Guy Reed

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Journals/Publications

• Gladysheva, I. P., Sullivan, R. D., & Reed, G. L. (2023). Falling corin and ANP activity levels accelerate development of heart failure and cardiac fibrosis. Frontiers in cardiovascular medicine, 10, 1120487.
• Gladysheva, I. P., Sullivan, R. D., Ramanathan, K., & Reed, G. L. (2022). Soluble (Pro)Renin Receptor Levels Are Regulated by Plasma Renin Activity and Correlated with Edema in Mice and Humans with HFrEF. Biomedicines, 10(8).
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  Symptomatic heart failure with reduced ejection fraction (HFrEF) is characterized by edema and chronic pathological activation of the classical renin-angiotensin-aldosterone system (RAAS). The soluble (pro)renin receptor (s(P)RR) is released into circulation by proteolytic cleavage of tissue expressed (P)RR and is a candidate biomarker of RAAS activation. However, previous studies linked elevated levels of s(P)RR in patients with HFrEF to renal dysfunction. Utilizing prospectively enrolled patients with comparable rEF, we show that increased plasma levels of s(P)RR are associated with symptomatic HF (characterized by edema), independent of chronic renal dysfunction. We also found that s(P)RR levels were positively correlated with patient plasma renin activity (PRA). Normotensive mice with dilated cardiomyopathy (DCM) and HFrEF, without renal dysfunction, showed plasma s(P)RR and PRA patterns similar to human HFrEF patients. Plasma s(P)RR levels positively correlated with PRA and systemic edema, but not with EF, resembling findings in patients with HFrEF without chronic kidney dysfunction. In female DCM mice with elevated PRA levels and plasma s(P)RR levels, a randomized, blinded trial comparing the direct renin inhibitor, aliskiren vs. vehicle control, showed that direct renin inhibition normalized PRA, lowered s(P)RR, and prevented symptomatic HFrEF. Considered in light of previous findings, these data suggest that, in HFrEF, in the absence of renal dysfunction, elevation of plasma s(P)RR levels is caused by increased PRA and associated with the development of systemic edema.
• Hernandez, M., Sullivan, R. D., McCune, M. E., Reed, G. L., & Gladysheva, I. P. (2022). Sodium-Glucose Cotransporter-2 Inhibitors Improve Heart Failure with Reduced Ejection Fraction Outcomes by Reducing Edema and Congestion. Diagnostics (Basel, Switzerland), 12(4).
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  Pathological sodium-water retention or edema/congestion is a primary cause of heart failure (HF) decompensation, clinical symptoms, hospitalization, reduced quality of life, and premature mortality. Sodium-glucose cotransporter-2 inhibitors (SGLT-2i) based therapies reduce hospitalization due to HF, improve functional status, quality, and duration of life in patients with HF with reduced ejection fraction (HFrEF) independently of their glycemic status. The pathophysiologic mechanisms and molecular pathways responsible for the benefits of SGLT-2i in HFrEF remain inconclusive, but SGLT-2i may help HFrEF by normalizing salt-water homeostasis to prevent clinical edema/congestion. In HFrEF, edema and congestion are related to compromised cardiac function. Edema and congestion are further aggravated by renal and pulmonary abnormalities. Treatment of HFrEF patients with SGLT-2i enhances natriuresis/diuresis, improves cardiac function, and reduces natriuretic peptide plasma levels. In this review, we summarize current clinical research studies related to outcomes of SGLT-2i treatment in HFrEF with a specific focus on their contribution to relieving or preventing edema and congestion, slowing HF progression, and decreasing the rate of rehospitalization and cardiovascular mortality.
• Sullivan, R. D., McCune, M. E., Hernandez, M., Reed, G. L., & Gladysheva, I. P. (2022). Suppression of Cardiogenic Edema with Sodium-Glucose Cotransporter-2 Inhibitors in Heart Failure with Reduced Ejection Fraction: Mechanisms and Insights from Pre-Clinical Studies. Biomedicines, 10(8).
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  In heart failure with reduced ejection fraction (HFrEF), cardiogenic edema develops from impaired cardiac function, pathological remodeling, chronic inflammation, endothelial dysfunction, neurohormonal activation, and altered nitric oxide-related pathways. Pre-clinical HFrEF studies have shown that treatment with sodium-glucose cotransporter-2 inhibitors (SGLT-2i) stimulates natriuretic and osmotic/diuretic effects, improves overall cardiac function, attenuates maladaptive cardiac remodeling, and reduces chronic inflammation, oxidative stress, and endothelial dysfunction. Here, we review the mechanisms and effects of SGLT-2i therapy on cardiogenic edema in various models of HFrEF. Overall, the data presented suggest a high translational importance of these studies, and pre-clinical studies show that SGLT-2i therapy has a marked effect on suppressing the progression of HFrEF through multiple mechanisms, including those that affect the development of cardiogenic edema.
• Gladysheva, I. P., Sullivan, R. D., & Reed, G. L. (2021). Neprilysin and Corin in HF: Does Combining 2 Biomarkers Double Our Insights?. JACC. Heart failure, 9(5), 406.
• Li, W., Kessinger, C. W., Orii, M., Lee, H., Wang, L., Weinberg, I., Jaff, M. R., Reed, G. L., Libby, P., Tawakol, A., Henke, P. K., & Jaffer, F. A. (2021). Time-Restricted Salutary Effects of Blood Flow Restoration on Venous Thrombosis and Vein Wall Injury in Mouse and Human Subjects. Circulation, 143(12), 1224-1238.
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  Up to 50% of patients with proximal deep vein thrombosis (DVT) will develop the postthrombotic syndrome characterized by limb swelling and discomfort, hyperpigmentation, skin ulcers, and impaired quality of life. Although catheter-based interventions enabling the restoration of blood flow (RBF) have demonstrated little benefit on postthrombotic syndrome, the impact on the acuity of the thrombus and mechanisms underlying this finding remain obscure. In experimental and clinical studies, we examined whether RBF has a restricted time window for improving DVT resolution.
• Nanda, A., Urban, A., Duong, V., Heckle, M., Ibebuogu, U. N., Reed, G., Jefferies, J., & Khouzam, R. N. (2021). The Paradoxical Impact of Insurance Status on Interfacility Transfer Times and Outcomes in Patients with ST-Elevation Myocardial Infarction. Current problems in cardiology, 46(3), 100414.
• Saleem, S., Wang, D., Zhao, T., Sullivan, R. D., & Reed, G. L. (2021). Matrix Metalloproteinase-9 Expression is Enhanced by Ischemia and Tissue Plasminogen Activator and Induces Hemorrhage, Disability and Mortality in Experimental Stroke. Neuroscience, 460, 120-129.
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  Matrix metalloproteinase-9 (MMP-9) degrades collagen and other cellular matrix proteins. After acute ischemic stroke, increased MMP-9 levels are correlated with hemorrhage, lack of reperfusion and stroke severity. Nevertheless, definitive data that MMP-9 itself causes poor outcomes in ischemic stroke are limited. In a model of experimental ischemic stroke with reperfusion, we examined whether ischemia and recombinant tissue plasminogen activator (r-tPA) therapy affected MMP-9 expression, and we used specific inhibitors to test if MMP-9 affects brain injury and recovery. After stroke, MMP-9 expression increased significantly in the ischemic vs. non-ischemic hemisphere of the brain (p 
• Tripathi, R., Sullivan, R. D., Fan, T. M., Mehta, R. M., Gladysheva, I. P., & Reed, G. L. (2021). A Low-Sodium Diet Boosts Ang (1-7) Production and NO-cGMP Bioavailability to Reduce Edema and Enhance Survival in Experimental Heart Failure. International journal of molecular sciences, 22(8).
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  Sodium restriction is often recommended in heart failure (HF) to block symptomatic edema, despite limited evidence for benefit. However, a low-sodium diet (LSD) activates the classical renin-angiotensin-aldosterone system (RAAS), which may adversely affect HF progression and mortality in patients with dilated cardiomyopathy (DCM). We performed a randomized, blinded pre-clinical trial to compare the effects of a normal (human-equivalent) sodium diet and a LSD on HF progression in a normotensive model of DCM in mice that has translational relevance to human HF. The LSD reduced HF progression by suppressing the development of pleural effusions ( < 0.01), blocking pathological increases in systemic extracellular water ( < 0.001) and prolonging median survival (15%, < 0.01). The LSD activated the classical RAAS by increasing plasma renin activity, angiotensin II and aldosterone levels. However, the LSD also significantly up-elevated the counter-regulatory RAAS by boosting plasma angiotensin converting enzyme 2 (ACE2) and angiotensin (1-7) levels, promoting nitric oxide bioavailability and stimulating 3'-5'-cyclic guanosine monophosphate (cGMP) production. Plasma HF biomarkers associated with poor outcomes, such as B-type natriuretic peptide and neprilysin were decreased by a LSD. Cardiac systolic function, blood pressure and renal function were not affected. Although a LSD activates the classical RAAS system, we conclude that the LSD delayed HF progression and mortality in experimental DCM, in part through protective stimulation of the counter-regulatory RAAS to increase plasma ACE2 and angiotensin (1-7) levels, nitric oxide bioavailability and cGMP production.
• Agarwal, M. A., Jain, N., Podila, P. S., Varadarajan, V., Patel, B., Shah, M., Garg, L., Khouzam, R. N., Ibebuogu, U., Reed, G. L., & Dagogo-Jack, S. (2020). Association of history of heart failure with hospital outcomes of hyperglycemic crises: Analysis from a University hospital and national cohort. Journal of diabetes and its complications, 34(1), 107466.
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  The impact of a history of heart failure (HF) on the outcomes of hospitalization for hyperglycemic crises (diabetic ketoacidosis and hyperosmolar hyperglycemic syndrome) is unknown. We aimed to test the hypothesis that a history of HF has a deleterious impact on the outcomes of hospitalization for hyperglycemic crises.
• Singh, S., Saleem, S., & Reed, G. L. (2020). Alpha2-Antiplasmin: The Devil You Don't Know in Cerebrovascular and Cardiovascular Disease. Frontiers in cardiovascular medicine, 7, 608899.
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  Alpha2-antiplasmin (α2AP), the fast-reacting, serine protease inhibitor (serpin) of plasmin, was originally thought to play a key role in protection against uncontrolled, plasmin-mediated proteolysis of coagulation factors and other molecules. However, studies of humans and mice with genetic deficiency of α2AP have expanded our understanding of this serpin, particularly in disease states. Epidemiology studies have shown an association between high α2AP levels and increased risk or poor outcome in cardiovascular diseases. Mechanistic studies in disease models indicate that α2AP stops the body's own fibrinolytic system from dissolving pathologic thrombi that cause venous thrombosis, pulmonary embolism, arterial thrombosis, and ischemic stroke. In addition, α2AP fosters the development of microvascular thrombosis and enhances matrix metalloproteinase-9 expression. Through these mechanisms and others, α2AP contributes to brain injury, hemorrhage and swelling in experimental ischemic stroke. Recent studies also show that α2AP is required for the development of stasis thrombosis by inhibiting the early activation of effective fibrinolysis. In this review, we will discuss the key role played by α2AP in controlling thrombosis and fibrinolysis and, we will consider its potential value as a therapeutic target in cardiovascular diseases and ischemic stroke.
• Sullivan, R. D., Houng, A. K., Gladysheva, I. P., Fan, T. M., Tripathi, R., Reed, G. L., & Wang, D. (2020). Corin Overexpression Reduces Myocardial Infarct Size and Modulates Cardiomyocyte Apoptotic Cell Death. International journal of molecular sciences, 21(10).
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  Altered expression of corin, a cardiac transmembrane serine protease, has been linked to dilated and ischemic cardiomyopathy. However, the potential role of corin in myocardial infarction (MI) is lacking. This study examined the outcomes of MI in wild-type vs. cardiac-specific overexpressed corin transgenic (Corin-Tg) mice during pre-MI, early phase (3, 24, 72 h), and late phase (1, 4 weeks) post-MI. Corin overexpression significantly reduced cardiac cell apoptosis ( < 0.001), infarct size ( < 0.001), and inhibited cleavage of procaspases 3, 9, and 8 ( < 0.05 to < 0.01), as well as altered the expression of Bcl2 family proteins, Bcl-xl, Bcl2 and Bak ( < 0.05 to < 0.001) at 24 h post-MI. Overexpressed cardiac corin also significantly modulated heart function (ejection fraction, < 0.0001), lung congestion (lung weight to body weight ratio, < 0.0001), and systemic extracellular water (edema, < 0.05) during late phase post-MI. Overall, cardiac corin overexpression significantly reduced apoptosis, infarct size, and modulated cardiac expression of key members of the apoptotic pathway in early phase post-MI; and led to significant improvement in heart function and reduced congestion in late phase post-MI. These findings suggest that corin may be a useful target to protect the heart from ischemic injury and subsequent post-infarction remodeling.
• Tripathi, R., Sullivan, R. D., Fan, T. M., Mehta, R. M., Gladysheva, I. P., & Reed, G. L. (2020). In Experimental Dilated Cardiomyopathy Heart Failure and Survival Are Adversely Affected by a Lack of Sexual Interactions. International journal of molecular sciences, 21(15).
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  Nearly one in three people in the U.S. will develop heart failure (HF), characterized by fluid retention (edema) in the lungs and elsewhere. This leads to difficult breathing, deterioration of physical capacity, restriction of normal activities and death. There is little data about the safety and effects of sexual interactions in patients with HF. We tested whether a lack of sexual interactions affected pathophysiological outcomes in a pre-clinical mouse model of dilated cardiomyopathy that recapitulates the progressive stages of human HF. Male mice were randomly given access to, or deprived from, sexual interactions with female mice, which were confirmed by videography and generation of offspring. Cohousing with access to sexual interactions markedly prolonged survival, while cohousing without access to sexual activity did not. Sexual interactions improved systolic function, reduced HF-associated edema, altered transcription of heart contractile protein genes and decreased plasma testosterone levels. To determine whether testosterone levels contributed to survival, testosterone levels were experimentally reduced. Reduction of testosterone levels significantly prolonged survival. Taken together, in mice with dilated cardiomyopathy, sexual activity altered cardiac contractile gene transcription, improved systolic function, reduced edema and prolonged survival which may be in part due to lower testosterone levels.
