurley, W. Q. (2004). The effect of dilution on plasma coagulation kinetics determined by thrombelastography is dependent on antithrombin activity and mode of activation.. Anesthesia and analgesia, 99(6), 1587-92, table of contents. doi:10.1213/01.ane.0000136843.58799.ab
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  Hemodilution-associated hypercoagulability has been the focus of several investigations because significant morbidity and mortality have been associated with perioperative thrombophilia. Because most investigations implicate imbalances in procoagulant/anticoagulant activity as the etiology of hemodilution-associated hypercoagulability, we determined the effects of dilution on coagulation kinetics and clot strength with thrombelastography (TEG(R)). Control plasma (+/-celite activation) and antithrombin (AT)-deficient (
• Urban, D. A., Nielsen, V. G., Hammontree, L. N., Geary, B. T., El-galley, R., Deierhoi, M. H., & Bums, J. R. (2004). 1851: Outcomes of Open Versus Laparoscopic Donor Nephrectomies in the Transplant Recipients. The Journal of Urology, 171(4S), 489-489. doi:10.1016/s0022-5347(18)39043-8
• Zhou, F., Parks, D. A., Nielsen, V. G., & Crow, J. P. (2004). Peroxynitrite inactivates tissue plasminogen activator.. Anesthesia and analgesia, 98(5), 1312-7, table of contents. doi:10.1213/01.ane.0000111105.38836.f6
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  Tissue plasminogen activator (tPA) has a prominent role in physiological fibrinolysis in vivo. Thrombosis has been associated with clinical scenarios (e.g., atherosclerotic disease) known to involve local decreases in tPA activity with concomitant formation of reactive nitrogen species such as peroxynitrite (OONO(-)), a molecule formed from nitric oxide and superoxide. We hypothesized that exposure of tPA to OONO(-) would result in a decrease in tPA activity. OONO(-) was generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide. Recombinant tPA was incubated at 37 degrees C for 60 min with 0 microM SIN-1; 100 microM SIN-1; 100 microM SIN-1 and 4000 U/mL recombinant human superoxide dismutase; or 4000 U/mL recombinant human superoxide dismutase (n = 8 separate reactions per condition). Changes in tPA activity were assessed by addition of tPA samples to tissue factor-exposed human plasma and measuring clot fibrinolysis with a thrombelastograph. Exposure to SIN-1 resulted in a decrease in tPA-mediated fibrinolysis (
• Zhou, F., Parks, D. A., Nielsen, V. G., Mogal, A., & Crow, J. P. (2004). Peroxynitrite decreases hemostasis in human plasma in vitro.. Anesthesia and analgesia, 99(1), 21-6. doi:10.1213/01.ane.0000116962.93953.70
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  Coagulopathy has been associated with clinical scenarios that involve reactive nitrogen species such as peroxynitrite (OONO-). Further, OONO- decreases tissue factor and fibrinogen function in vitro. Thus, we hypothesized that exposure of plasma to the OONO- generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide, would result in a decrease in hemostatic function via diminished coagulation protein activity. Hemostatic function of plasma exposed to SIN-1 (0, 1, 5, and 10 mM for 60 min at 37 degrees C) was assessed with thrombelastography, activated partial thromboplastin time, and prothrombin time in the presence or absence of superoxide dismutase (SOD) or an OONO- scavenger. SIN-1 exposure resulted in a significant (P < 0.05), dose-dependent decrease in plasma hemostatic function and concurrent significant (P < 0.05) decreases in activities of factor VII, factor VIII complex, and factor X. Fibrinogen concentration was not affected by SIN-1. Antithrombin and protein C activity also decreased significantly (P < 0.05). Coincubation with SOD or an OONO- scavenger significantly (P < 0.05) attenuated SIN-1 mediated changes in hemostasis and procoagulant/ anticoagulant activity. We conclude that OONO- may decrease hemostatic function in human plasma by nitration of key procoagulants and that OONO- may play a significant role in hemorrhagic states.
• Nielsen, V. G., Mccammon, A. T., Kieta, D. R., & Holman, W. L. (2003). Hemostatic analysis of a patient undergoing off-pump coronary artery bypass surgery with argatroban anticoagulation.. Anesthesia and analgesia, 96(4), 956-8, table of contents. doi:10.1213/01.ane.0000052381.47101.a2
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  This case describes the impact of argatroban and off-pump coronary revascularization on hemostasis as assessed by conventional hemostatic measures and Thrombelastography in a patient with heparin-induced thrombocytopenia.
• Nielsen, V. G. (2002). The detection of changes in heparin activity in the rabbit: a comparison of anti-xa activity, thrombelastography, activated partial thromboplastin time, and activated coagulation time.. Anesthesia and analgesia, 95(6), 1503-6, table of contents. doi:10.1097/00000539-200212000-00008
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  Thrombelastography (TEG) has been used to detect both exogenous and endogenous circulating heparin activity in clinical and laboratory settings. Thus, in this study I sought to compare the sensitivity of TEG, activated partial thromboplastin time (aPTT), and activated coagulation time (ACT) values with changes in anti-Xa activity after small-dose heparin administration in rabbits. Conscious rabbits (n = 11) had blood obtained from ear arteries for hematological analyses after the administration of 0, 10, 20, and 30 U/kg of IV heparin. Anti-Xa activities after the administration of 0, 10, 20, and 30 U/kg of heparin were, respectively, 38 +/- 9 mU/mL, 74 +/- 15 mU/mL, 105 +/- 14 mU/mL, and 134 +/- 17 mU/mL; all values were significantly different from each other. TEG variables (R and alpha) significantly (P < 0.05) changed between 0, 10, and 20 U/kg heparin doses, but a difference between 20 and 30 U/kg could not be discerned secondary to loss of a detectable clot. The aPTT was significantly (P < 0.05) different between 0, 20, and 30 U/kg doses. ACT values were significantly different between the 0 U/kg heparin dose and all other doses; however, there were no significant differences between the 10, 20, and 30 U/kg heparin doses. Changes in anti-Xa activity were significantly linearly related to R (r = 0.81, P < 0.0001), alpha (r = -0.85, P < 0.0001), aPTT (r = 0.74, P < 0.0001), and ACT (r = 0.41, P = 0.005). In this model of small-dose heparin administration, TEG variables were more sensitive to changes in heparin activity than aPTT and ACT..Changes in thrombelastography (TEG) variables more sensitively reflect changes in circulating heparin activity than activated partial thromboplastin time (aPTT) and activated coagulation time (ACT) after small-dose heparin administration in rabbits. Thus, TEG may be more helpful than aPTT and ACT in the detection of heparin in both laboratory and clinical settings wherein heparin may play a role in coagulopathy.
• Nielsen, V. G., & Geary, B. T. (2002). Coagulopathy mediated by hepatoenteric ischemia-reperfusion in rabbits: role of xanthine oxidase.. Transplantation, 74(8), 1181-3. doi:10.1097/00007890-200210270-00021
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  Hepatic transplantation may result in coagulopathy caused by the release of mast-cell-derived heparin, and xanthine oxidase (XO) inhibition stabilizes mast cells. Thus, XO inactivation could decrease coagulopathy after hepatoenteric ischemia-reperfusion. Rabbits were fed a standard or XO-inactivating diet before hepatoenteric ischemia for 35 min and before 30 min of reperfusion. Hemostasis was assessed by thrombelastography. Heparin activity was quantified by anti-IIa. XO inactivation resulted in clot formation after reperfusion in all animals, whereas only 37.5% of animals with XO activity clotted (P
• Stanley, A. W., Schmidt, F. E., Nielsen, V. G., Mcgiffin, D. C., Lell, W. A., & Kirklin, J. K. (2002). Glucose-insulin-potassium infusion for myocardial protection during off-pump coronary artery surgery.. The Annals of thoracic surgery, 73(4), 1246-51; discussion 1251-2. doi:10.1016/s0003-4975(01)03619-0
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  The purpose of this randomized, double-blind, placebo-controlled pilot study was to determine the effectiveness of an intravenous glucose-insulin-potassium (GIK) infusion in preventing myocardial damage and maintaining cardiac performance in patients undergoing "off-pump" myocardial revascularization..Forty-six adult patients undergoing elective off-pump coronary artery bypass received either normal saline or a GIK infusion immediately after the induction of anesthesia through the first 12 hours of intensive care unit convalescence. Measurements of blood glucose, circulating creatine kinase MB and troponin I concentrations, as well as cardiac index (CI) and mixed venous oxygen saturation (SvO2), were obtained immediately before starting the infusion (baseline) and at 6, 12, and 24 hours post-initial coronary artery occlusion..Five patients (8%) requiring cardiopulmonary bypass were excluded from data analysis. Twenty patients received saline. Twenty-one received GIK. Blood glucose was significantly higher in the GIK group. The concentration of circulating creatine kinase MB and troponin I significantly increased over time after off-pump coronary artery bypass, with no significant intergroup differences. Cardiac index and SvO2 did not differ significantly between groups..A GIK infusion protocol commonly used as an adjunct to reperfusion therapy for acute myocardial infarction causes insulin-resistant hyperglycemia in elective off-pump coronary artery bypass patients with no demonstrable benefit. The finding of significant release of cardio-specific enzymes in individual patients implies an ongoing need to develop more effective strategies for myocardial protection during off-pump coronary artery bypass.
