Injury to coronary, cerebral, or peripheral arteries evokes local platelet activation, recruitment, andthrombotic occlusion. Prevention and treatment of platelet-driven thrombus formation is a therapeuticchallenge, with major public health implications. In the presence of endothelial cells, platelets areunresponsive 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 andrecruitment. Recombinant, soluble human CD39 (solCD39) blocks human platelet aggregation in vitro, andinhibits porcine and murine platelet aggregation ex vivo. CD39 null mice exhibit a latent prothromboticphenotype with increased susceptibility to stroke and cardiac and pulmonary thrombosis. This is alleviatedin both wild-type and CD39 null mice by infusion of solCD39, demonstrating its critical role inthromboregulation. Long-term objectives and specific aims of this project include: I. Generation of largequantities of recombinant soluble CD39 for biochemical and functional studies, including mouse models ofvascular injury and occlusion, employing our CD39 null mice. These models include: a) Transluminal wireinjury 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 ligationleading to myocardial ischemia/reperfusion injury. This will delineate the effectiveness of solCD39 inmyocardial ischemia/reperfusion in short-term (comparable to effectiveness of solCD39 in reducing the sizeof brain infarct in CD39 null mice), as well as long-term conditions (LV remodeling and congestive heartfailure). [L Modulation of purinergic and adrenergic neurotransmission in the heart and peripheralsympathetic nervous system by NTPDase1/CD39 during ischemia. These studies will elucidate the role ofCD39 in sympathetic transmission in the heart and demonstrate the crucial modulatory role of CD39 atperipheral sympathetic neuro-effector junctions. Moreover, they will verify that overflow of ATP and NE, andseverity of reperfusion arrhythmias, can be attenuated. Thus, cardioprotection afforded by CD39 can berestored in CD39 null animals. IN. Structural analyses of soluble apyrases using CD and NMR will establishprotein-nucleotide interactions, kinetics of binding, and delineate important aspects of the active site ofsolCD39 - the principal thromboregulator. The proposed research represents a multidisciplinary andcollaborative approach to understanding the critical role of CD39 as the prime regulator of blood fluidity andof platelet-mediated occlusive thrombosis. The studies are based on compelling feasibility data andhistorical collaborative success, and will advance the understanding of NTPDase biology andthromborequlation. This should culminate in a unique and novel therapeutic aqent for thrombotic diatheses.
Showing the most recent 10 out of 226 publications