• Agarwal, M. A., Garg, L., Shah, M., Patel, B., Jain, N., Jain, S., Kabra, R., Kovesdy, C., Reed, G. L., & Lavie, C. J. (2019). Relation of Obesity to Outcomes of Hospitalizations for Atrial Fibrillation. The American journal of cardiology, 123(9), 1448-1452.
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  Obesity has been linked with increased incidence of atrial fibrillation (AF), but impact of presence of obesity on outcomes of hospitalizations for AF has not been investigated. We used the National Inpatient Sample database 2010 to 2014 to identify all adult hospitalizations aged ≥18years with a primary diagnosis of AF. Obese patients were identified using the co-morbidity variable for obesity, as defined in National Inpatient Sample databases. Multivariable logistic regression was used to compare in-hospital outcomes (mortality, acute stroke events) between obese and non-obese patients with AF. Of 431, 734 hospitalizations for AF, 66,138 (15.3%) were obese. Obese patients were younger and more likely to be African-Americans compared with non-obese patients. Despite being younger, obese patients had significantly higher prevalence of cardiovascular co-morbidities such as hypertension, diabetes mellitus, dyslipidemia, smoking, heart failure, and chronic renal failure (p
• Ifedili, I., Bob-Manuel, T., Kadire, S. R., Heard, B., John, L. A., Zambetti, B., Heckle, M. R., Thomas, F., Haji, S., Khouzam, R. N., Reed, G. L., & Ibebuogu, U. N. (2019). Cocaine Positivity in ST-Elevation Myocardial Infarction: A True or False Association. The Permanente journal, 23.
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  Every year, more than 500,000 US Emergency Department visits are associated with cocaine use. People who use cocaine tend to have a lower incidence of true ST-elevation myocardial infarction (STEMI).
• Reed, G. L., Podila, P. S., Kode, S., Khouzam, R. N., Jain, N., Ibebuogu, U. N., Dagogo-jack, S., & Agarwal, M. (2019). 1560-P: A History of CHF Predicts Adverse Outcomes of Hospitalizations for Hyperglycemic Crises—Data from National and Local Cohorts. Diabetes, 68(Supplement 1), 1560-P. doi:10.2337/db19-1560-p
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  Background: The impact of a history of congestive heart failure (CHF) on the outcomes of DKA and HHS hospitalizations is unknown, and current guidelines provide no clear recommendations for patients with CHF history. Objective: To test the hypothesis that a history of CHF impacts the outcomes of DKA/HHS hospitalizations. Design: Retrospective cohort study of DKA/HHS hospitalizations using Nationwide Inpatient Sample (NIS) database 2003 to 2014 (n= 1,793,983) and University Hospital cohort (n= 1,528). Outcomes: All cause in-hospital mortality, acute respiratory failure events, length of stay and post discharge utilization of nursing home. Results: In the NIS cohort, a history of CHF was present in 83,173 (4.6%) patients among DKA/HHS hospitalizations and was associated with higher adverse outcomes (mortality: 2.8% in CHF vs. 0.6% in no-CHF, adjusted odds ratio (AOR) = 1.5, 95% confidence interval (CI) 1.4 to 1.6; acute respiratory failure- 9.8% vs. 2.2%, AOR = 2.1, 95% CI 2.0 to 2.2). CHF history also predicted higher utilization of post discharge nursing home services (19.6% vs. 5.2%, AOR = 1.4, 95% CI 1.3 to 1.5) even after extensive adjustment for patient demographics, Elixhauser comorbidities and Charlson comorbidity index. In the University cohort, CHF history was present in 129 (8.4%) patients among 1,528 HGc hospitalizations and was associated with higher in-hospital complications (in-hospital mortality and acute respiratory failure [(13.2% vs. 5.2%, AOR = 2.1, 95% CI 1.2 to 3.7)] and higher utilization of post discharge nursing home (15.2% vs. 2.6%, AOR = 2.5, 95% CI 1.3 to 4.7) when compared with those without CHF. Mean length of stay was higher in patients with CHF history compared with no-CHF history in both datasets. Conclusions: A history of CHF is an under-recognized risk factor for adverse outcomes of hospitalizations for hyperglycemic crises. Prospective studies are warranted to better understand and mitigate these differences in outcomes. Disclosure M. Agarwal: None. N. Jain: None. S. Kode: None. P.S. Podila: None. R.N. Khouzam: None. U.N. Ibebuogu: None. G. Reed: Other Relationship; Self; Translational Sciences. S. Dagogo-Jack: Board Member; Self; Jana Care Inc. Consultant; Self; Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Sanofi-Aventis. Research Support; Self; AstraZeneca, Novo Nordisk Inc. Stock/Shareholder; Self; Dance Biopharm Holdings Inc.
• Singh, S., Houng, A. K., & Reed, G. L. (2019). Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin. Blood, 134(12), 970-978.
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  Stasis of venous blood triggers deep vein thrombosis by activating coagulation, yet its effects on the fibrinolytic system are not fully understood. We examined the relationship between stasis, fibrinolysis, and the development of experimental venous thrombosis. Effects of stasis-induced deep vein thrombosis and fibrinolysis on thrombosis were examined by inferior vena cava ligation in congenic mice with and without α2-antiplasmin (α2AP), the primary inhibitor of plasmin. Venous thrombus weights were measured and thrombus composition was determined by Martius scarlet blue and immunofluorescence staining. Venous thrombi from α2AP mice contained plasminogen activators, plasminogen activator inhibitor-1, plasminogen, and α2AP, which changed with thrombus age. Normal, α2AP mice developed large, occlusive thrombi within 5 hours after ligation; thrombi were even larger in plasminogen-deficient mice ( < .001). No significant thrombus formation was seen in α2AP mice ( < .0001) or in α2AP mice treated with an α2AP-inactivating antibody ( < .001). Venous stasis activated fibrinolysis, measured by D-dimer levels, in α2AP mice vs α2AP mice ( < .05). Inhibition of fibrinolysis by the indirect plasmin inhibitor ε-aminocaproic acid or by α2AP restored thrombosis in α2AP mice. In addition to its effects on acute thrombosis, thrombus formation was also markedly suppressed in α2AP mice vs α2AP mice ( < .0001) 1, 7, and 14 days after ligation. We conclude that experimental venous stasis activates the fibrinolytic system to block the development of venous thrombosis. Suppression of fibrinolysis by α2AP appears essential for stasis-induced thrombus development, which suggests that targeting α2AP may prove useful for preventing venous thrombosis.
• Sullivan, R. D., Mehta, R. M., Tripathi, R., Gladysheva, I. P., & Reed, G. L. (2019). Normalizing Plasma Renin Activity in Experimental Dilated Cardiomyopathy: Effects on Edema, Cachexia, and Survival. International journal of molecular sciences, 20(16).
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  Heart failure (HF) patients frequently have elevated plasma renin activity. We examined the significance of elevated plasma renin activity in a translationally-relevant model of dilated cardiomyopathy (DCM), which replicates the progressive stages (A-D) of human HF. Female mice with DCM and elevated plasma renin activity concentrations were treated with a direct renin inhibitor (aliskiren) in a randomized, blinded fashion beginning at Stage B HF. By comparison to controls, aliskiren treatment normalized pathologically elevated plasma renin activity ( < 0.001) and neprilysin levels ( < 0.001), but did not significantly alter pathological changes in plasma aldosterone, angiotensin II, atrial natriuretic peptide, or corin levels. Aliskiren improved cardiac systolic function (ejection fraction, < 0.05; cardiac output, < 0.01) and significantly reduced the longitudinal development of edema (extracellular water, < 0.0001), retarding the transition from Stage B to Stage C HF. The normalization of elevated plasma renin activity reduced the loss of body fat and lean mass (cachexia/sarcopenia), < 0.001) and prolonged survival ( < 0.05). In summary, the normalization of plasma renin activity retards the progression of experimental HF by improving cardiac systolic function, reducing the development of systemic edema, cachexia/sarcopenia, and mortality. These data suggest that targeting pathologically elevated plasma renin activity may be beneficial in appropriately selected HF patients.
• Sullivan, R. D., Mehta, R. M., Tripathi, R., Reed, G. L., & Gladysheva, I. P. (2019). Renin Activity in Heart Failure with Reduced Systolic Function-New Insights. International journal of molecular sciences, 20(13).
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  Regardless of the cause, symptomatic heart failure (HF) with reduced ejection fraction (rEF) is characterized by pathological activation of the renin-angiotensin-aldosterone system (RAAS) with sodium retention and extracellular fluid expansion (edema). Here, we review the role of active renin, a crucial, upstream enzymatic regulator of the RAAS, as a prognostic and diagnostic plasma biomarker of heart failure with reduced ejection fraction (HFrEF) progression; we also discuss its potential as a pharmacological bio-target in HF therapy. Clinical and experimental studies indicate that plasma renin activity is elevated with symptomatic HFrEF with edema in patients, as well as in companion animals and experimental models of HF. Plasma renin activity levels are also reported to be elevated in patients and animals with rEF before the development of symptomatic HF. Modulation of renin activity in experimental HF significantly reduces edema formation and the progression of systolic dysfunction and improves survival. Thus, specific assessment and targeting of elevated renin activity may enhance diagnostic and therapeutic precision to improve outcomes in appropriate patients with HFrEF.
• Tripathi, R., Sullivan, R. D., Fan, T. M., Houng, A. K., Mehta, R. M., Reed, G. L., & Gladysheva, I. P. (2019). Cardiac-Specific Overexpression of Catalytically Inactive Corin Reduces Edema, Contractile Dysfunction, and Death in Mice with Dilated Cardiomyopathy. International journal of molecular sciences, 21(1).
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  Humans with dilated cardiomyopathy (DCM) and heart failure (HF) develop low levels of corin, a multi-domain, cardiac-selective serine protease involved in natriuretic peptide cleavage and sodium and water regulation. However, experimental restoration of corin levels markedly attenuates HF progression. To determine whether the beneficial effects of corin in HF require catalytic activity, we engineered cardiac overexpression of an enzymatically inactive corin transgene (corin-Tg(i)). On a wild-type (WT) background, corin-Tg(i) had no evident phenotypic effects. However, in a well-established genetic model of DCM, corin-Tg(i)/DCM mice had increased survival ( < 0.01 to 0.001) vs. littermate corin-WT/DCM controls. Pleural effusion ( < 0.01), lung edema ( < 0.05), systemic extracellular free water ( < 0.01), and heart weight were decreased ( < 0.01) in corin-Tg(i)/DCM vs. corin-WT/DCM mice. Cardiac ejection fraction and fractional shortening improved ( < 0.01), while ventricular dilation decreased ( < 0.0001) in corin-Tg(i)/DCM mice. Plasma atrial natriuretic peptide, cyclic guanosine monophosphate, and neprilysin were significantly decreased. Cardiac phosphorylated glycogen synthase kinase-3β (pSer9-GSK3β) levels were increased in corin(i)-Tg/DCM mice ( < 0.01). In summary, catalytically inactive corin-Tg(i) decreased fluid retention, improved contractile function, decreased HF biomarkers, and diminished cardiac GSK3β activity. Thus, the protective effects of cardiac corin on HF progression and survival in experimental DCM do not require the serine protease activity of the molecule.
• Zhao, T., Houng, A., & Reed, G. L. (2019). Termination of bleeding by a specific, anticatalytic antibody against plasmin. Journal of thrombosis and haemostasis : JTH, 17(9), 1461-1469.
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  Excessive, plasmin-mediated fibrinolysis augments bleeding and contributes to death in some patients. Current therapies for fibrinolytic bleeding are limited by modest efficacy, low potency, and off-target effects.
• Agarwal, M. A., Garg, L., Lavie, C. J., Reed, G. L., & Khouzam, R. N. (2018). Impact of family history of coronary artery disease on in-hospital clinical outcomes in ST-segment myocardial infarction. Annals of translational medicine, 6(1), 3.
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  Patients with a family history of coronary artery disease (FHxCAD) are at increased risk for development of myocardial infarction (MI). However, the data on the influence of FHxCAD on in-hospital clinical outcomes post ST-segment myocardial infarction (STEMI) is limited. Hence, we evaluated the impact of FHxCAD on in-hospital clinical outcomes post STEMI in an unselected nationwide cohort.
• Agarwal, M. A., Shah, M., Patel, B., Nolan, V. G., Reed, G. L., Oudiz, R. J., Choudhary, G., & Maron, B. A. (2018). Association between Pulmonary Hypertension and Clinical Outcomes in Hospitalized Patients with Sickle Cell Disease. American journal of respiratory and critical care medicine, 198(4), 534-537.
• Agarwal, M., Agrawal, S., Garg, L., Reed, G. L., Khouzam, R. N., & Ibebuogu, U. N. (2018). Impact of smoking in patients undergoing transcatheter aortic valve replacement. Annals of translational medicine, 6(1), 2.
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  The paradox that smokers have better clinical outcomes in cardiovascular diseases remains controversial. No literature exists studying impact of smoking on outcomes following transcatheter aortic valve replacement (TAVR).
• Heckle, M. R., Efeovbokhan, N., Thomas, F., Blumer, M., Chumpia, M., Ibebuogu, U., Reed, G. L., & Khouzam, R. N. (2018). Accurate Prediction of False ST-Segment Elevation Myocardial Infarction: Ready for Prime Time?. Current problems in cardiology, 43(10), 400-412.
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  The incidence of inappropriate cardiac catheterization lab activation for treatment of a false ST-segment elevation myocardial infarction (STEMI) has been reported to be 2.6%-36%. Excessive inappropriate catheterization lab activation may be associated with risks to patients, provider fatigue and improper resource usage.
• Singh, S., Houng, A. K., & Reed, G. L. (2018). Matrix Metalloproteinase-9 Mediates the Deleterious Effects of α2-Antiplasmin on Blood-Brain Barrier Breakdown and Ischemic Brain Injury in Experimental Stroke. Neuroscience, 376, 40-47.