• Wright, J. P., Nielsen, V. G., Mccammon, A. T., & Figueroa, M. (2002). Hemodilution with albumin, but not Hextend, results in hypercoagulability as assessed by Thrombelastography in rabbits: role of heparin-dependent serpins and factor VIII complex.. Anesthesia and analgesia, 95(4), 844-50, table of contents. doi:10.1097/00000539-200210000-00010
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  Isovolemic hemodilution (IVHD) has been advocated as an effective method of reducing the need for transfusion but has been associated with hypercoagulability. We tested the hypothesis that IVHD enhances hemostatic function by decreasing circulating antithrombin activity in rabbits. Furthermore, it was determined whether different replacement solutions would affect hemostasis. Sedated rabbits were randomly assigned to groups that underwent IVHD (40% blood volume removed) with 5% human albumin (n = 10) or a 6% hetastarch solution (Hextend). Antithrombin and Factor VIII complex (VIII:C) activities were determined, and thrombelastography(R) was performed with or without platelet inhibition. IVHD resulted in a significant (P < 0.05) decrease in antithrombin (32%-39%) without fluid-specific differences observed. VIII:C did not change in the albumin group, whereas the hetastarch group had a significant (P < 0.05) decrease (43%) in VIII:C that was also significantly (P < 0.05) less than the albumin group. The time to clot initiation was decreased, and the rate of clot formation increased significantly via thrombelastography(R) in albumin animals. No significant change in clot kinetics was observed in hetastarch animals. In rabbits, the primary determinant of hemostasis after IVHD was the interaction of changes in antithrombin activity and VIII:C. These data serve as a rational basis to determine whether IVHD-mediated hypercoagulability encountered clinically may be attenuated or exacerbated by the choice of colloid administered..Isovolemic hemodilution (IVHD) is associated with hypercoagulability. Rabbits hemodiluted with albumin, but not Hextend, became hypercoagulable secondary to a loss of antithrombin activity with simultaneous maintenance of Factor VIII complex activity (VIII:C). Hextend-treated animals had proportionate decreases in both antithrombin activity and VIII:C. IVHD-mediated hypercoagulability encountered clinically may be attenuated or exacerbated by the choice of colloid administered.
• Nielsen, V. G. (2001). Endogenous heparin decreases the thrombotic response to hemorrhagic shock in rabbits.. Journal of critical care, 16(2), 64-8. doi:10.1053/jcrc.2001.26292
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  The purpose of this study was to determine if endogenous heparin release would modulate the hemostatic response to hemorrhagic shock in rabbits..Anesthetized rabbits (n = 13) underwent hemorrhagic shock (MAP 30-40 mm Hg) for 60 minutes. Blood samples obtained before and 60 minutes after hemorrhagic shock had thrombelasto-graphic variables (R, reaction time [min]; angle, alpha [ degrees ]; and G [dynes/cm(2)]) determined. Hemostatic function was assessed by modified thrombelastography under four conditions: (1) unmodified sample; (2) platelet inhibition with cytochalasin D; (3) heparinase I exposure; and (4) platelet inhibition and heparinase I exposure..Thrombelastographic variable values in samples without platelet inhibition or heparinase exposure did not significantly change after hemorrhage (before hemorrhage: R = 22.01 +/- 0.7 min, alpha 43.6 +/- 1.3 degrees, G 7,089 +/- 379 dyne/cm(2); after hemorrhage: R 22.1 +/- 2.4, alpha 41.6 +/- 3.9, G = 5,662 +/- 564; mean +/- SEM). However, blood samples exposed to heparinase after hemorrhage demonstrated enhanced hemostatic function with thrombelastographic values (R = 13.4 +/- 1.5, alpha 56.0 +/- 3.4, G = 7012 +/- 565) significantly different (P
• Nielsen, V. G. (2001). Nitric oxide decreases coagulation protein function in rabbits as assessed by thromboelastography.. Anesthesia & Analgesia, 92(2), 320-323. doi:10.1213/00000539-200102000-00006
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  Nitric oxide (NO) is administered via infusion of donors such as nitroglycerin or in inhaled form for treatment of ischemia and pulmonary hypertension, respectively. In rabbits, the NO donor, DETANONOate, decreases whole blood clotting function as assessed by thromboelastographic variables (R, reaction time; alpha, angle; and G, a measure of clot strength). I hypothesized that DETANONOate-derived NO would adversely affect coagulation protein and platelet function. Blood obtained from ear arteries of conscious rabbits (n = 8) anticoagulated with sodium citrate. The blood was then incubated with 0 or 10mM DETANONOate for 30 min. After incubation and recalcification, thromboelastography was performed for 60 min under four conditions: 1) 0mM DETANONOate, 2) 0mM DETANONOate with platelet inhibition with cytochalasin D, 3) 10mM DETANONOate, and 4) 10mM DETANONOate with platelet inhibition. DETANONOate significantly (P < 0.05) increased R and decreased alpha and G in samples with or without platelet inhibition, compared with samples not exposed to DETANONOate. Lastly, the percentage of total G (G(T)) attributable to platelet function (G(P)) was significantly more in the absence of DETANONOate (G(P) = 92.3% +/- 1.6%; mean +/- SD) than after exposure to DETANONOate (G(P) = 90.2% +/- 2.3%). DETANONOate-derived NO significantly decreased coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors.
• Nielsen, V. G. (2001). Resuscitation with Hextend decreases endogenous circulating heparin activity and accelerates clot initiation after hemorrhage in the rabbit.. Anesthesia and analgesia, 93(5), 1106-10. doi:10.1097/00000539-200111000-00007
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  Hemorrhagic shock can result in a hypercoagulable state and has been associated with both hemorrhagic and thrombotic complications in the perioperative period. The author hypothesized that hemorrhage and resuscitation could result in a hypercoagulable state via changes in the heparin-antithrombin III anticoagulant mechanism in rabbits. Rabbits sedated with ketamine underwent sham operation (n = 8) or hemorrhage (25 mL/kg blood shed) for 60 min, followed by resuscitation with an equal volume of 5% human albumin (n = 8) or Hextend (n = 8). Coagulation analysis with the Thrombelastograph analyzer and determination of endogenous heparin and antithrombin III activity were performed on arterial blood samples obtained before hemorrhage and 30 min after resuscitation. The reaction time significantly decreased by 34% after hemorrhage and resuscitation with Hextend, whereas no other significant changes in Thrombelastograph variables were noted. Antithrombin III activity was significantly less in the Albumin (83% +/- 8% of control, mean +/- SD) and Hextend (88% +/- 8%) Resuscitated groups compared with the Sham-Operated animals. Of interest, only the Hextend-Resuscitated animals demonstrated a significant decrease in heparin activity (53.4 +/- 13.6 mU/mL before hemorrhage, 42.3 +/- 5.6 mU/mL after resuscitation). A Hextend)-mediated decrease of both heparin and antithrombin III activity may explain the acceleration of clot initiation compared with albumin administration after hemorrhage in the rabbit..Hemorrhage may result in a hypercoagulable state after resuscitation. Decreases in both endogenous heparin and antithrombin III activity after hemorrhage and Hextend resuscitation in rabbits resulted in a significantly decreased time to clot coagulation analysis initiation without a significant change in the rate of clot formation or final clot strength.
