Injury to coronary, cerebral, and peripheral arteries evokes local platelet activation, recruitment, and thrombotic occlusion. Prevention and treatment of platelet-driven thrombus formation is a therapeutic challenge, with critical public health implications. In the presence of endothelial cells, platelets are unresponsive to agonists. This unresponsiveness is due to endothelial NTPDasel/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 routine 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. Broad, long-term objectives and specific aims include: I. Molecular, biochemical and functional studies of human solCD39: Determination of the antithrombotic efficacy of selected mutants with increased ADP specificity/activity, and evaluation of the contribution of disulfide bridges to CD39 activity. II. Studies of the effects of NTPDases on cell-cell signaling during ischemia: Modulation of norepinephrine release by NTPDases from cardiac sympathetic nerve endings (cSNE) and isolated hearts from guinea pigs, wild type and CD39 null mice. Identification of the ecto-nucleotidase complement in cSNE from mouse and human heart, and assessment of pneumoprotection by endogenous and exogenous CD39 during second organ perfusion injury, in wild-type and CD39 null mice. III. Studies of NTPDase activities in lymphocytes from patients with angiographically documented coronary artery disease (CAD) to determine whether an altered, prothrombotic nucleotidase profile is expressed in comparison to that of lymphocytes from healthy donors. IV. Metabolism of adenosine derived from AMP as generated by CD39 from ATP and ADP: Can PRP metabolize AMP and generate inhibition of platelet reactivity by production of adenosine via 5'-nucleotidase? If true, is this dependent on specific platelet agonists, or on other blood or endothelial cells? Dioxin toxicity may involve increased catabolism of adenosine via upregulation of adenosine deaminase, thus reducing availability of adenosine, a potent platelet inhibitor. The research represents a multidisciplinary approach to understanding the critical role of CD39 as the prime regulator of platelet-mediated occlusive thrombosis. It is based on compelling feasibility data and historical collaborative success, and will advance the understanding of NTPDase biology and thromboregulation, culminating in a unique and novel therapeutic agent for thrombotic diatheses.
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