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  During acute brain ischemia, α2-antiplasmin markedly enhances brain injury, blood-brain barrier breakdown and matrix metalloproteinase-9 (MMP-9) expression. Although α2-antiplasmin inhibits fibrin thrombus-degradation, and MMP-9 is a collagen-degrading enzyme altering blood-brain barrier, both have similar deleterious effects on the ischemic brain. We examined the hypothesis that MMP-9 is an essential downstream mediator of α2-antiplasmin's deleterious effects during brain ischemia. Middle cerebral artery thromboembolic stroke was induced in a randomized, blinded fashion in mice with increased blood levels of α2-antiplasmin. There was a robust increase in MMP-9 expression (immunofluorescence) in the ischemic vs. the non-ischemic hemisphere of MMP-9 but not MMP-9 mice, 24 h after stroke. Brain swelling and hemorrhage were significantly increased in the ischemic vs. the non-ischemic hemisphere of MMP-9 mice. By comparison to MMP-9 mice, the ischemic hemispheres of MMP-9 mice showed a ∼6-fold reduction in brain swelling (p 
• Wang, D., Gladysheva, I. P., Sullivan, R. D., Fan, T. M., Mehta, R. M., Tripathi, R., Sun, Y., & Reed, G. L. (2018). Increases in plasma corin levels following experimental myocardial infarction reflect the severity of ischemic injury. PloS one, 13(9), e0202571.
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  Following acute myocardial infarction, clinical studies show alterations in the blood levels of corin, a cardiac-selective activator of the natriuretic peptides pro-atrial natriuretic peptide (pro-ANP) and pro-B-type natriuretic peptide (pro-BNP). However, the temporal changes in circulating and cardiac corin levels and their relationships to the severity of myocardial infarction have not been studied. The main objective of this study was to examine the relationship between cardiac and circulating corin levels and their association with cardiac systolic function and infarct size during the early phase of acute myocardial infarction (
• Zaidi, S. S., Ward, R. D., Ramanathan, K., Yu, X., Gladysheva, I. P., & Reed, G. L. (2018). Possible Enzymatic Downregulation of the Natriuretic Peptide System in Patients with Reduced Systolic Function and Heart Failure: A Pilot Study. BioMed research international, 2018, 7279036.
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  In patients with reduced systolic function, the natriuretic peptide system affects heart failure (HF) progression, but the expression of key activating (corin) and degrading enzymes (neprilysin) is not well understood.
• Agarwal, M., Agrawal, S., Garg, L., Garg, A., Bhatia, N., Kadaria, D., & Reed, G. (2017). Effect of Chronic Obstructive Pulmonary Disease on In-Hospital Mortality and Clinical Outcomes After ST-Segment Elevation Myocardial Infarction. The American journal of cardiology, 119(10), 1555-1559.
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  There is controversy regarding in-hospital mortality, revascularization, and other adverse outcomes in patients with ST-segment elevation (STEMI) and chronic obstructive pulmonary disease (COPD). We queried the 2003 to 2011 Nationwide Inpatient Sample databases to identify patients aged ≥18 years with a primary diagnosis of STEMI. Univariate and multivariate analyses were performed to evaluate the association of COPD with in-hospital clinical outcomes. Patients with COPD comprised 13.2% of 2,120,005 patients with STEMI. COPD was associated with older age, Medicare insurance, greater co-morbidities, and lower socioeconomic status. Compared with non-COPD patients, patients with COPD had higher inpatient mortality even after adjustment for multiple potential other factors (12.5% vs 8.6%, adjusted odds ratio [AOR] 1.13, 95% CI 1.11 to 1.15, p
• Bob-Manuel, T., Ifedili, I., Reed, G., Ibebuogu, U. N., & Khouzam, R. N. (2017). Non-ST Elevation Acute Coronary Syndromes: A Comprehensive Review. Current problems in cardiology, 42(9), 266-305.
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  Non-ST elevation-acute coronary syndrome (NSTE-ACS) includes NSTE myocardial infarction and unstable angina. This patient population forms approximately two-thirds of all hospital admissions for ACS in the United States each year and is associated with an in-hospital mortality of 5%. NSTE-ACS is primarily due to an acute change in the supply and demand balance of coronary perfusion and myocardial oxygen consumption, because of the significant coronary artery obstruction presenting as plaque rupture or erosion. Nevertheless, nonobstructive causes may lead to that same phenomenon by excessive myocardial oxygen demand or reduced coronary supply from hypotension, anemia, or sepsis, including transient coronary vasospasm and endocardial dysfunction. The recent clinical application of high-sensitivity troponin biomarker assays and computer tomography angiography shows promise for improving the diagnosis and the risk stratification of patients with angina symptoms. Implementation of recent updates to the American College of Cardiology/American Heart Association (ACC/AHA) guidelines on NSTE-ACS, especially regarding the selection and duration of antiplatelet therapy, have led to improvement in management and outcomes of this disease. Additionally, new adjunctive therapies and approaches to diagnosis and treatment are discussed. Despite the progress made in recent years in the diagnosis and management of NSTE-ACS, morbidity remains high and mortality is significant. Such a fact suggests that future research targeting prevention, early diagnosis, and intervention in these patients is warranted. This article provides a detailed overview of the most recent information on the pathophysiology, diagnosis, treatment, and prognosis of NSTE-ACS.
• Reed, G. L., Garg, L., Aggarwal, S., & Agarwal, M. (2017). 5803Contemporary Trends of Incidence, Management and Outcomes of Non-acute Coronary Syndrome Associated Cardiogenic Shock: data from 2003 to 2011. European Heart Journal, 38(suppl_1). doi:10.1093/eurheartj/ehx493.5803
• Reed, G. L., Houng, A. K., Singh, S., & Wang, D. (2017). α2-Antiplasmin: New Insights and Opportunities for Ischemic Stroke. Seminars in thrombosis and hemostasis, 43(2), 191-199.
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  Thrombotic vascular occlusion is the leading cause of ischemic stroke. High blood levels of α-antiplasmin (a2AP), an ultrafast, covalent inhibitor of plasmin, have been linked in humans to increased risk of ischemic stroke and failure of tissue plasminogen activator (tPA) therapy. Consistent with these observations, a2AP neutralizes the therapeutic benefit of tPA therapy in experimental stroke. In addition, a2AP has deleterious, dose-related effects on ischemic brain injury in the absence of therapy. Experimental therapeutic inactivation of a2AP markedly reduces microvascular thrombosis, ischemic brain injury, brain swelling, brain hemorrhage, and death after thromboembolic stroke. These data provide new insights into the critical importance of a2AP in the pathogenesis of ischemic brain injury and suggest that transiently inactivating a2AP may have therapeutic value in ischemic stroke.
• Shah, R., Rashid, A., Hwang, I., Fan, T. M., Khouzam, R. N., & Reed, G. L. (2017). Meta-Analysis of the Relative Efficacy and Safety of Oral P2Y12 Inhibitors in Patients With Acute Coronary Syndrome. The American journal of cardiology, 119(11), 1723-1728.
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  A cornerstone of medical therapy for patients with acute coronary syndrome (ACS) is dual antiplatelet therapy, which includes aspirin and a P2Y12 inhibitor. Randomized controlled trials (RCTs) have shown that prasugrel and ticagrelor are superior to clopidogrel, but none directly compared these 3 commonly used oral P2Y12 inhibitors for safety and efficacy. Therefore, we performed a Bayesian network meta-analysis of RCTs to compare the efficacies and safeties of 3 commonly used oral P2Y12 inhibitors in patients with ACS. Scientific databases and websites were searched for relevant RCTs. We included data from 9 RCTs that enrolled 106,288 patients. Clopidogrel decreased the rates of major adverse cardiac event, recurrent myocardial infarction, and all-cause mortality compared with placebo. Both ticagrelor and prasugrel decreased the rates for major adverse cardiac event and recurrent myocardial infarction compared with clopidogrel, but there was no difference between the 2. Both also decreased the stent thrombosis rate compared with clopidogrel, but prasugrel was more effective than ticagrelor. Ticagrelor use was also associated with improved all-cause and CV mortalities compared with clopidogrel. There was no difference in CV mortality or all-cause mortality between clopidogrel and prasugrel. Prasugrel use was also associated with significantly increased risk of major bleeding compared with clopidogrel but showed a nonsignificant trend toward increasing the risk of bleeding compared with ticagrelor. In treatment ranking, ticagrelor was the most efficacious, and prasugrel was the least safe. In conclusion, this meta-analysis shows that in patients with ACS, adding P2Y12 inhibitors to aspirin and other standard treatments reduces ischemic events and all-cause mortality. Among the commonly used oral P2Y12 inhibitors, ticagrelor has the best net efficacy and safety profile.
• Singh, S., Houng, A., & Reed, G. L. (2017). Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation. Circulation, 135(11), 1011-1020.
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  In patients with hemodynamically significant pulmonary embolism, physiological fibrinolysis fails to dissolve thrombi acutely and r-tPA (recombinant tissue-type plasminogen activator) therapy may be required, despite its bleeding risk. To examine potential mechanisms, we analyzed the expression of key fibrinolytic molecules in experimental pulmonary emboli, assessed the contribution of α2-antiplasmin to fibrinolytic failure, and compared the effects of plasminogen activation and α2-antiplasmin inactivation on experimental thrombus dissolution and bleeding.
• Tripathi, R., Sullivan, R., Fan, T. M., Wang, D., Sun, Y., Reed, G. L., & Gladysheva, I. P. (2017). Enhanced heart failure, mortality and renin activation in female mice with experimental dilated cardiomyopathy. PloS one, 12(12), e0189315.
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  Dilated cardiomyopathy (DCM) is the major cause of heart failure affecting both women and men. Limited clinical studies show conflicting data in sex-related differences in the progression of dilated cardiomyopathy and heart failure (HF) outcomes. We examined the comparative sex-related progression of cardiomyopathy and the development of HF (at 4, 7, 13 weeks of age) in a well-established, transgenic mouse model of DCM that recapitulates the progressive stages of human HF. By 13 weeks of age, female mice with DCM had more severe left ventricular systolic dysfunction, left ventricular dilation and wall thinning (P
• Wang, D., & Reed, G. L. (2017). Potential value of circulating corin levels in acute and chronic myocardial infarction. Journal of laboratory and precision medicine, 2(6).
• Yu, H., Huang, Y., Chen, X., Nie, W., Wang, Y., Jiao, Y., Reed, G. L., Gu, W., & Chen, H. (2017). High-sensitivity C-reactive protein in stroke patients - The importance in consideration of influence of multiple factors in the predictability for disease severity and death. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia, 36, 12-19.
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  High sensitivity C-reactive protein (hsCRP) has been evaluated as a biomarker in stroke and relevant pathological diseases. While its predictive values in several pathological phenotypes have been confirmed, controversy exists among different studies. This review summarizes reports of the predictive values of hsCRP for the diagnosis, etiology, prognosis and mortality of stroke diseases. The current literature suggests that CRP expression is influenced by multiple factors, such as polymorphisms, the genomic backgrounds and gender. However, few reported studies analyzed data based on all these multiple factors. Future studies should focus on comprehensive analysis based on multiple factors.
• Zambetti, B. R., Thomas, F., Hwang, I., Brown, A. C., Chumpia, M., Ellis, R. T., Naik, D., Khouzam, R. N., Ibebuogu, U. N., & Reed, G. L. (2017). A web-based tool to predict acute kidney injury in patients with ST-elevation myocardial infarction: Development, internal validation and comparison. PloS one, 12(7), e0181658.
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  In ST-elevation myocardial infarction (STEMI), acute kidney injury (AKI) may increase subsequent morbidity and mortality. Still, it remains difficult to predict AKI risk in these patients. We sought to 1) determine the frequency and clinical outcomes of AKI and, 2) develop, validate and compare a web-based tool for predicting AKI.
• Bolorunduro, O., Smith, B., Chumpia, M., Valasareddy, P., Heckle, M. R., Khouzam, R. N., Reed, G. L., & Ibebuogu, U. N. (2016). Racial Difference in Symptom Onset to Door Time in ST Elevation Myocardial Infarction. Journal of the American Heart Association, 5(10).
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  There are poorer outcomes following ST elevation myocardial infarction in blacks compared to white patients despite comparable door-to-reperfusion time. We hypothesized that delays to hospital presentation may be contributory.
• Singh, S., Houng, A. K., Wang, D., & Reed, G. L. (2016). Physiologic variations in blood plasminogen levels affect outcomes after acute cerebral thromboembolism in mice: a pathophysiologic role for microvascular thrombosis. Journal of thrombosis and haemostasis : JTH, 14(9), 1822-32.
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  Essentials Physiologic variations in blood plasminogen (Pg) levels may affect ischemic stroke outcomes. We tested Pg effects in a model with translational relevance to human thromboembolic stroke. A dose-response exists between Pg levels and brain injury, fibrinolysis, barrier breakdown. Higher Pg levels reduce microvascular thrombosis and improve outcomes in ischemic stroke.
• Tripathi, R., Wang, D., Sullivan, R., Fan, T. H., Gladysheva, I. P., & Reed, G. L. (2016). Depressed Corin Levels Indicate Early Systolic Dysfunction Before Increases of Atrial Natriuretic Peptide/B-Type Natriuretic Peptide and Heart Failure Development. Hypertension (Dallas, Tex. : 1979), 67(2), 362-7.
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  Dilated cardiomyopathy is a major cause of heart failure (HF) that affects millions. Corin cleaves and biologically activates pro-atrial natriuretic peptide (pro-ANP) and pro-B-type natriuretic peptide (pro-BNP). High corin levels reduce the development of systolic dysfunction and HF in experimental dilated cardiomyopathy. Yet, patients with significant HF unexpectedly show low corin levels with high plasma ANP/BNP levels. Therefore, we examined the relationship between cardiac corin expression, ANP/BNP levels, and the stages of HF. We used a well-established, dilated cardiomyopathy model to evaluate gene and protein expression as mice longitudinally developed Stages A-D HF. Cardiac systolic function (ejection fraction) continuously declined over time (P
• Ibebuogu, U. N., Bolorunduro, O., Giri, S., Dagogo-Jack, S., Smith, B. G., Kar, S., & Reed, G. L. (2015). Bivalirudin Versus Heparin Plus Glycoprotein IIb/IIIa Inhibitors in Patients with Diabetes Mellitus Undergoing Percutaneous Coronary Intervention: A Meta-Analysis of Randomized Controlled Trials. American journal of cardiovascular drugs : drugs, devices, and other interventions, 15(4), 275-85.