• Nielsen, V. G., & Chaney, J. D. (2001). Considerations for the hemophiliac patient with inhibitors to factor VIII.. Anesthesia and analgesia, 92(3), 785-6. doi:10.1097/00000539-200103000-00044
• Nielsen, V. G., Mcgiffin, D. C., Lell, W. A., Chaney, J. D., & Adair, T. M. (2001). Hemostatic analysis of a patient with hereditary angioedema undergoing coronary artery bypass grafting.. Anesthesia and analgesia, 93(6), 1480-2, table of contents. doi:10.1097/00000539-200112000-00025
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  Hereditary angioedema is a disease associated with acute complement-mediated inflammation and swelling of the airway and other vital organs. This case describes the impact of hereditary angioedema and cardiopulmonary bypass on hemostasis as assessed by thrombelastography.
• Opentanova, I. L., Nielsen, V. G., Geary, B. T., & Armstead, V. E. (2001). Pentalyte does not decrease heparinoid release but does decrease circulating thrombotic mediator activity associated with aortic occlusion-reperfusion in rabbits.. Anesthesia and analgesia, 92(2), 314-9. doi:10.1097/00000539-200102000-00005
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  Hemorrhage and thrombosis are associated with major vascular and trauma surgery. Release of heparinoids and thrombotic mediators may contribute to these complications and have been described in rabbits after aortic occlusion-reperfusion. We hypothesized that the resuscitative fluid used could reduce heparinoid and thrombotic mediator release after aortic occlusion-reperfusion in rabbits as assessed by thromboelastographic variables (R, reaction time; alpha, angle; and G, a measure of clot strength). Anesthetized rabbits were administered lactated Ringer's solution (n = 8) or PentaLyte (n = 8) at reperfusion after 30 min of ischemia. Blood was obtained before ischemia and after 30 min of reperfusion for thromboelastography under four conditions: 1) unmodified sample, 2) platelet inhibition, 3) heparinase, and 4) platelet inhibition and heparinase. During reperfusion, unmodified samples demonstrated a significant increase in R and decrease in alpha and G that was not affected by PentaLyte. In the presence of heparinase, no significant fluid-specific thromboelastographic differences were noted. However, thrombotic mediator release (discerned by a decrease in R and an increase in alpha) during reperfusion in samples with platelet inhibition and heparinase was significantly attenuated by PentaLyte. PentaLyte administration does not decrease heparinoid release but does decrease thrombotic mediator release after aortic occlusion-reperfusion.
• Nielsen, V. G. (2000). Anemia and Arterial Partial Pressure of Oxygen. Anesthesiology, 92(1), 284-284. doi:10.1097/00000542-200001000-00053
• Nielsen, V. G., & Baird, M. S. (2000). Extreme hemodilution in rabbits: an in vitro and in vivo Thrombelastographic analysis.. Anesthesia and analgesia, 90(3), 541-5. doi:10.1097/00000539-200003000-00008
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  Isovolemic hemodilution is used to decrease the incidence of blood transfusions. However, the effects of the degree of hemodilution and the fluid used on hemostasis are controversial. We tested the hypothesis that hemodilution and the fluid administered would adversely alter Thrombelastographic(R) (Haemoscope, Skokie, IL) variables (reaction time, alpha angle and maximal amplitude). Conscious rabbits had blood sampled from ear arteries and diluted 0% or 75% in vitro with one of four solutions: 6% hetastarch in 0.9% NaCl, 5% human albumin in 0.9% NaCl, or balanced electrolyte solutions containing either 6% pentastarch or 6% hetastarch. Isoflurane-anesthetized rabbits were randomly assigned to groups (n = 9 per group) that underwent in vivo isovolemic hemodilution (75% of estimated blood volume removed), with blood replaced with one of the four solutions mentioned previously. In vitro hemodilution resulted in a significant (P < 0.05) decrease in hemostatic function (increase in reaction time, decrease in alpha angle and maximal amplitude) that was largest after hemodilution with albumin. However, although in vivo hemodilution significantly (P < 0.05) decreased reaction time, increased the alpha angle, and decreased maximal amplitude, there were no significant fluid-dependent effects..The effects of hemodilution and the fluid used on Thrombelastographic(R) (Haemoscope, Skokie, IL) variables are markedly different between in vitro and in vivo hemodilution studies.
• Nielsen, V. G., & Geary, B. T. (2000). Hepatoenteric ischemia-reperfusion increases circulating heparinoid activity in rabbits.. Journal of critical care, 15(4), 142-6. doi:10.1053/jcrc.2000.19230
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  The purpose of this study was to determine if an increase in circulating heparinoid activity contributes to the hemostatic abnormalities associated with hepatoenteric ischemia-reperfusion..Anesthetized rabbit (n = 18) underwent thoracic aorta occlusion for 30 minutes with a balloon catheter, followed by 30 minutes of reperfusion. Blood samples were obtained after 30 minutes of equilibration and 30 minutes of reperfusion. Hemostatic function was assessed by changes in the thrombelastographic variables R (reaction time), alpha (a measure of the speed of clot formation), and G (a measure of clot strength). Thrombelastography was performed on blood without platelet inhibition in the presence or absence of heparinase (n = 9 rabbits). Additional samples (n = 9) were exposed to cytochalasin D (platelet inhibitor) with or without heparinase..Compared with preischemic values, blood samples with intact platelet function obtained during reperfusion demonstrated a decrease in hemostatic function evidenced by a significant (P
• Nielsen, V. G., & Geary, B. T. (2000). Thoracic aorta occlusion-reperfusion decreases hemostasis as assessed by thromboelastography in rabbits.. Anesthesia & Analgesia, 91(3), 517-521. doi:10.1213/00000539-200009000-00003
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  UNLABELLED Perioperative hemorrhage and thrombosis are serious complications associated with major vascular surgery. We hypothesized that thoracic aortic occlusion-reperfusion in rabbits would adversely affect hemostasis as assessed by thromboelastographic variables (reaction time, alpha angle and G [a measure of clot strength]). Isoflurane-anesthetized rabbits underwent either sham operation (n = 10) or 30 min of aortic occlusion followed by 90 min of reperfusion (n = 10). Blood samples (350 microL) were exposed to 10 microL of either 0.9% NaCl or cytochalasin D (a platelet inhibitor, 10 microM final concentration) and analyzed for 1 h by using thromboelastography after 30 min of postpreparation equilibration and at 30 and 90 min of reperfusion. Aortic occlusion-reperfusion resulted in a significant (P: < 0.05) increase in reaction time, decrease in alpha angle, and decrease in G at 30 and 90 min of reperfusion compared with the sham-operated group. The decrease in hemostatic function after aortic occlusion-reperfusion was observed to the same degree in samples with or without platelet inhibition. There were no significant differences in platelet concentration between the sham-operated and aortic occlusion-reperfusion groups. Aortic occlusion-reperfusion decreased hemostatic function in rabbits primarily by decreasing the coagulation factor-dependent, platelet-independent contribution to clotting. IMPLICATIONS Thoracic aortic occlusion-reperfusion decreased hemostatic function in rabbits primarily by decreasing the coagulation factor-dependent, platelet-independent contribution to clotting. This decrease in hemostatic function may contribute to hemorrhagic complications associated with major vascular surgery.
• Nielsen, V. G., Geary, B. T., & Baird, M. S. (2000). Effects of DETANONOate, a nitric oxide donor, on hemostasis in rabbits: an in vitro and in vivo thrombelastographic analysis.. Journal of critical care, 15(1), 30-5. doi:10.1053/jcrc.2000.0150030
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  The purpose of this study was to determine if whole blood thrombelastographic variables (reaction time, K, alpha, and maximum amplitude) would be adversely effected by exposure to the nitric oxide (NO) donor, DETANONOate, in vitro or after alveolar instillation in vivo..Conscious rabbits (n = 10) had blood sampled from ear arteries anticoagulated with sodium citrate. The blood was then incubated with 0, 1, 5, 10, or 20 mmol/L DETANONOate for 30 minutes. Arterial blood from anesthetized rabbits (n = 4) was obtained and anticoagulated before and 60 minutes after 1 mmol/L DETANONOate (2 mL/kg) was instilled into the right lung. After incubation, all samples were placed in a thrombelastograph and recalcified, with thrombelastographic variables measured for 45 minutes..In vitro, 10 mmol/L DETANONOate significantly (P < .05) increased reaction time, K, and decreased alpha compared with values observed after incubation with 0, 1, and 5 mmol/L DETANONOate. Twenty mmol/L DETANONOate significantly (P < .05) increased reaction time, K, and decreased alpha and maximum amplitude values compared with all other concentrations. In vivo, DETANONOate administration did not significantly affect thrombelastographic variables..DETANONOate significantly decreased hemostatic function in vitro in a dose-dependent fashion but did not significantly affect hemostatic function in vivo.
