Injury to coronary, cerebral, or peripheral arteries evokes local platelet activation, recruitment, and thrombotic occlusion. Prevention and treatment of platelet-driven thrombus formation is a therapeutic challenge, with major public health implications. In the presence of endothelial cells, platelets are unresponsive to agonists. This unresponsiveness is due to endothelial NTPDase1/CD39/ecto-ADPase, which rapidly metabolizes ATP and ADP released from activated platelets. This abolishes aggregation and recruitment. Recombinant, soluble human CD39 (solCD39) blocks human platelet aggregation in vitro, and inhibits porcine and murine platelet aggregation ex vivo. CD39 null mice exhibit a latent prothrombotic phenotype with increased susceptibility to stroke and cardiac and pulmonary thrombosis. This is alleviated in both wild-type and CD39 null mice by infusion of solCD39, demonstrating its critical role in thromboregulation. Long-term objectives and specific aims of this project include: I. Generation of large quantities of recombinant soluble CD39 for biochemical and functional studies, including mouse models of vascular injury and occlusion, employing our CD39 null mice. These models include: a) Transluminal wire injury of mouse femoral artery, in which we will ascertain the role of CD39 in both the initial platelet- dependent stage, as well as the late proliferative stage of this vascular injury;and b} murine LAD ligation leading to myocardial ischemia/reperfusion injury. This will delineate the effectiveness of solCD39 in myocardial ischemia/reperfusion in short-term (comparable to effectiveness of solCD39 in reducing the size of brain infarct in CD39 null mice), as well as long-term conditions (LV remodeling and congestive heart failure). [L Modulation of purinergic and adrenergic neurotransmission in the heart and peripheral sympathetic nervous system by NTPDase1/CD39 during ischemia. These studies will elucidate the role of CD39 in sympathetic transmission in the heart and demonstrate the crucial modulatory role of CD39 at peripheral sympathetic neuro-effector junctions. Moreover, they will verify that overflow of ATP and NE, and severity of reperfusion arrhythmias, can be attenuated. Thus, cardioprotection afforded by CD39 can be restored in CD39 null animals. IN. Structural analyses of soluble apyrases using CD and NMR will establish protein-nucleotide interactions, kinetics of binding, and delineate important aspects of the active site of solCD39 - the principal thromboregulator. The proposed research represents a multidisciplinary and collaborative approach to understanding the critical role of CD39 as the prime regulator of blood fluidity and of platelet-mediated occlusive thrombosis. The studies are based on compelling feasibility data and historical collaborative success, and will advance the understanding of NTPDase biology and thromborequlation. This should culminate in a unique and novel therapeutic aqent for thrombotic diatheses.
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