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  Diabetes mellitus (DM) is a pro-thrombotic state with enhanced thrombin generation and platelet reactivity. For most patients undergoing percutaneous coronary intervention (PCI), bivalirudin demonstrates efficacy comparable with that of heparin and glycoprotein IIb/IIIa inhibitors (GPIs). Yet, because of their pro-thrombotic condition, we hypothesized that patients with DM may benefit from more aggressive dual antithrombin and antiplatelet therapy. The aim of this paper was to provide a systematic review comparing outcomes of PCI with bivalirudin versus heparin plus GPI in patients with DM using meta-analytical techniques. Eligible studies needed to have reported a subgroup analysis of outcomes among diabetic patients. Six trials comprising 5924 diabetic patients were eligible. At 30 days, bivalirudin was associated with a reduction in net adverse cardiac events [relative risk (RR) 0.81, 95 % confidence interval (CI) 0.70-0.93, p = 0.002] and major bleeds (RR 0.68, 95 % CI 0.49-0.95; p = 0.02), with no difference in composite ischemia (RR 0.92, 95 % CI 0.74-1.14; p = 0.43) or mortality (RR 0.71, 95 % CI 0.45-1.13; p = 0.15). At 1 year, bivalirudin was associated with a significant reduction in all-cause mortality (RR 0.73, 95 % CI 0.54-1.00, p = 0.05) despite similar composite ischemia (RR 1.02, 95 % CI 0.56-1.21, p = 0.811). In conclusion, thrombin inhibition with bivalirudin alone was associated with reduced 30-day major bleeding and 1-year all-cause mortality compared with heparin plus GPI in diabetic patients undergoing PCI.
• Ibebuogu, U. N., Bolorunduro, O., Giri, S., Dagogo-Jack, S., Smith, B. G., Kar, S., & Reed, G. L. (2015). Erratum to: Bivalirudin Versus Heparin Plus Glycoprotein IIb/IIIa Inhibitors in Patients with Diabetes Mellitus Undergoing Percutaneous Coronary Intervention: A Meta-Analysis of Randomized Controlled Trials. American journal of cardiovascular drugs : drugs, devices, and other interventions, 15(4), 287.
• Stein-Merlob, A. F., Kessinger, C. W., Erdem, S. S., Zelada, H., Hilderbrand, S. A., Lin, C. P., Tearney, G. J., Jaff, M. R., Reed, G. L., Henke, P. K., McCarthy, J. R., & Jaffer, F. A. (2015). Blood Accessibility to Fibrin in Venous Thrombosis is Thrombus Age-Dependent and Predicts Fibrinolytic Efficacy: An In Vivo Fibrin Molecular Imaging Study. Theranostics, 5(12), 1317-27.
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  Fibrinolytic therapy of venous thromboembolism (VTE) is increasingly utilized, yet limited knowledge is available regarding in vivo mechanisms that govern fibrinolytic efficacy. In particular, it is unknown how age-dependent thrombus organization limits direct blood contact with fibrin, the target of blood-based fibrinolytic agents. Utilizing high-resolution in vivo optical molecular imaging with FTP11, a near-infrared fluorescence (NIRF) fibrin-specific reporter, here we investigated the in vivo interrelationships of blood accessibility to fibrin, thrombus age, thrombus neoendothelialization, and fibrinolysis in murine venous thrombosis (VT). In both stasis VT and non-stasis VT, NIRF microscopy showed that FTP11 fibrin binding was thrombus age-dependent. FTP11 localized to the luminal surface of early-stage VT, but only minimally to subacute VT (p
• Houng, A. K., Wang, D., & Reed, G. L. (2014). Reversing the deleterious effects of α2-antiplasmin on tissue plasminogen activator therapy improves outcomes in experimental ischemic stroke. Experimental neurology, 255, 56-62.
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  High blood levels of α2-antiplasmin have been associated with failed tissue plasminogen activator (TPA) therapy for ischemic stroke. Yet, other data suggests that α2-antiplasmin may be protective in stroke, because it defends against bleeding and excitotoxicity. To address this paradox, we examined the effects of high α2-antiplasmin levels and α2-antiplasmin inactivation in mice treated with TPA 0.5-2.5h after middle cerebral artery (MCA) thromboembolism. Brain infarction, swelling, hemorrhage, blood brain barrier breakdown and neuronal apoptosis were measured by a blinded observer. Thrombus dissolution was determined by gamma counting. During TPA treatment, high α2-antiplasmin blood levels increased brain infarction (2.2-fold) and swelling (3.7-fold), but decreased MCA thrombus dissolution. Conversely, α2-antiplasmin inactivation during TPA treatment reduced brain infarction, hemorrhage and swelling, but increased MCA thrombus dissolution. Inactivation of α2-antiplasmin during TPA treatment reduced neuronal apoptosis and blood brain barrier breakdown. Inactivation of α2-antiplasmin also reduced short-term mortality. Taken together these data show that α2-antiplasmin opposes the effects of TPA therapy and contributes to enhanced brain injury after experimental thromboembolic stroke. Conversely, α2-antiplasmin inactivation during TPA treatment improves thrombus dissolution and reduces brain infarction, swelling and hemorrhage. Consistent with clinical observations, these data suggest that α2-antiplasmin exerts deleterious effects that reduce the efficacy and safety of TPA therapy for ischemic stroke.
• Reed, G. L., Houng, A. K., & Wang, D. (2014). Microvascular thrombosis, fibrinolysis, ischemic injury, and death after cerebral thromboembolism are affected by levels of circulating α2-antiplasmin. Arteriosclerosis, thrombosis, and vascular biology, 34(12), 2586-93.
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  Ischemic stroke is primarily attributable to thrombotic vascular occlusion. Elevated α2-antiplasmin (a2AP) levels correlate with increased stroke risk, but whether a2AP contributes to the pathogenesis of stroke is unknown. We examined how a2AP affects thrombosis, ischemic brain injury, and survival after experimental cerebral thromboembolism.
• Speich, H. E., Bhal, V., Houser, K. H., Caughran, A. T., Lands, L. T., Houng, A. K., Bäckstrom, J., Enerbäck, M., Reed, G. L., & Jennings, L. K. (2014). Signaling via P2Y12 may be critical for early stabilization of platelet aggregates. Journal of cardiovascular pharmacology, 63(6), 520-7.
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  P2Y(12) receptor antagonism inhibits platelet aggregation by preventing adenosine diphosphate (ADP)-mediated amplification of activation pathways downstream of primary agonists, such as thrombin and collagen. However, the role of ADP signaling in maintaining aggregate stability and the effects of P2Y(12) antagonists on preestablished aggregates in vitro and arterial thrombus in vivo are not well understood. This study evaluated the impact of P2Y(12) signaling on platelet aggregate stability and early thrombotic occlusion using a reversible P2Y(12) antagonist, ticagrelor. There were 2 study objectives: (1) to determine if there was a time-dependent factor on the capacity of a P2Y(12) antagonist to affect human platelet aggregate stability in vitro using light transmission aggregometry and (2) to evaluate the extent of arterial thrombus reversal in a preclinical model upon administration of ticagrelor in vivo. Platelet aggregates were exposed to ticagrelor after ADP or collagen activation, monitored for stability by aggregometry, and visualized by microscopy. Freshly formed ADP- and collagen-induced platelet aggregates were more rapidly dispersed by a P2Y(12) antagonist than drug carrier control at clinically relevant concentrations (P < 0.05). However, stable aggregates were not noticeably affected. A murine arterial thrombosis model was used to evaluate thrombus stability in an in vivo mouse model. Thrombotic occlusion was induced by FeCl(3), followed by a bolus intravenous administration of ticagrelor or vehicle control. Doppler blood flow was monitored before injury and 30 minutes after bolus administration. Arteries were retrieved for inspection for residual thrombus. Early arterial thrombotic occlusion in vivo was partially reversed by ticagrelor administration. Blood flow through the injured artery increased, and thrombus size within the artery decreased (P < 0.05, n = 3). In conclusion, P2Y(12) antagonism disrupts the stability of newly formed platelet aggregates, promoting disaggregation, and reverses thrombotic vascular occlusion. Thus, in addition to activating platelets, signaling via P2Y(12) seems to be required for stabilizing platelet thrombi.
• Wang, D., Gladysheva, I. P., Fan, T. H., Sullivan, R., Houng, A. K., & Reed, G. L. (2014). Atrial natriuretic peptide affects cardiac remodeling, function, heart failure, and survival in a mouse model of dilated cardiomyopathy. Hypertension (Dallas, Tex. : 1979), 63(3), 514-9.
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  Dilated cardiomyopathy is a frequent cause of heart failure and death. Atrial natriuretic peptide (ANP) is a biomarker of dilated cardiomyopathy, but there is controversy whether ANP modulates the development of heart failure. Therefore, we examined whether ANP affects heart failure, cardiac remodeling, function, and survival in a well-characterized, transgenic model of dilated cardiomyopathy. Mice with dilated cardiomyopathy with normal ANP levels survived longer than mice with partial ANP (P
• Gladysheva, I. P., Wang, D., McNamee, R. A., Houng, A. K., Mohamad, A. A., Fan, T. M., & Reed, G. L. (2013). Corin overexpression improves cardiac function, heart failure, and survival in mice with dilated cardiomyopathy. Hypertension (Dallas, Tex. : 1979), 61(2), 327-32.
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  Heart failure, caused by dilated cardiomyopathy and other cardiac disorders such as hypertension, is a major public health problem with high morbidity and mortality. Corin, a cardiac enzyme that cleaves natriuretic peptides, is a promising biomarker of cardiomyopathy and heart failure, but its functional role in these processes is not understood. We evaluated the potential effects of corin in mice with a well-characterized model of dilated cardiomyopathy. Mice with dilated cardiomyopathy developed heart failure, reduced contractile function, cardiac fibrosis, and accelerated mortality in the setting of low corin expression. In wild-type mice, transgenic, cardiac-targeted, overexpression of corin enhanced cyclic guanosine monophosphate and blood pressure responses to pro-atrial natriuretic peptide, but did not affect heart size, contractility, body weights, survival, and blood pressure. In mice with dilated cardiomyopathy, corin overexpression significantly reduced the development of myocardial fibrosis (P
• Aluoch, A. O., Jessee, R., Habal, H., Garcia-Rosell, M., Shah, R., Reed, G., & Carbone, L. (2012). Heart failure as a risk factor for osteoporosis and fractures. Current osteoporosis reports, 10(4), 258-69.
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  Although heart failure (HF) and osteoporosis are common diseases, particularly in elderly populations, patients with HF have an increased risk for osteoporosis. The relationship of HF with osteoporosis is modified by gender and the severity of HF. In addition, shared risk factors, medication use, and common pathogenic mechanisms affect both HF and osteoporosis. Shared risk factors for these 2 conditions include advanced age, hypovitaminosis D, renal disease, and diabetes mellitus. Medications used to treat HF, including spironolactone, thiazide diuretics, nitric oxide donors, and aspirin, may protect against osteoporosis. In contrast, loop diuretics may make osteoporosis worse. HF and osteoporosis appear to share common pathogenic mechanisms, including activation of the renin-angiotensin-aldosterone system, increased parathyroid hormone levels, and/or oxidative/nitrosative stress. HF is a major risk factor for mortality following fractures. Thus, in HF patients, it is important to carefully assess osteoporosis and take measures to reduce the risk of osteoporotic fractures.
• McCarthy, J. R., Sazonova, I. Y., Erdem, S. S., Hara, T., Thompson, B. D., Patel, P., Botnaru, I., Lin, C. P., Reed, G. L., Weissleder, R., & Jaffer, F. A. (2012). Multifunctional nanoagent for thrombus-targeted fibrinolytic therapy. Nanomedicine (London, England), 7(7), 1017-28.
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  Current thrombolytic therapies utilize exogenous plasminogen activators (PAs) to effectively lyse clots, restoring blood flow, and preventing tissue and organ death. These PAs may also impair normal hemostasis, leading to life-threatening bleeding, including intracerebral hemorrhage.
• Zhang, Y., Gladysheva, I. P., Houng, A. K., & Reed, G. L. (2012). Streptococcus uberis plasminogen activator (SUPA) activates human plasminogen through novel species-specific and fibrin-targeted mechanisms. The Journal of biological chemistry, 287(23), 19171-6.
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  Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.
• Ibebuogu, U. N., Gladysheva, I. P., Houng, A. K., & Reed, G. L. (2011). Decompensated heart failure is associated with reduced corin levels and decreased cleavage of pro-atrial natriuretic peptide. Circulation. Heart failure, 4(2), 114-20.
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  By promoting salt and water excretion, the corin and the atrial natriuretic peptide (ANP) system should help to maintain fluid balance in heart failure. Yet, the development of fluid retention despite high levels of ANP-related peptides suggests that this compensatory system is limited.
• Gentry, M. B., Dias, J. K., Luis, A., Patel, R., Thornton, J., & Reed, G. L. (2010). African-American women have a higher risk for developing peripartum cardiomyopathy. Journal of the American College of Cardiology, 55(7), 654-9.
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  The purpose of this study was to assess whether African-American women are at increased risk of having peripartum cardiomyopathy.
• Anea, C. B., Zhang, M., Stepp, D. W., Simkins, G. B., Reed, G., Fulton, D. J., & Rudic, R. D. (2009). Vascular disease in mice with a dysfunctional circadian clock. Circulation, 119(11), 1510-7.
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  Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. A profound pattern exists in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Although the molecular components of circadian rhythms rhythmically oscillate in blood vessels, evidence of a direct function for the "circadian clock" in the progression to vascular disease is lacking.
• Houng, A. K., McNamee, R. A., Kerner, A., Sharma, P., Mohamad, A., Tronolone, J., & Reed, G. L. (2009). Atrial natriuretic peptide increases inflammation, infarct size, and mortality after experimental coronary occlusion. American journal of physiology. Heart and circulatory physiology, 296(3), H655-61.