• Nielsen, V. G., Geary, B. T., & Baird, M. S. (2000). Evaluation of the contribution of platelets to clot strength by thromboelastography in rabbits: the role of tissue factor and cytochalasin D.. Anesthesia and analgesia, 91(1), 35-9. doi:10.1097/00000539-200007000-00007
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  The contribution of platelets and soluble clotting components to clot strength has been the focus of several clinical studies using thromboelastography; it would, therefore, be beneficial to develop an animal model with which to mechanistically approach hemostatic disorders. Thus, we proposed to determine if the contribution of platelet function (G(P), dyne/cm(2)) and soluble components of the coagulation pathway to total clot strength (G(T)) in rabbits were similar to those in humans. Blood was sampled from the ear arteries of conscious rabbits (n = 12); 350 microL of the blood was placed in a thromboelastograph. Ten microliters of normal saline, cytochalasin D (an inhibitor of microtubule function, 10 microM final concentration), or tissue factor (a potent stimulator of platelet function, 0.00625% final concentration) was added to the blood sample, and thromboelastography performed for 1 h. The G(T) (mean +/- SD) was significantly (P < 0.001) different among samples exposed to normal saline, cytochalasin D, or tissue factor, with G(T) values of 7238 +/- 1432, 937 +/- 372, and 16,556 +/- 3314, respectively. G(P) was responsible for 87% and 94% of G(T) in the absence or presence of tissue factor, respectively. G(P) did not significantly correlate with platelet concentration in the absence or presence of tissue factor. The contribution of G(P) to G(T) is similar to that observed in humans..Rabbits may serve as a model of hemostasis that closely approximates human situations to mechanistically determine the etiology of coagulopathy. The contribution of platelet function to total clot strength is similar to that observed in humans.
• Nielsen, V. G., Matalon, S., & Lazrak, A. (2000). Mechanisms of increased Na(+) transport in ATII cells by cAMP: we agree to disagree and do more experiments.. American journal of physiology. Lung cellular and molecular physiology, 278(2), L233-8. doi:10.1152/ajplung.2000.278.2.l233
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  Existing evidence supports the presence of active transport of Na(+) across the mammalian alveolar epithelium and its upregulation by agents that increase cytoplasmic cAMP levels. However, there is controversy regarding the mechanisms responsible for this upregulation. Herein we present the results of various patch-clamp studies indicating the presence of 25- to 27-pS, amiloride-sensitive, moderately selective Na(+) channels (Na(+)-to-K(+) permeability ratio = 7:1) located on the apical membranes of rat alveolar type II (ATII) cells maintained in primary culture. The addition of terbutaline to the bath solution increased the open probability of single channels present in cell-attached patches of ATII cells without affecting their conductance. A similar increase in open probability was seen after the addition of protein kinase A, ATP, and Mg(2+) to the cytoplasmic side of inside-out patches. Measurement of short-circuit currents across confluent monolayers of rat or rabbit ATII cells indicates that terbutaline and 8-(4-chlorophenylthio)-cAMP increase vectorial Na(+) transport and activate Cl(-) channels. Currently, there is a controversy as to whether the cAMP-induced increase in Na(+) transport is due solely to hyperpolarization of the cytoplasmic side of the ATII cell membrane due to Cl(-) influx or whether it results from simultaneous stimulation of both Cl(-) and Na(+) conductive pathways. Additional studies are needed to resolve this issue.
• Nielsen, V. G., Matalon, S., Chen, L., & Baird, M. S. (2000). DETANONOate, a nitric oxide donor, decreases amiloride-sensitive alveolar fluid clearance in rabbits.. American journal of respiratory and critical care medicine, 161(4 Pt 1), 1154-60. doi:10.1164/ajrccm.161.4.9907033
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  Inhaled nitric oxide (NO) has been administered to animals to selectively reduce pulmonary hypertension via NO donors such as the NONOates. However, vectorial Na(+) transport across confluent monolayers of alveolar type II (ATII) pneumocytes has been decreased by NO. We tested the hypothesis that administration of the NO donor, DETANONOate, would decrease alveolar fluid clearance (AFC) in the rabbit in vivo. We instilled a solution of 5% albumin in 0.9% NaCl with 3 mM DETANONOate into anesthetized rabbits. Two hours later, similar AFC values were measured in the presence and absence of 3 mM DETANONOate (38 +/- 12% versus 43 +/- 13%; mean +/- SD). However, animals coadministered 1 mM amiloride with one of three doses of DETANONOate (100 microM, 300 microM, or 3 mM) had significantly (p < 0.05) greater AFC values (23 +/- 8, 20 +/- 14, 28 +/- 12%, respectively) than those administered amiloride alone (10 +/- 7%). When 5% albumin in a Cl(-)-free solution was administered in the presence or absence of 100 microM DETANONOate, neither AFC values nor alveolar Cl(-) concentrations were different. DETANONOate decreases the amiloride-sensitive fraction of AFC but does not decrease total AFC. DETANONOate does not influence total AFC secondary to an increase in the amiloride-insensitive fraction of AFC that is not associated with a decrease in alveolar Cl(-) secretion.
• Nielsen, V. G., Matalon, S., Geary, B. T., & Baird, M. S. (2000). Halothane does not decrease amiloride-sensitive alveolar fluid clearance in rabbits.. Anesthesia and analgesia, 90(6), 1445-9. doi:10.1097/00000539-200006000-00036
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  Halothane decreases alveolar fluid clearance (AFC), a function required for efficient gas exchange in the rat. Further, halothane decreases amiloride-sensitive Na(+) transport in rat alveolar type II cells, a process responsible for a significant portion of AFC. We tested the hypothesis that halothane would decrease amiloride-sensitive AFC in rabbits. Rabbits anesthetized with 1.8% halothane had 5% albumin in 0.9% NaCl instilled into the right lung with (n = 11) or without (n = 11) 1 mM amiloride present in the instillate. Similarly, animals anesthetized with IV fentanyl and droperidol were administered 5% albumin solution with (n = 11) or without (n = 11) amiloride. At 90 min after instillation, alveolar fluid samples were obtained, and AFC was determined by changes in fluid protein concentration. Rabbits anesthetized with halothane or fentanyl and droperidol in the absence of amiloride had similar AFC values (35% +/- 12% and 35% +/- 7%, respectively, mean +/- SD). Rabbits anesthetized with halothane or fentanyl and droperidol in the presence of amiloride had similar AFC values (20% +/- 10% and 16% +/- 12%, respectively) that were significantly less than the groups not administered amiloride (P < 0.01). Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia..Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia.
• Peterson, E. D., Pacifico, A. D., Nielsen, V. G., Mcgiffin, D. C., Li, Q., Kiefe, C. I., Holman, W. L., & Allman, R. M. (2000). Prophylactic value of preincision intra-aortic balloon pump: analysis of a statewide experience.. The Journal of thoracic and cardiovascular surgery, 120(6), 1112-9. doi:10.1067/mtc.2000.110459
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  The objective of this study was to determine whether preincision use of an intra-aortic balloon pump improves survival and shortens postoperative length of stay in hemodynamically stable, high-risk patients undergoing coronary artery bypass grafting..A post hoc analysis of the Alabama CABG Cooperative Project database was performed by using propensity scores to model the likelihood of receiving a prophylactic preincision intra-aortic balloon pump. Every patient receiving a prophylactic preincision balloon pump was matched with another patient of similar propensity score who did not receive one. We then compared outcomes for matched pairs..There were 7581 patients of whom 592 received a prophylactic preincision balloon pump. Patients with preoperative renal insufficiency, heart failure, or left main coronary artery disease, or who had undergone previous bypass grafting were significantly more likely to receive a prophylactic preincision balloon pump. By using propensity scores, we matched 550 patients who received a prophylactic preincision balloon pump with 550 who did not. Survival did not significantly differ by whether a prophylactic preincision balloon pump was used. However, surviving patients who received a preincision balloon pump had a significantly shorter postbypass length of stay (7 +/- 7.3 days) than did matched patients not receiving a balloon pump (8 +/- 6.2 days; P
• Swenson, E. R., Nielsen, V. G., & Deem, S. (2000). Anemia and arterial partial pressure of oxygen [4] (multiple letters). Anesthesiology, 92(1), 284-285.