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  Acute coronary artery occlusion triggers the release of atrial natriuretic peptide (ANP) from the heart. ANP affects vasodilation, natriuresis, and inflammation, but the integrated biological effects of ANP on myocardial infarction are unknown. To elucidate these effects, the left anterior coronary artery was ligated in anesthetized, ANP-deficient (ANP(-/-)) and congenic wild-type (ANP(+/+)) mice. The survival of ANP(-/-) mice was markedly better (56%) at 30 days postinfarction than the survival of ANP(+/+) mice (20%, P < 0.01). Surviving mice were comparable initially, but ANP(-/-) mice developed more cardiac hypertrophy (P < 0.001) and had lower contractility indexes 30 days after infarction (P < 0.05). An analysis 24 h after coronary occlusion showed that ANP(-/-) mice had smaller infarcts than ANP(+/+) mice (62.6 +/- 12.1 vs. 100.8 +/- 3.8%, P < 0.001) adjusted for comparable areas at risk for ischemia. The administration of ANP to ANP(-/-) mice via osmotic minipumps significantly enlarged infarct size to levels comparable with those observed in ANP(+/+) mice (P < 0.05). There was no difference in neutrophil migration into the noninfarcted myocardium of ANP(-/-) mice undergoing actual versus sham-operated coronary occlusion. By comparison, after coronary occlusion, the neutrophil infiltration into the myocardium was enhanced in ANP(+/+) (P < 0.0005) and ANP(-/-) mice administered ANP (P < 0.0005). The expression of P-selectin, a molecule that mediates neutrophil adhesion, was significantly greater after coronary occlusion in the vasculature of ANP(+/+) or ANP(-/-) mice treated with ANP than in ANP(-/-) mice (P < 0.002). Taken together, these results indicate that ANP increases P-selectin, neutrophil infiltration, infarct size, and mortality following experimental coronary occlusion.
• King, S. M., McNamee, R. A., Houng, A. K., Patel, R., Brands, M., & Reed, G. L. (2009). Platelet dense-granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice. Circulation, 120(9), 785-91.
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  Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet dense-granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury.
• Sazonova, I. Y., McNamee, R. A., Houng, A. K., King, S. M., Hedstrom, L., & Reed, G. L. (2009). Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator. Journal of thrombosis and haemostasis : JTH, 7(8), 1321-8.
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  Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface.
• Dodani, S., Kaur, R., Reddy, S., Reed, G. L., Navab, M., & George, V. (2008). Can dysfunctional HDL explain high coronary artery disease risk in South Asians?. International journal of cardiology, 129(1), 125-32.
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  Coronary artery disease (CAD) is the leading cause of mortality and morbidity in United States, and South Asian immigrants (SAIs) have a higher risk for CAD compare to Caucasians. Traditional risk factors do not completely explain high risk, and some of the unknown risk factors need to be explored. We assessed dysfunctional pro-inflammatory high density lipoprotein (HDL) in SAIs and assessed its association with sub-clinical CAD using carotid intima-media thickness (IMT) as a surrogate marker for atherosclerosis.
• Ibebuogu, U. N., Thornton, J. W., & Reed, G. L. (2008). An unrecognized cause of acute abdomen in peripartum cardiomyopathy. Southern medical journal, 101(4), 447-8.
• Gladysheva, I. P., Sazonova, I. Y., Houng, A., Hedstrom, L., & Reed, G. L. (2007). Regulation of nonproteolytic active site formation in plasminogen. Biochemistry, 46(30), 8879-87.
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  Streptokinase may be less effective at saving lives in patients with heart attacks because it explosively generates plasmin in the bloodstream at sites distant from fibrin clots. We hypothesized that this rapid plasmin generation is due to SK's singular capacity to nonproteolytically generate the active protease SK x Pg*, and we examined whether the kringle domains regulate this process. An SK mutant lacking Ile-1 (deltaIle1-SK) does not form SK x Pg*, although it will form complexes with plasmin that can activate plasminogen. When compared to SK, deltaIle1-SK diminished the generation of plasmin in plasma by more than 30-fold, demonstrating that the formation of SK x Pg* plays an important role in SK activity in the blood. The rate of SK x Pg* formation (measured by an active site titrant) was much slower in Glu-Pg, which contains five kringle domains, than in Pg forms containing one kringle (mini-Pg) or no kringles (micro-Pg). In a similar manner, Streptococcus uberis Pg activator (SUPA), an SK-like molecule, generated SUPA x Pg* much slower with bovine Pg than bovine micro-Pg. The velocity of SK x Pg* formation was regulated by agents that influence the conformation of Pg through interactions with the kringle domains. Chloride ions, which maintain the compact Pg conformation, hindered SK x Pg* formation. In contrast, epsilon-aminocaproic acid, fibrin, and fibrinogen, which induce an extended Pg conformation, accelerated the formation of SK x Pg*. In summary, the explosive generation of plasmin in blood or plasma, which diminishes SK's therapeutic effects, is attributable to the formation of SK x Pg*, and this process is governed by kringle domains.
• Huggins, G. S., Lepore, J. J., Greytak, S., Patten, R., McNamee, R., Aronovitz, M., Wang, P. J., & Reed, G. L. (2007). The CREB leucine zipper regulates CREB phosphorylation, cardiomyopathy, and lethality in a transgenic model of heart failure. American journal of physiology. Heart and circulatory physiology, 293(3), H1877-82.
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  Signaling through cAMP plays an important role in heart failure. Phosphorylation of cAMP response element binding protein (CREB) at serine-133 regulates gene expression in the heart. We examined the functional significance of CREB-S133 phosphorylation by comparing transgenic models in which a phosphorylation resistant CREB-S133A mutant containing either an intact or a mutated leucine zipper domain (CREB-S133A-LZ) was expressed in the heart. In vitro, CREB-S133A retained the ability to interact with wild-type CREB, whereas CREB-S133A-LZ did not. In vivo, CREB-S133A and CREB-S133A-LZ were expressed at comparable levels in the heart; however, CREB-S133A markedly suppressed the phosphorylation of endogenous CREB, whereas CREB-S133A-LZ had no effect. The one-year survival of mice from two CREB-S133A-LZ transgenic lines was equivalent to nontransgenic littermate control mice (NTG), whereas transgenic CREB-S133A mice died with heart failure at a median 30 wk of age (P < 0.0001). CREB-S133A mice had an altered gene expression characteristic of the failing heart, whereas CREB-S133A-LZ mice did not. Left ventricular contractile function was substantially reduced in CREB-S133A mice versus NTG mice and only modestly reduced in CREB-S133A-LZ mice (P < 0.02). When considered in light of other studies, these findings indicate that overexpression of the CREB leucine zipper is required for both inhibition of endogenous CREB phosphorylation and cardiomyopathy in this murine model of heart failure.
• Ibebuogu, U. N., Thornton, J. W., & Reed, G. L. (2007). An unusual case of peripartum cardiomyopathy manifesting with multiple thrombo-embolic phenomena. Thrombosis journal, 5, 18.
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  Peripartum cardiomyopathy (PPCM) is a rare form of heart failure with a reported incidence of 1 per 3000 to 1 per 4000 live births and a fatality rate of 20%-50%. Onset is usually between the last month of pregnancy and up to 5 months postpartum in previously healthy women. Although viral, autoimmune and idiopathic factors may be contributory, its etiology remains unknown. PPCM initially presents with signs and symptoms of congestive heart failure and rarely with thrombo-embolic complications. We report an unusual case of PPCM in a previously healthy postpartum woman who presented with an acute abdomen due to unrecognized thromboemboli of the abdominal organs. This case illustrates that abdominal pain in PPCM may not always result from hepatic congestion as previously reported, but may occur as a result of thromboemboli to abdominal organs. Further research is needed to determine the true incidence of thromboemboli in PPCM.
• Sazonova, I. Y., Thomas, B. M., Gladysheva, I. P., Houng, A. K., & Reed, G. L. (2007). Fibrinolysis is amplified by converting alpha-antiplasmin from a plasmin inhibitor to a substrate. Journal of thrombosis and haemostasis : JTH, 5(10), 2087-94.
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  alpha(2)-Antiplasmin (alpha(2)-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against alpha(2)-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of alpha(2)-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to alpha(2)-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured alpha(2)-AP. Binding studies with chimeric alpha(2)-APs revealed that none of the mAbs bound to sites in alpha(2)-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of alpha(2)-AP, or the reactive center loop in the serpin domain of alpha(2)-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of alpha(2)-AP (L(13)GNQEPGGQTALKSPPGVCS(32)) near the site at which alpha(2)-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of alpha(2)-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by alpha(2)-AP (from 1.1 +/- 0.1 to 51 +/- 4 and 67 +/- 7) indicating that they convert alpha(2)-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of alpha(2)-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known alpha(2)-AP-plasmin contacts to enhance fibrinolysis by converting alpha(2)-AP from an inhibitor to a plasmin substrate.
• Nahrendorf, M., Hu, K., Frantz, S., Jaffer, F. A., Tung, C. H., Hiller, K. H., Voll, S., Nordbeck, P., Sosnovik, D., Gattenlöhner, S., Novikov, M., Dickneite, G., Reed, G. L., Jakob, P., Rosenzweig, A., Bauer, W. R., Weissleder, R., & Ertl, G. (2006). Factor XIII deficiency causes cardiac rupture, impairs wound healing, and aggravates cardiac remodeling in mice with myocardial infarction. Circulation, 113(9), 1196-202.
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  Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data.
• Jaffer, F. A., Tung, C. H., Wykrzykowska, J. J., Ho, N. H., Houng, A. K., Reed, G. L., & Weissleder, R. (2004). Molecular imaging of factor XIIIa activity in thrombosis using a novel, near-infrared fluorescent contrast agent that covalently links to thrombi. Circulation, 110(2), 170-6.
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  Activated factor XIII (FXIIIa) mediates fibrinolytic resistance and is a hallmark of newly formed thrombi. In vivo imaging of FXIIIa activity could further elucidate the role of this molecule in thrombosis and other biological processes and aid in the clinical detection of acute thrombi.
• Reed, G. L. (2004). Platelet secretory mechanisms. Seminars in thrombosis and hemostasis, 30(4), 441-50.
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  Platelet granule secretion or exocytosis is required for normal platelet function and plays an important role in the pathogenesis of cardiovascular diseases. Platelets secrete molecules that amplify thrombosis, induce vascular remodeling, recruit and activate cells. The platelet secretory process begins in megakaryocytes where molecules are targeted to developing granules through specific vesicle trafficking and transporter mechanisms. Secretory granules may continue to mature in the circulation after the platelet has been released from the megakaryocyte. The platelet secretory process culminates when ligands interact with specific platelet receptors to trigger exocytosis. A convergence of new insights from several different organisms has begun to illuminate the molecular mechanisms responsible for the platelet secretory process, from granule development through membrane fusion and exocytosis.
• Sazonova, I. Y., Robinson, B. R., Gladysheva, I. P., Castellino, F. J., & Reed, G. L. (2004). alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator. The Journal of biological chemistry, 279(24), 24994-5001.
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  The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.
• Gladysheva, I. P., Turner, R. B., Sazonova, I. Y., Liu, L., & Reed, G. L. (2003). Coevolutionary patterns in plasminogen activation. Proceedings of the National Academy of Sciences of the United States of America, 100(16), 9168-72.
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  The generation of plasmin by plasminogen (Pg) activators (PAs) is a physiologic process in animals that dissolves blood clots and promotes wound healing, blood vessel growth, and the migration of normal and cancerous cells. Pathogenic bacteria have evolved PAs [e.g., streptokinase (SK) and staphylokinase] that exploit the Pg system to infect animals. Animal PAs have a conserved ability to cleave a wide spectrum of animal Pgs, but the ability of bacterial PAs to cleave different animal Pgs is surprisingly restricted. We show that the spectrum of activity of an archetypal bacterial PA (SK) with animal Pgs can be profoundly altered by mutations that affect intermolecular complementarity at sites that participate in complex formation or substrate binding. Comparative sequence analysis of animal plasmins vs. close structural homologues (trypsin and chymotrypsin) that are not molecular targets for invading bacteria indicates that the sites in plasmin that interact with SK are preferentially targeted for mutation. Conversely, intermolecular contact sites in SKs that activate human Pg are more highly conserved than other loci in the molecule or than the same sites in other SKs that activate non-human Pgs. We propose that active modulation of intermolecular complementarity at sites of contact between SK and Pg may represent a competitive evolutionary strategy in a survival battle, whereby animals seek to evade bacterial invasion, and bacteria endeavor to invade their animal hosts.
• Go, A. S., Reed, G. L., Hylek, E. M., Phillips, K. A., Liu, L., Henault, L. E., Selby, J. V., & Singer, D. E. (2003). Factor V Leiden and risk of ischemic stroke in nonvalvular atrial fibrillation: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study. Journal of thrombosis and thrombolysis, 15(1), 41-6.
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  Atrial fibrillation is a major cause of cardioembolic stroke. Since atrial and venous pressures are similar, genetic variants that promote venous thromboembolism may increase the risk of atrial thrombi and subsequent stroke in atrial fibrillation.
• Houng, A., Polgar, J., & Reed, G. L. (2003). Munc18-syntaxin complexes and exocytosis in human platelets. The Journal of biological chemistry, 278(22), 19627-33.
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  The Sec1-Munc18 (SM) proteins are required for cellular exocytosis, but their mechanistic function remains poorly understood. We examined SM-syntaxin complexes in human platelets, which are terminally differentiated, anuclear cells that secrete the contents of their intracellular granules through syntaxin 2- and syntaxin 4-dependent mechanisms. Munc18a, Munc18b, and Munc18c were detected in human platelets by immunoblotting and/or PCR. The SM proteins and syntaxin 2 were found in the membrane and cytosolic fractions of cells, whereas syntaxin 4 was detected only in the membrane. Platelet membranes contain Munc18c-syntaxin 4 complexes, but minimal if any Munc18c-syntaxin 2 complexes were found. No significant amounts of Munc18a or Munc18b complexes were seen with either syntaxin. Munc18c-syntaxin 4 complexes were dissociated when cells were activated to secrete. Two potential inhibitors of Munc18c-syntaxin 4 complexes were generated to examine whether complex dissociation may lead to exocytosis. Peptides that mimic the projected intermolecular contact sites of Munc18c with syntaxin enhanced Ca2+-triggered dense granule exocytosis in permeabilized cells. Similarly, an anti-Munc18c monoclonal antibody that inhibited the Munc18c-syntaxin complex potently amplified Ca2+-induced platelet granule secretion. In summary, Munc18 proteins bind to specific syntaxin isoforms in platelets despite the presence of other potential binding partners. Acute inhibition of the SM-syntaxin complex promotes Ca2+-induced exocytosis, suggesting that complex formation per se has a regulatory effect on triggered secretion.
• Naimark, W. A., Lepore, J. J., Klugherz, B. D., Wang, Z., Guy, T. S., Osman, H., Moainie, S. L., Gorman, R. C., Reed, G., Gorman, J. H., Palasis, M., Parmacek, M. S., & Wilensky, R. L. (2003). Adenovirus-catheter compatibility increases gene expression after delivery to porcine myocardium. Human gene therapy, 14(2), 161-6.
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  Endomyocardial injection of adenoviral gene vectors enables localized delivery to comprised myocardial tissue. However, many materials used in endomyocardial delivery catheters may not be compatible with adenoviral gene vectors. In this study, a series of catheter-based endocardial and epicardial (direct visualization) procedures were performed to assess catheter-adenovirus compatibility in an in vivo model. A standard Nitinol-stainless steel (Ni-SS) catheter was compared with a novel Stiletto catheter designed for improved biocompatibility. In 4 animals 40 endocardial injections of adenovirus encoding beta-galactosidase (beta-Gal) were performed with the 2 catheters. After sectioning of the hearts only 8 of 20 Ni-SS beta-Gal+ sites could be identified (40% retrieval) whereas 16 of the 20 Stiletto injection sites were identified (80%). Within these areas successful transfection was observed (12.2 +/- 4.0 beta-Gal+ cells/high-power field [HPF] in the Ni-SS group vs. 30.1 +/- 6.8 beta-Gal+ cells/HPF in the Stiletto group; p = 0.03). After epicardial delivery to distinct areas of the myocardium adenoviral delivery as assayed by beta-galactosidase protein activity was >21 +/- 16-fold (range, 5 to >43-fold) greater than after Stiletto delivery. In conclusion, this study highlights the importance of adenovirus-material compatibility in gene delivery to the myocardium. Efficiency was greater when using the catheter designed to enhance biocompatibility.
• Polgár, J., Lane, W. S., Chung, S. H., Houng, A. K., & Reed, G. L. (2003). Phosphorylation of SNAP-23 in activated human platelets. The Journal of biological chemistry, 278(45), 44369-76.
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  Phosphorylation of SNARE proteins may provide a critical link between cell activation and secretory processes. Platelets contain all three members of the SNAP-23/25/29 gene family, but by comparison to brain tissue, SNAP-23 is the most highly enriched of these proteins in platelets. SNAP-23 function is required for exocytosis from platelet alpha, dense, and lysosomal granules. SNAP-23 was phosphorylated largely on serine residues in platelets activated with thrombin. Phosphorylation kinetics paralleled or preceded granule secretion. Inhibition studies suggested that SNAP-23 phosphorylation proceeds largely through a protein kinase C (PKC) mechanism and purified PKC directly phosphorylated recombinant (r-) SNAP-23 (up to 0.3 mol of phosphate/mol of protein). Five major tryptic phosphopeptides were identified in cellular SNAP-23 isolated from activated platelets; three phosphopeptides co-migrated with those identified in PKC-phosphorylated r-SNAP-23. In contrast, only one major phosphopeptide was identified when SNAP-23, engaged in a ternary SNARE complex, was phosphorylated by PKC. Ion trap mass spectrometry revealed that platelet SNAP-23 was phosphorylated at Ser23/Thr24 and Ser161, after cell activation by thrombin; these sites were also identified in PKC-phosphorylated r-SNAP-23. SNAP-23 mutants that mimic phosphorylation at Ser23/Thr24 inhibited syntaxin 4 interactions, whereas a phosphorylation mutant of Ser161 had only minor effects. Taken together these studies show that SNAP-23 is phosphorylated in platelets during cell activation through a PKC-related mechanism at two or more sites with kinetics that parallel or precede granule secretion. Because mutants that mimic SNAP-23 phosphorylation affect syntaxin 4 interactions, we hypothesize that SNAP-23 phosphorylation may be important for modulating SNARE-complex interactions during membrane trafficking and fusion.
• Reed, G. L. (2003). Good breeding matters: in-bred rodents provide genetic insights into platelet secretion. Thrombosis and haemostasis, 89(6), 951-2.
• Tung, C. H., Ho, N. H., Zeng, Q., Tang, Y., Jaffer, F. A., Reed, G. L., & Weissleder, R. (2003). Novel factor XIII probes for blood coagulation imaging. Chembiochem : a European journal of chemical biology, 4(9), 897-9.
• Dale, G. L., Friese, P., Batar, P., Hamilton, S. F., Reed, G. L., Jackson, K. W., Clemetson, K. J., & Alberio, L. (2002). Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface. Nature, 415(6868), 175-9.
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  Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.
• Gladysheva, I. P., Sazonova, I. Y., Chowdhry, S. A., Liu, L., Turner, R. B., & Reed, G. L. (2002). Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation, substrate, and inhibitor interactions. The Journal of biological chemistry, 277(30), 26846-51.
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  Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.
• King, S. M., & Reed, G. L. (2002). Development of platelet secretory granules. Seminars in cell & developmental biology, 13(4), 293-302.
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  Platelet granule exocytosis plays a critical role in thrombosis and wound healing. Platelets have three major types of secretory granules that are defined by their unique molecular contents, kinetics of exocytosis and morphologies. Although the ontogeny of platelet granules is poorly understood, a convergence of new insights into megakaryocyte development, the molecular mechanisms of vesicle trafficking and the genetic basis of platelet granule defects, is beginning to define the cellular and molecular pathways responsible for platelet granule ontogeny.
• Polgár, J., Chung, S. H., & Reed, G. L. (2002). Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion. Blood, 100(3), 1081-3.
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  Secretion of platelet granules is necessary for normal hemostasis. Platelet secretion requires soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation between different members of the syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP) gene families. Using microcapillary reverse-phase high-performance liquid chromatography-nano-electrospray tandem mass spectrometry, we identified VAMP-3 and VAMP-8 as VAMP isoforms coimmunoprecipitated from platelets with syntaxin 4. Immunoblotting experiments confirmed the presence of VAMP-3 and VAMP-8 but not VAMP-1 or VAMP-2 in platelets. To examine the effect of VAMP proteins on platelet secretion, soluble recombinant (r) VAMP-2, rVAMP-3, and rVAMP-8 were incubated with streptolysin O-permeabilized platelets. Secretion of alpha granules (monitored by flow cytometric measurement of P-selectin) was blocked, and dense-granule secretion (assessed by release of carbon 14-serotonin) was almost completely inhibited by rVAMP-3, whereas rVAMP-8 inhibited secretion of dense granules but not alpha granules. In contrast, rVAMP-2, which formed SNARE complexes in vitro, had no effect on platelet exocytosis. We conclude that VAMP-3 and VAMP-8 form SNARE complexes with platelet syntaxin 4 and are required for platelet granule secretion.
• Turner, R. B., Liu, L., Sazonova, I. Y., & Reed, G. L. (2002). Structural elements that govern the substrate specificity of the clot-dissolving enzyme plasmin. The Journal of biological chemistry, 277(36), 33068-74.
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  There is remarkable homology between the core structures of plasmin, a fibrin clot-degrading enzyme, and factor D, a complement-activating enzyme, despite markedly different biological functions. We postulated that sequence divergence in the loop structures between these two enzymes mediated the unique substrate and inhibitor interactions of plasmin. Recombinant microplasminogens chimerized with factor D sequences at loops 3, 5, and 7 were cleaved by the plasminogen activator urokinase and developed titratable active sites. Chimerization abolished functional interactions with the plasminogen activator streptokinase but did not block complex formation. The microplasmin chimeras showed enhanced resistance (k(i) decreased up to two to three times) to inactivation of microplasmin by alpha(2)-antiplasmin. Microplasmin chimerization had minimal ( approximately 2 fold) effects on the catalytic efficiency for cleavage of small substrates and did not alter the cleavage of fibrin. However, microplasmin and the microplasmin chimeras showed enhanced abilities to degrade fibrin in plasma clots suspended in human plasma. These studies indicate that loop regions of the protease domain of plasmin are important for interactions with substrates, regulatory molecules, and inhibitors. Because modification of these regions affected substrate and inhibitor interactions, loop chimerization may hold promise for improving the clot dissolving properties of this enzyme.
• Sazonova, I. Y., Houng, A. K., Chowdhry, S. A., Robinson, B. R., Hedstrom, L., & Reed, G. L. (2001). The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase. The Journal of biological chemistry, 276(16), 12609-13.
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  The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.
• Chung, S. H., Polgar, J., & Reed, G. L. (2000). Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets. The Journal of biological chemistry, 275(33), 25286-91.
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  We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.
• Fitzgerald, M. L., Moore, K. J., Freeman, M. W., & Reed, G. L. (2000). Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages. Journal of immunology (Baltimore, Md. : 1950), 164(5), 2692-700.
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  Gene deletion studies indicate that the macrophage scavenger receptor A (SR-A) protects mice from LPS-induced endotoxemia. Paradoxically, cultured human monocyte-derived macrophages down-regulate SR-A expression when exposed to LPS. We found that human THP-1 monocyte/macrophages decrease SR-A expression in response to LPS independent of their differentiation status. In contrast, primary and elicited mouse peritoneal macrophages as well as the J774A.1 and RAW264.7 mouse macrophage lines increase SR-A expression in response to LPS. Exposure to LPS caused J774A.1 and RAW264.7 cells to increase SR-A transcripts by 3- and 5-fold, respectively. LPS caused a concomitant 3-fold increase in SR-A protein levels and increased cell membrane expression of the receptor. RAW264.7 cells increased SR-A transcript levels in response to LPS at concentrations as low as 1 ng/ml, and the response was saturated at 10 ng/ml. The LPS induction of SR-A transcripts required continual protein synthesis and began at 8 h, peaked by 16 h, and persisted for at least 48 h. LPS induction did not increase SR-A gene transcription or affect alternative transcript splicing, but mildly increased mature transcript stability and proceeded in the presence of actinomycin D. Finally, treatment of RAW264.7 cells with TNF-alpha did not induce SR-A transcript levels, indicating that a TNF-alpha autocrine/paracrine signaling mechanism alone is not sufficient to recapitulate the LPS induction of SR-A transcripts. The induction of SR-A expression by LPS-stimulated mouse macrophages is the opposite of the down-regulation of SR-A reported in human monocyte-derived macrophages and may have implications for the observed resistance mice show toward endotoxemia.
• Lin, L. F., Houng, A., & Reed, G. L. (2000). Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms. Biochemistry, 39(16), 4740-5.
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  Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.
• Liu, L., Sazonova, I. Y., Turner, R. B., Chowdhry, S. A., Tsai, J., Houng, A. K., & Reed, G. L. (2000). Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation. The Journal of biological chemistry, 275(48), 37686-91.
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  The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.
• Reed, G. L., Fitzgerald, M. L., & Polgár, J. (2000). Molecular mechanisms of platelet exocytosis: insights into the "secrete" life of thrombocytes. Blood, 96(10), 3334-42.
• Robinson, B. R., Houng, A. K., & Reed, G. L. (2000). Catalytic life of activated factor XIII in thrombi. Implications for fibrinolytic resistance and thrombus aging. Circulation, 102(10), 1151-7.
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  Because the increased fibrinolytic resistance of older thrombi may be caused by the continuous cross-linking action of fibrin-bound activated factor XIII (FXIIIa), we examined the persistence of FXIIIa catalytic activity in clots of various ages.
• Wang, S., Reed, G. L., & Hedstrom, L. (2000). Zymogen activation in the streptokinase-plasminogen complex. Ile1 is required for the formation of a functional active site. European journal of biochemistry, 267(13), 3994-4001.
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  Plasminogen (Plgn) is usually activated by proteolysis of the Arg561-Val562 bond. The amino group of Val562 forms a salt-bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK.Plgn) which subsequently rearranges to an active complex (SK.Plgn*) although the Arg561-Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (muPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK.Plgn* by 100-fold (Ile1Val) to >/= 104-fold (Ile1-->Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK.Plgn but is necessary for SK.Plgn*. The substitution of Lys698 of muPlgn decreases the activity of SK.Plgn* by only 10-60-fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15-50-fold. These observations suggest that Lys698 is involved in formation of the initial SK.Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740.
• Fitzgerald, M. L., & Reed, G. L. (1999). Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution. The Biochemical journal, 342 ( Pt 2)(Pt 2), 353-60.
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  In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.
• Polgár, J., & Reed, G. L. (1999). A critical role for N-ethylmaleimide-sensitive fusion protein (NSF) in platelet granule secretion. Blood, 94(4), 1313-8.
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  The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both alpha-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant alpha-SNAP-stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant alpha-SNAP-stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion.
• Reed, G. L., & Houng, A. K. (1999). The contribution of activated factor XIII to fibrinolytic resistance in experimental pulmonary embolism. Circulation, 99(2), 299-304.
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  The resistance of thrombi to fibrinolysis induced by plasminogen activators remains a major impediment to the successful treatment of thrombotic diseases. This study examines the contribution of activated factor XIII (factor XIIIa) to fibrinolytic resistance in experimental pulmonary embolism.
• Reed, G. L., Houng, A. K., & Fitzgerald, M. L. (1999). Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion. Blood, 93(8), 2617-26.
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  In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.
• Reed, G. L., Houng, A. K., Liu, L., Parhami-Seren, B., Matsueda, L. H., Wang, S., & Hedstrom, L. (1999). A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator. Proceedings of the National Academy of Sciences of the United States of America, 96(16), 8879-83.
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  Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH(2)-terminal "catalytic switch" that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSKDelta59) fusion protein lacking the 59 NH(2)-terminal residues was no longer capable of fibrin-independent Pg activation (k(cat)/K(m) decreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH(2)-terminal peptide rSK1-59. Deletion of the NH(2) terminus made rSKDelta59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSKDelta59 activator complex apparently resulted from selective catalytic processing of fibrin-bound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSKDelta59) of the SK NH(2)-terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSKDelta59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH(2) terminus are responsible for SK's unique mechanism of fibrin-independent Pg activation. Because deletion of the NH(2) terminus alters SK's mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK's therapeutic efficacy.