• Nielsen, V. G., Matalon, S., Brix, A. E., & Baird, M. S. (1999). Extreme, progressive isovolemic hemodilution with 5% human albumin, PentaLyte, or Hextend does not cause hepatic ischemia or histologic injury in rabbits.. Anesthesiology, 90(5), 1428-35. doi:10.1097/00000542-199905000-00028
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  Physicians and their patients are greatly concerned about perioperative blood administration. Although isovolemic hemodilution is utilized to decrease the incidence of transfusion, it is unclear at what degree of hemodilution hepatoenteric ischemia and injury occurs. The authors hypothesized that hepatic ischemia, systemic ischemia, and tissue injury would occur during hemodilution in rabbits, and that the severity of ischemia and injury may be dependent on the fluid administered..Rabbits anesthetized with isoflurane were assigned randomly to a sham-operated group (n = 8) or groups that underwent four isovolemic hemodilutions (25% of the blood volume removed at hourly intervals), with blood replaced with one of three solutions: balanced electrolyte solutions containing 6% pentastarch (n = 8), 6% hetastarch (n = 9), or 5% human albumin in normal saline (n = 8). Arterial ketone body ratio and plasma lactate, respectively, served as measures of hepatic and systemic ischemia. Gastric, duodenal, and hepatic histologic injury was assessed post mortem..Hemodilution from a baseline hematocrit of about 33% to about 8% (third hemodilution) with all three colloids did not result in a significant increase in plasma lactate concentration or decrease in arterial ketone body ratio. At a hematocrit of about 5% (fourth hemodilution), the hetastarch group had a significantly (P < 0.05) greater plasma lactate concentration than the sham-operated and 5% human albumin groups. There were no significant differences in arterial ketone body ratio or histologic injury between the groups..Isovolemic hemodilution (approximately 5% hematocrit) with albumin, pentastarch, or hetastarch solutions does not result in significant hepatic ischemia or injury assessed by histology.
• Tan, S., Zhou, F., Wang, Z., Tan, S., Parks, D. A., Nielsen, V. G., & Gladson, C. L. (1999). Increased injury following intermittent fetal hypoxia-reoxygenation is associated with increased free radical production in fetal rabbit brain.. Journal of neuropathology and experimental neurology, 58(9), 972-81. doi:10.1097/00005072-199909000-00007
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  Hypoxia associated with perinatal events can result in brain damage in the neonate. In labor and eclampsia, hypoxia can be intermittent, which may result in more severe damage than sustained hypoxia. The pathogenesis of brain injury in sustained ischemia involves free radical production; therefore, we investigated whether higher levels of free radicals contribute to the greater injury induced by repetitive ischemia. Brains were obtained from fetuses of near-term, pregnant rabbits subjected to repetitive ischemia-reperfusion (RIR), sustained uterine ischemia-reperfusion (IR), or a control protocol. Compared with controls, fetal brains from RIR or IR groups had more brain edema. Brains from RIR fetuses exhibited higher levels of lipid peroxidation, 3-nitrotyrosine, and nitrogen oxides, and lower total antioxidant capacity and cortical cellular viability than those of IR or control fetuses. Maternal administration of antioxidants following RIR and fetal bradycardia resulted in lower levels of fetal cortical and hippocampal cell death. Coadministration of Trolox and ascorbic acid resulted in less brain edema and liquefaction, and fewer hippocampal ischemic nuclei as compared with the saline control. Higher free radical production may be responsible for the greater fetal brain injury following repetitive hypoxia-reoxygenation. Maternal antioxidant treatment resulted in transplacental passage of antioxidants and amelioration of brain injury, and may be a viable clinical option following diagnosis of fetal distress.
• Nielsen, V. G. (1998). Anesthesia for Cardiac Surgery, 2nd edition. Anesthesiology, 89(5), 1302-1303. doi:10.1097/00000542-199811000-00064
• Nielsen, V. G., Lang, J. D., Brix, A. E., Baird, M. S., & Axon, R. N. (1998). PentaLyte decreases lung injury after aortic occlusion-reperfusion.. American journal of respiratory and critical care medicine, 157(6 Pt 1), 1982-90. doi:10.1164/ajrccm.157.6.9708094
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  Lung injury often occurs after hepatoenteric ischemia, with xanthine oxidase (XO, an oxidant-generating enzyme), released from reperfusing liver and intestines, mediating a significant component of this injury. Since pentastarch administration decreases intestinal reperfusion injury, we determined whether resuscitation with PentaLyte (a pentastarch-containing solution) would decrease hepatoenteric reperfusion injury, xanthine oxidase release, and concomitant lung injury after aortic occlusion- reperfusion. Aortic occlusion was established in rabbits for 40 min, and was followed by 3 h of reperfusion, during which either PentaLyte or lactated Ringer's solution-based resuscitation was administered. Sham-operated animals served as controls. Hepatoenteric reperfusion injury, as manifested by release of the enzyme aspartate aminotransferase and decreased gastric intramucosal pH, was significantly (p < 0.0167) attenuated by PentaLyte administration after aortic occlusion-reperfusion, as compared with its occurrence in animals given lactated Ringer's solution. The release of XO after aortic occlusion-reperfusion was 4-fold smaller after PentaLyte administration than after resuscitation with lactated Ringer's solution (p < 0.05). Pulmonary injury, as defined by an increase in bronchoalveolar lavage fluid (BALF) protein content and lactate dehydrogenase (LDH) activity, was 4-fold less after PentaLyte administration following aortic occlusion-reperfusion than after administration of lactated Ringer's solution (p < 0.05). We conclude that remote pulmonary injury is significantly decreased by concomitant PentaLyte-mediated reduction of hepatoenteric reperfusion injury and XO release.
• Nielsen, V. G., Matalon, S., Duvall, M. D., & Baird, M. S. (1998). cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium.. The American journal of physiology, 275(6), L1127-33. doi:10.1152/ajplung.1998.275.6.l1127
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  Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl- transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl--free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl- and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl- secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+ absorptive pathways are damaged, transepithelial Cl- secretion and the consequent intra-alveolar fluid influx may be upregulated.
• Nielsen, V. G., Mcadams, M. L., Freeman, B. A., & Baird, M. S. (1998). Desflurane increases pulmonary alveolar-capillary membrane permeability after aortic occlusion-reperfusion in rabbits: evidence of oxidant-mediated lung injury.. Anesthesiology, 88(6), 1524-34. doi:10.1097/00000542-199806000-00017
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  Pulmonary injury occurs after vascular surgery, with xanthine oxidase (an oxidant generator) released from reperfusing liver and intestines mediating a significant component of this injury. Because halogenated anesthetics have been observed to enhance oxidant-mediated injury in vitro, the authors hypothesized that desflurane would increase alveolar-capillary membrane permeability mediated by circulating xanthine oxidase after thoracic occlusion and reperfusion..Rabbits were assigned to one of five groups: aorta occlusion groups administered desflurane (n=14), desflurane and tungstate (xanthine oxidase inactivator, n=12), fentanyl plus droperidol (n=13), and two sham-operated groups (desflurane, n=7 and fentanyl plus droperidol, n=7). Aortic occlusion was maintained for 45 min with a balloon catheter, followed by 3 h of reperfusion. Alveolar-capillary membrane permeability was assessed by measurement of bronchoalveolar lavage fluid protein. Xanthine oxidase activity was determined in plasma and lung tissue. Ascorbic acid content (an antioxidant) was determined in lung tissue..Desflurane was associated with significantly increased alveolar-capillary membrane permeability after aortic occlusion-reperfusion when compared with the fentanyl plus droperidol anesthesia or sham-operated groups (P < 0.05). Inactivation of xanthine oxidase abrogated the alveolar-capillary membrane compromise associated with desflurane. Although significantly greater than for sham-operated animals, plasma xanthine oxidase activities released after aortic occlusion-reperfusion were not different between the two anesthetics. There were no anesthetic-associated differences in lung tissue xanthine oxidase activity. However, desflurane anesthesia resulted in a significant reduction in lung ascorbic acid after aortic occlusion-reperfusion compared with the sham-operated animals..Desflurane anesthesia increased xanthine oxidase-dependent alveolar-capillary membrane compromise after aortic occlusion-reperfusion in concert with depletion of a key tissue antioxidant.
• Zhou, F., Wang, Z., Tan, S., Parks, D. A., Nielsen, V. G., & Gladson, C. L. (1998). Sustained hypoxia-ischemia results in reactive nitrogen and oxygen species production and injury in the premature fetal rabbit brain.. Journal of neuropathology and experimental neurology, 57(6), 544-53. doi:10.1097/00005072-199806000-00002
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  Free radical-mediated injury is implicated in hypoxic-ischemic encephalopathy observed in neonates. We investigated in utero free radical production and injury following hypoxia-ischemia to premature fetal brain utilizing a rabbit model of acute placental insufficiency. Pregnant rabbits at 29 days gestation were randomized to uterine ischemia for 50 minutes (min) (hypoxia) or nonischemic controls. Fetal brains were obtained immediately after ischemia for oxidative and acute-injury markers or 24 hours (h) post-ischemia for histopathology. Nitrotyrosine formation, a marker of NO-derived species such as peroxynitrite, was observed only in hypoxic brains. Hypoxia resulted in a significant increase in nitrogen oxides, lipid peroxidation, and protein oxidation, with a concomitant decrease in total antioxidant capacity, compared with controls. Peroxynitrite addition to brain homogenate increased nitrogen oxides linearly (1:1), although protein carbonyls were unchanged. Concomitantly, in vitro cortical and hippocampal cell viability and ATP levels decreased, with an increase in brain edema in hypoxic brains. Fetuses delivered 24 h post-ischemia had increased hippocampal nuclear karyorrhexis on histology compared with controls. Antioxidant administration (ascorbic acid and Trolox) intraperitoneally ameliorated changes in cellular viability and brain edema. Acute fetal hypoxia-ischemia without reoxygenation results in increased nitrogen and oxygen free radical production that may cause brain injury. The merits of the described model are discussed.
• Zhou, F., Yokoyama, Y., Wang, Z., Tan, S., Parks, D. A., Nielsen, V. G., Murdoch, A. D., & Adams, C. P. (1998). Hypoxia-reoxygenation is as damaging as ischemia-reperfusion in the rat liver.. Critical care medicine, 26(6), 1089-95. doi:10.1097/00003246-199806000-00034
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  We hypothesized that the extent of injury and release of xanthine oxidase, an oxidant generator, into the circulation would be less in normal-flow hypoxia-reoxygenation than in equal duration no-flow ischemia-reperfusion..Randomized study..University-based animal research facility..Male Sprague-Dawley rats..The livers were isolated, perfused, and then randomly subjected to 2 hrs of hypoxia (normal flow, low oxygen) or ischemia (no flow, no oxygen), and 2 hrs of reperfusion. Hepatocytes were also isolated, and were subjected to either: a) hypoxia (0, 2, 4, and 6 hrs); or b) hypoxia (2 and 4 hrs) with reoxygenation (2 hrs)..The extent of liver injury (as assessed by release of hepatocellular enzymes) and the release of xanthine oxidase were measured from isolated-perfused rat livers and cultured hepatocytes. The pattern of release of xanthine oxidase in isolated-perfused liver effluent was different in hypoxia-reoxygenation compared with ischemia-reperfusion. During hypoxia, xanthine oxidase gradually increased in the effluent; then, the xanthine oxidase decreased to low concentrations during reoxygenation. After ischemia, there was a sharp spike in xanthine oxidase at 1 min of reperfusion, with a rapid decrease to low concentrations. The total release of xanthine oxidase during hypoxia-reoxygenation was similar to that during ischemia-reperfusion. Lactate dehydrogenase and other markers of liver injury showed a pattern of release that was similar to that of xanthine oxidase, but the total release of markers was not different between the two groups. In hepatocytes, most of the release of enzymes occurred in hypoxia, and the rate of release was not different between hypoxia and hypoxia-reoxygenation..Hypoxia-reoxygenation results in as much damage to the liver as ischemia-reperfusion, and results in the release of a similar amount of oxidant-producing xanthine oxidase into the circulation.
• Tan, S., Parks, D. A., Nielsen, V. G., Brix, A. E., & Baird, M. S. (1997). Hextend (hetastarch solution) decreases multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion in rabbits.. Critical care medicine, 25(9), 1565-74. doi:10.1097/00003246-199709000-00026
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  We hypothesized that multiple organ injury and concentrations of xanthine oxidase (an oxidant-generating enzyme released after hepatoenteric ischemia) would be decreased by the administration of a bolus of a colloid solution at reperfusion..Randomized, masked, controlled animal study..University-based animal research facility..Fifty-four New Zealand white male rabbits, weighing 2 to 3 kg..Anesthetized rabbits were assigned to either the hepatoenteric ischemia-reperfusion group (n = 27) or the sham-operated group (n = 27). Hepatoenteric ischemia was maintained for 40 mins with a balloon catheter in the thoracic aorta, followed by 3 hrs of reperfusion. Each group was randomly administered a bolus of one of three fluids at the beginning of reperfusion: Hextend (hetastarch solution); 5% human albumin; or lactated Ringer's solution. The investigators were masked as to the identity of the fluid administered..Multiple organ injury was assessed by the release of lactate dehydrogenase activity into the plasma and by indices of gastric and pulmonary injury. Circulating lactate dehydrogenase activity was significantly greater (p < .001) in animals receiving lactated Ringer's solution than in rabbits receiving either colloid solution. Gastric injury (tissue edema, Histologic injury Score) was significantly decreased (p < .01) by administration of both colloid solutions. Lung injury (bronchoalveolar lavage lactate dehydrogenase activity) was significantly decreased (p < .05) by the hetastarch solution administration. The hetastarch solution administration resulted in 50% less xanthine oxidase activity release during reperfusion compared with albumin or lactated Ringer's solution administration (p < .001)..We conclude that multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion are decreased by colloid administration.
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1997). Xanthine oxidase mediates myocardial injury after hepatoenteric ischemia-reperfusion.. Critical care medicine, 25(6), 1044-50. doi:10.1097/00003246-199706000-00023
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  To determine if myocardial injury results from hepatoenteric ischemia-reperfusion. We also proposed to determine if this remote heart injury is mediated by a xanthine oxidase-dependent mechanism..Randomized, controlled animal study..University-based animal research facility..Thirty-six New Zealand white male rabbits, weighing 1.8 to 3 kg..Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) a sham-operated group; b) a sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) an aorta occlusion group; and d) an aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion..Myocardial injury, manifested by increased circulating creatine kinase-MB fraction activity, was significantly associated with aortic occlusion and reperfusion (p < .05). Sodium tungstate pretreatment significantly (p < .05) reduced circulating and myocardial xanthine oxidase activity. Xanthine oxidase inactivation by sodium tungstate significantly decreased circulating creatine kinase-MB fraction activity after hepatoenteric ischemia-reperfusion (p < .05). Finally, circulating creatine kinase-MB fraction activity was significantly associated with circulating xanthine oxidase activity (r2 = .85; p < .001)..We conclude that remote myocardial injury is caused by hepatoenteric ischemia-reperfusion. The pathoetiology of this myocardial injury involves a xanthine oxidase-dependent mechanism.
• Tan, S., Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., Kirk, K. A., & Baird, M. S. (1997). Halothane and xanthine oxidase increase hepatocellular enzyme release and circulating lactate after ischemia-reperfusion in rabbits.. Anesthesiology, 87(4), 908-17. doi:10.1097/00000542-199710000-00026
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  Multiple-organ injury often occurs after aortic occlusion-reperfusion. Oxidants derived from xanthine oxidase have been implicated as a source of injury after aortic occlusion-reperfusion. Halogenated anesthetics modify oxidant-mediated injury. The current study determined if halothane modifies hepatocellular enzyme release (e.g., alanine aminotransferase) and circulating lactate after aortic occlusion-reperfusion..Rabbits were randomly assigned to one of four groups that underwent 40 min of thoracic aortic occlusion and 2 h of reperfusion: Two groups were given either halothane or fentanyl plus droperidol anesthesia and two groups were given either anesthetic and sodium tungstate (xanthine oxidase inactivator). Each of the four groups was then matched with a similarly treated group that did not undergo aortic occlusion..Halothane anesthesia was associated with significantly (P < 0.05) increased release of alanine aminotransferase (34 +/- 9 U/l at baseline and 539 +/- 370 U/l at 120 min of reperfusion; mean +/- SD) and increased plasma lactate concentrations (2.8 +/- 2.0 mM at baseline and 12.1 +/- 9.7 mM at 120 min of reperfusion) after aortic occlusion-reperfusion compared with fentanyl plus droperidol anesthesia (alanine aminotransferase, 33 +/- 12 U/l and 148 +/- 109 U/l; lactate, 3.4 +/- 2.0 mM and 3.8 +/- 1.2 mM at baseline and 120 min of reperfusion, respectively). Inactivation of xanthine oxidase significantly decreased the release of hepatocellular enzymes (P < 0.05) and decreased circulating lactate in animals anesthetized with halothane after aortic occlusion-reperfusion..Halothane increased hepatocellular enzyme release and circulating lactate after aortic occlusion-reperfusion compared with fentanyl plus droperidol anesthesia. Xanthine oxidase activity inactivation also decreased hepatocellular enzyme activity release during reperfusion. These findings justify further investigations to determine if halogenated anesthetics modify tissue injury in clinical settings involving oxidant stress.