• Shi, C., Patel, A., Zhang, D., Wang, H., Carmeliet, P., Reed, G. L., Lee, M. E., Haber, E., & Sibinga, N. E. (1999). Plasminogen is not required for neointima formation in a mouse model of vein graft stenosis. Circulation research, 84(8), 883-90.
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  Recent studies of mice that lack plasminogen have identified a critical role for this zymogen in arterial remodeling. To permit the use of these (and other) genetically modified mice in the analysis of venous injury, we developed a model in which a patch cut from the external jugular vein of a mouse is grafted to repair a surgically created defect in its carotid artery. In wild-type mice, the venous graft showed initial endothelial denudation and formation of a neointima that progressively and reproducibly expanded in a manner analogous to human vein graft disease, albeit at an accelerated pace. This neointima occupied 37+/-4.6% of the vessel lumen at day 7 and 66+/-5.7% at day 20. The proliferative index of neointimal cells assessed by proliferating cell nuclear antigen staining was 50.6+/-3. 6% at day 7 and 15.2+/-2.0% at day 20. CD45-positive leukocytes and alpha-actin-positive smooth muscle cells accounted for 9.5+/-1.0% and 9.9+/-1.1% of intimal area at day 7, respectively, with the latter increasing to 40.9+/-2.6% at day 20. Collagen accounted for 6.8+/-0.7% of intimal area at day 7 and 20.7+/-1.8% at day 20. Surprisingly, even though arterial neointima formation due to electrostatic and immune-mediated injury is impaired in plasminogen -/- mice, in our study vein graft neointima formation in these mice was not significantly different from that in controls (70.9+/-6.4 versus 65.6+/-4.4% luminal occlusion, P=NS). Thus, plasmin proteolysis, although critical in extracellular matrix degradation and cellular migration after arterial injury, does not appear to be so important in vein graft neointima formation, perhaps because of the relative lack of structural barriers to cellular migration in the normal vein wall. This novel model of vein graft injury should be useful for further studies of differences in the response to injury of arterial and venous tissues.
• Wang, S., Reed, G. L., & Hedstrom, L. (1999). Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis. Biochemistry, 38(16), 5232-40.
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  Plasminogen (Plgn) is usually activated by proteolytic cleavage of Arg561-Val562. The new N-terminal amino group of Val562 forms a salt bridge with Asp740, creating the active protease plasmin (Pm). However, streptokinase (SK) binds to Plgn, generating an active protease in a poorly understood, nonproteolytic process. We hypothesized that the N-terminus of SK, Ile1, substitutes for the N-terminal Val562 of Pm, forming an analogous salt bridge with Asp740. SK initially forms an inactive complex with Plgn, which subsequently rearranges to create an active complex; this rearrangement is rate limiting at 4 degrees C. SK.Plgn efficiently hydrolyzes amide substrates at 4 degrees C, although DeltaIle1-SK. Plgn has no amidolytic activity. DeltaIle1-SK prevents formation of wild-type SK.Plgn. These results indicate that DeltaIle1-SK forms the initial inactive complex with plasminogen, but cannot form the active complex. However, when the experiment is performed at 37 degrees C, amidolytic activity is observed when DeltaIle1-SK is added to plasminogen. SDS-PAGE analysis demonstrates that the amidolytic activity results from the formation of DeltaIle1-SK.Pm. To further demonstrate that the activity of DeltaIle1-SK requires the conversion of Plgn to Pm, we characterized the reaction of SK with a mutant microplasminogen, Arg561Ala-microPlgn, that cannot be converted to microplasmin. Amidolytic activity is observed when Arg561Ala-microPlgn is incubated with wild-type SK at 37 degrees C; however, no amidolytic activity is observed in the presence of DeltaIle1-SK. These observations demonstrate that the amidolytic activity of DeltaIle1-SK at 37 degrees C requires the conversion of Plgn to Pm. Our findings indicate that Ile1 of SK is required for the nonproteolytic activation of Plgn by SK and are consistent with the hypothesis that Ile1 of SK substitutes for Val562 of Pm.
• Boeve, T. J., Reed, G. L., de Oliveira NC, ., Titus, J., Janssens, S., Giugliano, R. P., Torchiana, D., Daggett, W., Schwarz, R., & Jang, I. K. (1998). Comparison of Argatroban and Hirudin for the Reperfusion of Thrombotic Arterial Occlusion by Tissue Plasminogen Activator. Journal of thrombosis and thrombolysis, 6(2), 103-108.
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  Despite theoretical advantages of direct thrombin inhibitors, recent clinical studies failed to show the superiority of hirudin over heparin in patients with acute coronary syndromes. However, these inhibitors have important in vitro differences for the inhibition of clot-bound thrombin that may translate into different in vivo relative efficacy. The effects of two direct thrombin inhibitors, argatroban and hirudin, on the reperfusion of thrombotic arterial occlusion by t-PA were compared. In anesthetized rabbits thrombotic occlusion was induced in the femoral artery. t-PA, aspirin, and various doses of argatroban (1.25, 2.5, and 5.0 mg/kg/h) or hirudin (2.5 and 5.0 mg/kg/h) were administered (six animals in each group). Blood flow was measured for 4 hours. Animals treated with 2.5 mg argatroban more rapidly achieved full reperfusion than those treated with high-dose argatroban or hirudin (P < 0.05). At the doses that induced comparable prolongation of bleeding time, argatroban showed a significantly faster and higher level of reperfusion than hirudin. In animals treated with hirudin, there was a positive correlation between the aPTT and the mean reperfusion blood flow (r = 0.70, P < 0.05). In animals treated with argatroban, this correlation did not exist and the high-dose argatroban was paradoxically less effective in promoting thrombolysis despite greater anticoagulation effects. In this animal model of arterial thrombosis, argatroban was more effective than hirudin in inducing rapid, full reperfusion with t-PA. Although they are both direct thrombin inhibitors, these two agents showed important dose-related differences in efficacy and anticoagulant effects.
• Reed, G. L., Houng, A. K., & Bianchi, C. (1998). Comparative biochemical and ultrastructural studies of P-selectin in rabbit platelets. Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology, 119(4), 729-38.
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  The role of platelets in thrombotic vascular disease has been widely studied in rabbits. Yet, in rabbit platelets, there is little known about the alpha-granules, which contain many of the key effector molecules for thrombosis. In this comparative study of rabbit platelets, we have characterized the structure and expression of P-selectin, an alpha-granule membrane protein that mediates leukocyte adhesion and thrombus propagation. The sequences of tryptic peptides of rabbit P-selectin show an overall sequence identity of 74% with human P-selectin, and 69-77% identity with cow, dog, mouse, rat and sheep P-selectins. The mean (+/- S.D.) apparent molecular mass of reduced rabbit P-selectin is 117 +/- 7 kDa which is approximately 8 kDa larger than the unreduced protein (109 +/- 5 kDa). Rabbit P-selectin appears smaller than human P-selectin, but is comparable to other species P-selectins, that have fewer 'complement regulatory protein' repeat domains. Cell membrane labeling experiments and antibody binding studies indicate that rabbit P-selectin is nearly absent from the surface of platelets (290 +/- 30 molecules cell-1). However, cellular activation with thrombin causes nearly a 30-fold increase in expression to 14,200 +/- 1100 molecules cell-1. P-selectin is also be expressed on the surface of rabbit platelets activated by other agonists like ADP, A23817 and epinephrine. This selective expression is explained by immunoelectronmicroscopic studies, which show that rabbit P-selectin is sequestered in the intracellular granules of resting platelets. After cell activation by thrombin, P-selectin is found decorating the external membranes of platelet pseudopodia and the surface connected canalicular system. In summary, these studies of P-selectin in rabbit platelets indicate that it is similar in structure, cell localization and expression to human and other species P-selectins. This suggests that studies of P-selectin in thrombosis in rabbits are likely to provide useful insights into the role of this molecule in human thrombotic vascular disease and related conditions.
• Butte, A. N., Houng, A. K., Jang, I. K., & Reed, G. L. (1997). Alpha 2-antiplasmin causes thrombi to resist fibrinolysis induced by tissue plasminogen activator in experimental pulmonary embolism. Circulation, 95(7), 1886-91.
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  In patients with pulmonary embolism, thrombi resist fibrinolysis induced by plasminogen activators. Because the molecular basis of this thrombus resistance is poorly understood, we used a potent inhibitor to examine the potential role of alpha 2-antiplasmin (alpha 2AP) in experimental pulmonary embolism.
• Houng, A. K., Maggini, L., Clement, C. Y., & Reed, G. L. (1997). Identification and structure of activated-platelet protein-1, a protein with RNA-binding domain motifs that is expressed by activated platelets. European journal of biochemistry, 243(1-2), 209-18.
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  Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated-platelet protein-1 (APP-1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP-I was expressed on the platelet surface. APP-1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15 900 +/- 2800 molecules APP-1 were expressed/platelet. A 2.3-kb cDNA fragment containing a partial coding sequence for APP-1 was isolated from a rabbit bone marrow library by expression cloning with the anti-APP-1 monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, APP-1 bound specifically to poly(A)-Sepharose. The full-length cDNA coding for human APP-1, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP-1 identified a 3.7-kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP-1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP-1 is structurally related to polyadenylate-binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes.
• Parhami-Seren, B., Keel, T., & Reed, G. L. (1997). Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage. Journal of molecular biology, 271(3), 333-41.
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  Though streptokinase (SK) is widely used to treat humans with thrombotic disease, it is antigenic and anti-SK antibody causes allergic reactions and neutralizes SK's therapeutic effects. To pinpoint the fine structure of two immunodominant, continuous epitopes in SK, we used unconstrained 15 and 6-mer random peptide libraries displayed on phage (theoretical complexity of 3.2 x 10(19) and 0.64 x 10(8) unique sequences). The first epitope, recognized by both human Ab and murine monoclonal (m)Abs, was previously localized to the amino terminus of SK. Repeated panning and selection experiments against a 15-mer peptide phage library, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthetic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibited the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer library, an epitope sequence spanning residues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the second epitope in SK, which is immunodominant for murine but not human antibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide binding experiments. This epitope is cleaved and destroyed when SK reacts with human but not murine plasminogen. Thus, pinpointing the sequences of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the overlapping fine structure of epitopes recognized by competitive mAbs, (3) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in patients treated with SK.
• Reed, G. L. (1997). Functional characterization of monoclonal antibody inhibitors of alpha 2-antiplasmin that accelerate fibrinolysis in different animal plasmas. Hybridoma, 16(3), 281-6.
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  In humans with acute thrombotic disease, thrombi often appear to resist fibrinolysis induced by plasminogen activators. To examine the potential role of alpha 2-antiplasmin (alpha 2AP) in thrombus resistance in vivo, we generated monoclonal antibody inhibitors of alpha 2AP. In a somatic cell fusion, 99 hybridomas were obtained that produced MAbs that bound to human 125I-alpha 2AP in a capture assay. Screening assays showed that 3 of these MAbs, 49, 70, and 77, neutralized the function of alpha 2AP. Immunoblotting experiments indicated that these MAbs recognized an epitope present in native alpha 2AP that was destroyed by denaturation with SDS. Each of these MAbs fully inhibited the binding of the other MAbs to alpha 2AP, but none of them competed with the binding of another anti-alpha 2AP MAb, RWR. When tested for their binding to nonhuman alpha 2APs in plasmas, all three MAbs were strongly crossreactive with all primate plasmas tested but showed an idiosyncratic pattern of binding to alpha 2AP in other plasmas, suggesting unique fine epitope specificities. In human plasma, all three MAbs amplified the lysis of human plasma clots induced by plasminogen activators, increasing the potency of urokinase by nearly 50- to 100-fold. These MAbs also markedly amplified the lysis of clots from baboon, cynomolgus, african green monkey plasmas, and to a lesser extent, ferret and dog. By virtue of their ability to potently inhibit alpha 2AP in other animal plasmas, these MAbs should be useful for examining the role of alpha 2AP in thrombus resistance to fibrinolysis in vivo.
• Lin, L. F., Oeun, S., Houng, A., & Reed, G. L. (1996). Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex. Biochemistry, 35(51), 16879-85.
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  Through a unique but poorly understood mechanism, streptokinase (SK) interacts with human plasminogen to generate an "activator complex" that efficiently cleaves substrate plasminogen molecules. Previous studies have suggested that lysine residues in SK may play a role in the binding and function of the activator complex. To investigate this hypothesis, 10 different lysine residues in the plasminogen binding region of SK were altered to construct 8 recombinant (r) SK mutants. Only one double mutant, rSKK256,257A (replacing Lys with Ala at residues 256 and 257), showed a statistically significant reduction (63%) in binding affinity for Glu-plasminogen. This mutant also displayed a lagtime in the appearance of maximal activity, and modest impairments (2-5-fold) in kinetic parameters for amidolytic and plasminogen activator activity compared to rSK. In contrast, another mutant, rSKK332,334A, formed an activator complex with profound and nearly selective defects in the catalytic processing of substrate plasminogen molecules. When compared to rSK in kinetic assays of plasminogen activation, the rSKK332,334A mutant formed an activator complex that bound substrate plasminogens normally (normal K(m), but its ability to activate or cleave these molecules (kcat) was reduced by 34-fold. In contrast, in amidolytic assays, the kinetic parameters of rSKK332,334A showed only minor differences (< 2-fold) from rSK. Similarly, the binding affinity of this mutant to human Glu-plasminogen was indistinguishable from rSK [(2.6 +/- 0.8) x 10(9) vs (2.4 +/- 0.2) x 10(9) M-1, respectively]. In summary, these experiments have identified lysine residues in a plasminogen binding region of SK which appear to be necessary for normal high-affinity binding to plasminogen, and for the efficient catalytic processing of substrate plasminogen molecules by the activator complex.
• Parhami-Seren, B., Keel, T., & Reed, G. L. (1996). Structural characterization of immunodominant regions of streptokinase recognized by murine monoclonal antibodies. Hybridoma, 15(3), 169-76.
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  In order to determine the nature of the antigenic and functional determinants of streptokinase (SK), we produced monoclonal antibodies (MAbs) by immunizing A/J mice with native SK protein. By virtue of their differential binding to a large panel of recombinant SK truncated proteins, and their effect on the formation of functional SK-plasminogen activator complex (SKPAC), these MAbs were found to recognize 6 unique and minimally overlapping epitopes on the SK protein. The fine epitope specificity of the anti-SK MAbs derived from A/J mice was compared with that of MAbs derived from BALB/c mice. A number of MAbs from both inbred strains of mice were directed against the same sequences of SK (1-13, 1-253, 120-352) suggesting that these regions of the molecule contain peptide sequences that are immunodominant. Two of the "immunodominant" sequences of SK protein appeared to be important for SK function, since the formation of SKPAC could be inhibited by MAbs against these sequences.
• Parhami-Seren, B., Lynch, M., White, H. D., & Reed, G. L. (1995). Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy. Molecular immunology, 32(10), 717-24.
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  Streptokinase saves lives in patients suffering a myocardial infarction. However, because nearly all humans tested show antibodies against streptokinase, allergic reactions to streptokinase are common and may be severe. In this report we have analysed antibodies purified from normal blood donors and patients, before and after streptokinase therapy, to identify antigenic regions of the streptokinase molecule. Antibody to streptokinase was seen in all subjects, but there were 20-30-fold differences between individuals in the antibody titer. These individual differences in titer persisted after SK treatment, though the titer for all patients rose an average of 7-fold 1 week after streptokinase therapy. To identify the regions of streptokinase to which the antibody bound, we employed a panel of well-characterized murine monoclonal antibodies and recombinant streptokinase truncated fragments. Antibodies to three discrete regions of streptokinase could be detected in all patients. Antibodies to two other regions, at the amino terminal and carboxyl terminus of the molecule, were found in many but not in all patients. However, antibodies to a sixth region of streptokinase were uncommon and of very low titer. Interestingly, individuals receiving streptokinase tended to show the same pattern of immunoreactivity after treatment as they had prior to streptokinase. We conclude that although individual differences exist in the titers of streptokinase antibody, certain regions of streptokinase appear to be more antigenic or immunodominant.
• Reed, G. L., & Lukacova, D. (1995). Generation and mechanism of action of a potent inhibitor of factor XIII function. Thrombosis and haemostasis, 74(2), 680-5.
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  Although known to play a critical role in thrombosis, the precise role of factor XIII in other processes like wound healing and gestation remains to be elucidated. Because a specific, potent inhibitor could help define the function of factor XIII in these processes, or determine the potential benefits of factor XIII suppression in thrombotic disease, we have derived and characterized a monoclonal antibody inhibitor of factor XIII activation. This immunoinhibitor, 9C11, reacts specifically with both plasma and platelet forms of factor XIII. When added to human plasma, either as whole immunoglobulin or as Fab fragments, 9C11 completely prevented thrombin-activated factor XIII activity with small molecular weight substrates like 14C-putrescine. In addition, when clotted with plasma, 9C11 ablated the gamma chain crosslinking of fibrin catalyzed by factor XIII and markedly accelerated the fibrinolysis of plasma clots by urokinase. Studies of the mechanism of action showed that 9C11 inhibited the cleavage and activation of factor XIII by thrombin, but did not affect the catalytic function of previously activated factor XIII. Although it inhibited thrombin activation, experiments indicated that it bound comparably to both factor XIII zymogen and the thrombin-cleaved zymogen, and did not bind to the thrombin cleavage site of the molecule. Taken together these experiments indicated that 9C11 acted as an inhibitor of the thrombin cleavage and activation of the factor XIII A-subunit. Its potency and specificity make it a useful agent for studying thrombin-activatable factor XIII function in biological systems.
• Reed, G. L., Lin, L. F., Parhami-Seren, B., & Kussie, P. (1995). Identification of a plasminogen binding region in streptokinase that is necessary for the creation of a functional streptokinase-plasminogen activator complex. Biochemistry, 34(32), 10266-71.
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  Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.
• Lukacova, D., & Reed, G. L. (1993). Single step purification of platelet factor XIII using an immobilized factor XIII a-subunit monoclonal antibody. Thrombosis and haemostasis, 69(4), 397-400.
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  To facilitate the analysis of the catalytic subunit of factor XIII we have developed a method for the immunoaffinity purification of this protein from platelets. This method employs a monoclonal antibody that binds to the a-subunit of factor XIII. The anti-factor XIII antibody was immobilized on agarose and then incubated with platelet lysate. Subsequently factor XIII was isolated from the platelet lysate in a single step with a 41% yield as measured by enzyme assay. The purified platelet factor XIII appeared nearly homogeneous when analyzed by polyacrylamide electrophoresis and by immunoblotting with another factor XIII monoclonal antibody.
• Reed, G. L., Kussie, P., & Parhami-Seren, B. (1993). A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments. Journal of immunology (Baltimore, Md. : 1950), 150(10), 4407-15.
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  Streptokinase (SK), a bacterial product of pathogenic Streptococcus species, is now widely used as an effective therapy for the treatment of heart attacks. Because naturally occurring antibody to SK is ubiquitous, serious allergic reactions to SK therapy are common. To begin to identify regions of the molecule that are important for the antigenicity of SK we performed studies using a panel of 51 hybridomas producing anti-SK antibodies, recombinant SK fragments, and assays of SK activity. Antibodies generated from mice hyperimmunized with wild-type SK were shown to fall into six distinct complementation groups by competitive binding studies. Recombinant SK fragments were used to determine the peptide regions recognized by these complementation groups. Correlation of the effects of the mAb on SK function, with knowledge of their SK fragment-binding pattern, suggested regions of the SK molecule that are important for the construction and the catalytic function of the SK-plasminogen activator complex.
• Haber, E., Bode, C., Matsueda, G. R., Reed, G. L., & Runge, M. S. (1992). Antibody targeting as a thrombolytic strategy. Annals of the New York Academy of Sciences, 667, 365-81.
• Reed, G. L. (1992). Generation of species-specific, rat monoclonal antibodies that bind to the kappa chain of mouse immunoglobulin. Journal of immunological methods, 147(1), 111-7.
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  Large numbers of mouse monoclonal antibodies (MAbs) with exquisite binding specificities have become available. However the study of these primary antibodies in biological fluids by second, anti-mouse antibody techniques, has been confounded by the large quantity of other animal immunoglobulin which is often present in these fluids. To overcome this problem, we have derived high affinity rat MAbs which bind specifically to the antigen binding fragments (Fab) of mouse antibody and display minimal or no crossreactivity with human or rabbit immunoglobulin at the concentrations which are usually found in plasma. Equilibrium binding studies demonstrated that these antibodies had binding affinity constants for mouse Fab that ranged from 4.3 x 10(8) to 4.1 x 10(9) M-1. All of these rat MAbs bound to the kappa chain of mouse immunoglobulin, though competition binding studies amongst these MAbs indicated that they probably recognized three different epitopes. Because of their specificity and affinity, these rat anti-mouse kappa MAbs may be useful in a wide variety of experimental biological systems both in vitro and in vivo.
• Reed, G. L., Matsueda, G. R., & Haber, E. (1992). Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin. Thrombosis and haemostasis, 68(3), 315-20.
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  Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.
• Lukacova, D., Matsueda, G. R., Haber, E., & Reed, G. L. (1991). Inhibition of factor XIII activation by an anti-peptide monoclonal antibody. Biochemistry, 30(42), 10164-70.
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  As the final enzyme in the coagulation cascade, activated fibrin stabilizing factor or factor XIII catalyzes the intermolecular cross-linking of fibrin chains. To study this enzyme in plasma, we derived a monoclonal antibody (MAb 309) against a peptide sequence (NH2-G-V-N-L-Q-E-F-C-COOH) in the thrombin activation site of factor XIII. Radioimmunoassays indicate that MAb 309 binds specifically to both platelet and plasma factor XIII. Peptide inhibition studies demonstrate that the MAb binds equally well to the factor XIII (FXIII) zymogen and the active form of FXIII (FXIIIa). In immunoblots of whole platelet lysates, MAb 309 binds only to FXIII and does not cross-react with other proteins. In saturation binding studies, the antibody shows a binding avidity of (1.75 +/- 0.35) x 10(9) M-1. MAb 309 also inhibited 99% of apparent FXIIIa activity in a standard transglutaminase assay. SDS-PAGE analysis of fibrin clots showed that MAb 309 inhibited fibrin gamma-gamma cross-linking. Moreover, MAb 309 accelerated the lysis of plasma clots, consistent with inhibition of fibrin-fibrin and fibrin-alpha 2-antiplasmin cross-linking. Immunoblotting experiments revealed that MAb 309 affected apparent FXIIIa activity by inhibiting the thrombin activation of the FXIII zymogen. In addition to its utility as a specific probe for the FXIII a-subunit, the strategy used to obtain MAb 309 may be used to generate MAbs that inhibit the activation of other coagulation factor zymogens.
• Reed, G. L., Matsueda, G. R., & Haber, E. (1991). Fibrin-fibrin and alpha 2-antiplasmin-fibrin cross-linking by platelet factor XIII increases the resistance of platelet clots to fibrinolysis. Transactions of the Association of American Physicians, 104, 21-8.
• Reed, G. L., Matsueda, G. R., & Haber, E. (1990). Inhibition of clot-bound alpha 2-antiplasmin enhances in vivo thrombolysis. Circulation, 82(1), 164-8.
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  Recent experiments in vitro have shown that inhibition of human alpha 2-antiplasmin by a monoclonal antibody (MAb RWR) markedly enhances clot lysis by plasminogen activators. To extend these studies in vivo, we tested whether inhibition of clot or fibrin-bound alpha 2-antiplasmin by MAb RWR could enhance the lysis of a human clot by tissue-type plasminogen activator (t-PA) in a rabbit jugular vein thrombosis model. Compared with a saline placebo or a control antibody, MAb RWR significantly increased thrombolysis by endogenous plasminogen activator in rabbits to which no t-PA was administered (p less than 0.05). In rabbits that received t-PA, the combination of MAb RWR and t-PA caused significantly greater thrombolysis than equivalent doses of t-PA alone (p less than 0.05). However, compared with equipotent doses of t-PA alone, the combination of MAb RWR and t-PA did not increase the nonspecific consumption of fibrinogen. These experiments suggest that the combination of an alpha 2-antiplasmin inhibitor and a plasminogen activator could be a more potent thrombolytic strategy.
• Reed, G. L., Matsueda, G. R., & Haber, E. (1990). Synergistic fibrinolysis: combined effects of plasminogen activators and an antibody that inhibits alpha 2-antiplasmin. Proceedings of the National Academy of Sciences of the United States of America, 87(3), 1114-8.
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  To improve the efficacy of plasminogen activators, we produced a monoclonal antibody (RWR) that inhibits human alpha 2-antiplasmin (alpha 2AP). In addition to inhibiting alpha 2AP in plasma, RWR binds to and inhibits fibrin cross-linked alpha 2AP and reproduces the "spontaneous" clot lysis that is the hallmark of human alpha 2AP deficiency. By inhibiting the inactivation of plasmin by alpha 2AP, RWR interacts synergistically with plasminogen activators to increase the potency (for 50% clot lysis) of urokinase by 80-fold, tissue plasminogen activator by 27-fold, and streptokinase by 20-fold. Yet, for a given amount of fibrinolysis, the combination of RWR and lower doses of plasminogen activator leads to less fibrinogen consumption than is obtained with higher, equipotent doses of plasminogen activator alone. These results suggest a strategy for increasing the efficacy of plasminogen activators. More generally, this approach to amplifying enzymatic activity by immunoneutralizing an inhibitor may be useful in other biologic processes that are rigidly governed by inhibitors.
• Reed, G. L., Matsueda, G. R., & Haber, E. (1988). Acceleration of plasma clot lysis by an antibody to alpha 2-antiplasmin. Transactions of the Association of American Physicians, 101, 250-6.
• Reed, G. L., Singer, D. E., Picard, E. H., & DeSanctis, R. W. (1988). Stroke following coronary-artery bypass surgery. A case-control estimate of the risk from carotid bruits. The New England journal of medicine, 319(19), 1246-50.
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  The causes of stroke following coronary-artery bypass surgery are largely unknown. To determine whether carotid bruits increase the risk of these events, we compared 54 patients with postoperative stroke or transient ischemic attacks with 54 randomly selected control patients. Both groups were drawn from 5915 consecutive patients who had coronary bypass surgery at our hospital from 1970 to 1984. Carotid bruits were noted preoperatively in 13 patients with postoperative stroke and in 4 control patients. Case-control analysis showed that the presence of carotid bruits increased the risk of stroke or transient ischemic attacks by 3.9-fold (95 percent confidence interval, 1.2 to 12.8; P less than 0.05). This increased risk remained essentially unchanged after adjustment for potentially confounding variables in a multiple logistic regression analysis. Other factors associated with a significantly increased risk (P less than 0.05) of these neurologic deficits were a history of stroke or transient ischemic attack (odds ratio, 6.0; 95 percent confidence interval, 1.6 to 22.1), a history of congestive heart failure (odds ratio, 5.3; confidence interval, 1.6 to 17.0), mitral regurgitation (odds ratio, 4.3; confidence interval, 1.4 to 12.9), postoperative atrial fibrillation (odds ratio, 3.0; confidence interval, 1.4 to 6.7), a cardiopulmonary-bypass pump time of more than two hours (odds ratio, 2.7; confidence interval, 1.1 to 6.7), and a previous myocardial infarction (odds ratio, 2.3; confidence interval, 1.1 to 5.1). We conclude that the presence of carotid bruits increases the risk of stroke after coronary-artery bypass surgery. However, the absolute magnitude of this risk, 2.9 percent, is small and comparable to the reported risk of stroke from carotid endarterectomy.
• Reed, G. L., & Eagle, K. A. (1986). Difficulties in studying silent ischemia. The New England journal of medicine, 315(17), 1094-5.

Presentations

• Jannenga, H., Bond, M. T., Hilligoss, B., Reed, G., Betlach, T., & Gilliland, S. W. (2018, May). Future of Healthcare. Future of Healthcare Panel. Phoenix, AZ: University of Arizona Eller College of Management and UA Phoenix College of Medicine.