• Tan, S., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1996). Gastric intramucosal pH and multiple organ injury: impact of ischemia-reperfusion and xanthine oxidase.. Critical care medicine, 24(8), 1339-44. doi:10.1097/00003246-199608000-00012
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  To determine if gastric intramucosal pH is affected by hepatoneteric ischemia-reperfusion. We additionally proposed to determine if changes in gastric mucosal hydrogen ion concentration are associated with liver and lung injury following hepatoenteric ischemia-reperfusion. Finally, we hypothesized that gastric intramucosal pH is influenced by xanthine oxidase, an oxidant-generating enzyme released after hepatoenteric ischemia-reperfusion..Randomized, controlled, animal study..University-based animal research facility..Thirty-six New Zealand white male rabbits (2 to 3 kg)..Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) sham-operated group; b) sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) aorta occlusion group; and d) aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion..Gastric tonometry was performed after completion of the surgical preparation (30-min equilibration) and at 30, 60, 90, and 120 mins of reperfusion. Plasma alanine aminotransferase activity was determined at 120 mins of reperfusion to assess hepatic injury. Bronchoalveolar lavage of the right lung was performed after 120 mins of reperfusion, and the protein content was determined as a measure of pulmonary alveolar-capillary membrane compromise. Descending thoracic aorta occlusion resulted in a significant decrease in gastric intramucosal pH as compared with sham-operated rabbits (p < .001). The change in gastric mucosal hydrogen ion concentration was significantly associated with plasma alanine aminotransferase activity (r2 = .48, p < .01) and bronchoalveolar protein content (r2 = .51, p < .01). Xanthine oxidase inactivation significantly improved gastric intramucosal pH after aortic occlusion and reperfusion (p < .001), with a concomitant attenuation of the release of plasma alanine aminotransferase (p < .05) and accumulation of bronchoalveolar protein (p < .05) during reperfusion..Gastric intramucosal pH was significantly decreased after hepatoenteric ischemia-reperfusion. Furthermore, an increase in gastric intramucosal hydrogen ion concentration was associated with a concomitant increase in tissue injury, a presumed harbinger of multiple organ failure. Gastric intramucosal pH values improved during reperfusion after xanthine oxidase inactivation, concomitant with attenuation of hepatic and pulmonary injury. Gastric tonometry is an important clinical tool that can provide critical insight into the pathogenesis of multiple organ injury after hepatoenteric ischemia-reperfusion. Gastric tonometry may aid in the rapid assessment of pharmacologic interventions designed to attenuate multiple organ injury in similar clinical settings (e.g., trauma, shock, major vascular surgery).
• Tan, S., Skinner, K. A., Parks, D. A., Nielsen, V. G., Liu, Y. Y., Kirk, K. A., & Baldwin, S. T. (1996). Maternal infusion of antioxidants (Trolox and ascorbic acid) protects the fetal heart in rabbit fetal hypoxia.. Pediatric research, 39(3), 499-503. doi:10.1203/00006450-199603000-00019
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  The antioxidants, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water soluble analog of vitamin E) and ascorbic acid (AA), protect the heart from ischemia-reperfusion injury. We hypothesized that maternal infusion of Trolox and AA, would reduce the fetal bradycardia and myocardial damage observed in fetal hypoxia and increase the total antioxidant activity in fetal plasma. Either i.v. saline (control group) or Trolox + AA (drug group) was randomly administered to 29-d-old pregnant rabbits. Fetal hypoxia was induced by uterine ischemia. Fetal heart rate, plasma CK-MB activity, and plasma total radical antioxidant potential (TRAP) were measured in different sets of animals. Fetal heart rate in the drug group was higher than in the control group for the first 35 min (p < 0.05 at every 5-min interval). Fetal bradycardia ( or = 2.0 mM) were associated with lower CK-MB levels (
• Tan, S., Weinbroum, A., Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Gelman, S. (1996). Lung injury after hepatoenteric ischemia-reperfusion: role of xanthine oxidase.. American journal of respiratory and critical care medicine, 154(5), 1364-9. doi:10.1164/ajrccm.154.5.8912749
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  Oxidant stress plays a major role in the pathophysiologic processes associated with ischemia-reperfusion injury. Xanthine oxidase (XO) is often implicated as a significant source of oxidants and increases in the circulation after hepatoenteric ischemia-reperfusion. We hypothesized that pulmonary injury is associated with hepatic ischemia-reperfusion resulting from descending thoracic aorta occlusion-reperfusion (AoOR). We also proposed that this remote pulmonary injury is attenuated through inactivation of circulating and tissue XO by tungstate, implicating an XO-dependent mechanism. Aortic occlusion was established in rabbits (standard or tungstate diet) for 40 min by 2 h reperfusion. Sham operated rabbits (standard or tungstate diet) served as controls. Hepatic reperfusion injury, as manifested by release of the hepatocellular enzyme alanine aminotransferase (ALT), was markedly increased after AoOR. Suprarenal-infrahepatic occlusion failed to increase ALT release. Tungstate pretreatment significantly (p < 0.05) reduced XO activity and ameliorated liver and intestinal injury (p < 0.05). Lung injury, manifested by increased bronchoalveolar lavage (BAL) protein concentration, BAL lactate dehydrogenase (LDH) activity and increased lung edema was significantly associated with liver injury (p < 0.05) and circulating XO activity (p < 0.001). XO inactivation significantly decreased BAL protein concentration, BAL LDH activity, and lung edema (p < 0.05). We conclude that remote pulmonary injury is significantly influenced by the extent of liver injury and circulating XO activity.
• Tan, S., Zhou, F., Tan, S., Parks, D. A., & Nielsen, V. G. (1996). INCREASED FREE RADICAL PRODUCTION AND INJURY IN THE FETAL RABBIT BRAIN FOLLOWING FETAL HYPOXIA. † 1475. Pediatric Research, 39(4), 248-248. doi:10.1203/00006450-199604001-01498
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  INCREASED FREE RADICAL PRODUCTION AND INJURY IN THE FETAL RABBIT BRAIN FOLLOWING FETAL HYPOXIA. † 1475
• Tan, S., Zhou, F., Tan, S., Parks, D. A., Nielsen, V. G., & Cifuentes, J. (1996). REPETITIVE FETAL HYPOXIA-REOXYGENATION INCREASES FREE RADICAL PRODUCTION AND INJURY IN THE FETAL RABBIT BRAIN. ▴ 1476. Pediatric Research, 39, 249-249. doi:10.1203/00006450-199604001-01499
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  REPETITIVE FETAL HYPOXIA-REOXYGENATION INCREASES FREE RADICAL PRODUCTION AND INJURY IN THE FETAL RABBIT BRAIN. ▴ 1476
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1995). ANESTHETIC-OXIDANT INTERACTIONS AND MYOCARDIAL INJURY FOLLOWING THORACIC AORTA OCCLUSION AND REPERFUSION IN RABBITS. Anesthesia & Analgesia, 80(Supplement), SCA61. doi:10.1213/00000539-199504001-00060
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1995). ANESTHETIC-OXIDANT INTERACTIONS INCREASE HEPATIC AND INTESTINAL REPERFUSION INJURY AFTER THORACIC AORTA OCCLUSION IN RABBITS. Anesthesia & Analgesia, 80(Supplement), SCA38. doi:10.1213/00000539-199504001-00037
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1995). ANESTHETIC-OXIDANT INTERACTIONS INCREASE PULMONARY INJURY FOLLOWING THORACIC AORTA OCCLUSION AND REPERFUSION IN RABBITS. Anesthesia & Analgesia, 80(Supplement), SCA39. doi:10.1213/00000539-199504001-00038
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1995). GASTRIC TONOMETRY AND ANESTHETIC-OXIDANT INTERACTIONS FOLLOWING THORACIC AORTA OCCLUSION AND REPERFUSION IN RABBITS. Anesthesia & Analgesia, 80(Supplement), SCA60. doi:10.1213/00000539-199504001-00059
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Baird, M. S. (1995). SYSTEMIC ISCHEMIA AND ANESTHETIC-OXIDANT INTERACTIONS FOLLOWING THORACIC AORTA OCCLUSION AND REPERFUSION IN RABBITS. Anesthesia & Analgesia, 80(Supplement), SCA62. doi:10.1213/00000539-199504001-00061
• Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., Mccammon, A. T., & Kirk, K. A. (1995). Xanthine oxidase inactivation attenuates postocclusion shock after descending thoracic aorta occlusion and reperfusion in rabbits.. The Journal of thoracic and cardiovascular surgery, 110(3), 715-22. doi:10.1016/s0022-5223(95)70103-6
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  "Declamping shock" is observed after aortic crossclamping, with hypovolemia, hypotension, and metabolic acidemia invariably present. We hypothesized that oxidants derived from xanthine oxidase influence the resuscitative interventions required to maintain baseline hemodynamic and acid-base status after aortic occlusion and reperfusion in rabbits. We also hypothesized that inactivation of xanthine oxidase with sodium tungstate could reduce systemic injury as assessed by the release of lactate dehydrogenase and alkaline phosphatase. To test these hypotheses, we established aortic occlusion in rabbits (n = 10, standard diet; n = 8, tungstate diet) for 40 minutes by inflation of a 4F Fogarty catheter in the descending thoracic aorta followed by 2 hours of reperfusion. Sham-operated rabbits (n = 10, standard diet; n = 9, tungstate diet) served as controls. Tungstate-pretreated rabbits required significantly less Ringer's solution (28%), phenylephrine (68%), and sodium bicarbonate (30%) during reperfusion (p < 0.005). Lactate dehydrogenase and alkaline phosphatase release during reperfusion was significantly attenuated by tungstate pretreatment (p < 0.05). Tungstate pretreatment resulted in plasma xanthine oxidase activities significantly lower than those in the sham group administered a standard diet (p = 0.007). Resuscitation requirements and systemic injury were reduced by inactivation of xanthine oxidase in a rabbit model that simulates the situation of human thoracic aorta operations.
• Weinbroum, A., Tan, S., Skinner, K. A., Parks, D. A., Nielsen, V. G., Matalon, S., Gelman, S., & Bradley, E. L. (1995). Liver ischemia-reperfusion increases pulmonary permeability in rat: role of circulating xanthine oxidase.. The American journal of physiology, 268(6 Pt 1), G988-96. doi:10.1152/ajpgi.1995.268.6.g988
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  Reactive oxygen species play an important role in pathogenesis of a variety of pathological processes, e.g., ischemia-reperfusion, acute viral infections, thermal injury, hepatic diseases, and acute lung injury. Xanthine oxidase (XO) may be a significant source of these cytotoxic oxygen species. We tested the hypothesis that hepatic ischemia-reperfusion releases xanthine dehydrogenase + XO (XDH + XO) into the circulation and that circulating XO damages isolated perfused lung. Isolated liver + lung preparation was perfused with Krebs-Henseleit buffer to minimize confounding effects of circulating neutrophils. In one group, livers were rendered globally ischemic for 2 h and then reperfused (I/R). In another group, livers were pretreated with allopurinol and perfused with buffer containing additional allopurinol (I/R + Allo). After 2 h of ischemia, an isolated lung was connected to liver, and liver + lung preparation was reperfused in series for 15 min. Liver reperfusion was terminated, and lung was recirculated with liver effluent for 45 min. Capillary filtration coefficient (ml.min-1.cmH2O-1.100 g lung dry wt-1) was 2.0 +/- 0.3 and 1.9 +/- 0.4 in control and I/R + Allo lungs, respectively, and 9.0 +/- 1.2 in I/R lungs (P < 0.001). Lung wet-to-dry weight ratio in control and I/R + Allo lungs was 8.6 +/- 0.3 and 9.1 +/- 0.5, respectively, and 14.9 +/- 1.1 in I/R lungs (P < 0.01). Control and I/R + Allo bronchoalveolar lavage protein content was < 1.0 mg/ml compared with 32.6 +/- 8.4 mg/ml in I/R group.(ABSTRACT TRUNCATED AT 250 WORDS)
• Weinbroum, A., Tan, S., Samuelson, P. N., Parks, D. A., Nielsen, V. G., & Gelman, S. (1994). Xanthine oxidoreductase release after descending thoracic aorta occlusion and reperfusion in rabbits. The Journal of Thoracic and Cardiovascular Surgery, 107(5), 1222-1227. doi:10.1016/s0022-5223(94)70041-9
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  Cardiopulmonary and other organ dysfunction often occurs after operation on the descending thoracic aorta. Though there are multiple causes of organ dysfunction in this setting, free radical injury may play a prominent role. Xanthine oxidoreductase, an enzyme that generates oxidants after exposure to ischemia, could be released from ischemic liver and intestine during reperfusion. To test this hypothesis, we created aortic occlusion in eight rabbits for 40 minutes by inflation of a 4F Fogarty balloon catheter in the descending thoracic aorta. Eight sham-operated rabbits served as a control group. Two hours of reperfusion followed removal of the balloon catheter. Hemodynamic and acid-base status were maintained near baseline values during reperfusion. Plasma samples were obtained for determination of the activity of the hepatocellular enzymes xanthine oxidoreductase, aspartate aminotransferase, alanine transferase, and lactate dehydrogenase. Plasma xanthine oxidoreductase activity increased significantly ( p
• Tan, S., Weinbroum, A., Wang, Z., Tan, S., Skinner, K. A., Parks, D. A., Nielsen, V. G., Morris, R., Liu, Y., & Baldwin, S. T. (1993). Maternal infusion of trolox and ascorbic acid affects fetal heart rate in fetal rabbit hypoxia. Free Radical Biology and Medicine, 15(5), 533. doi:10.1016/0891-5849(93)90417-s
• Webster, R. O., & Nielsen, V. G. (1987). Inhibition of human polymorphonuclear leukocyte functions by ibuprofen.. Immunopharmacology, 13(1), 61-71. doi:10.1016/0162-3109(87)90027-0
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  We have found that pretreatment of human neutrophils with ibuprofen (0.10-1.0 mg/ml) results in an irreversible, concentration-dependent inhibition of superoxide anion generation and release of lysosomal enzymes (myeloperoxidase, lysozyme) stimulated by the synthetic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, and to a lesser extent by serum opsonized zymosan. Inhibition of granule exocytosis and oxygen radical generation at ibuprofen concentrations less than 5 mg/ml was not due to drug cytotoxicity since release of the cytoplasmic enzyme lactate dehydrogenase was not affected by ibuprofen. In contrast to neutrophil responses mediated by C5a or FMLP, ibuprofen did not inhibit either enzyme release or superoxide anion generation by neutrophils stimulated with phorbol myristate acetate. Ibuprofen did not function as an oxygen radical scavenger in a cell-free system in which superoxide anion was generated by the aerobic action of xanthine oxidase on hypoxanthine. Ibuprofen also inhibited in a concentration-dependent fashion both directed migration (chemotaxis) and stimulated random migration (chemokinesis) of neutrophils exposed to either FMLP or C5a. Inhibition of neutrophil adherence to plastic surfaces and bovine pulmonary artery endothelial cells was equally effective when the neutrophils were treated with ibuprofen before stimulation with FMLP or phorbol myristate acetate. The inhibitory effects of ibuprofen pretreatment of neutrophils could not be overcome by addition of prostaglandins E1 or E2 (0.3-300 nM). These results demonstrate that ibuprofen is capable of suppressing many functions thought to be important in neutrophil-mediated acute pulmonary inflammatory processes. Results of these experiments further suggest that ibuprofen may inhibit neutrophil functions by acting on cellular components separate from membrane receptors or by blockade of cyclo-oxygenase products which may be involved in these neutrophil functions.

Poster Presentations

• DaDeppo, A. J., Hadley, H. A., CHen, A., Matika, R. W., & Nielsen, V. G. (2015, May). Chronic Migraineurs Form Carboxyhemeifrinogen and Iron-Bound Fibrinogen. Western Anesthesia Residents Conference. Seattle, WA.

Others

• Nielsen, V. G., Galvani, C. A., Boyle, P. K., Nielsen, V. G., Galvani, C. A., & Boyle, P. K. (2013, December 3). Obesity-Mediated Hypercoagulability: Role of Hemeoxygenase-1. IRB 13-0745.