Local injury to coronary, cerebral, and peripheral arteries promotes platelet activation, recruitment and thrombotic occlusion. Prevention and reversal of platelet thrombus formation represents a major therapeutic challenge with important public health implications. In the presence of endothelial cells (EC), platelets are unresponsive to agonists, even when eicosanoid and nitric oxide production are blocked. This unresponsiveness is due to EC CD39/ecto-ADPase, which rapidly metabolizes ADP released from activated platelets, thereby abolishing aggregation and recruitment. Recombinant, soluble human CD39 (""""""""solCD39"""""""") potently blocks agonist-induced human platelet aggregation in vitro, and prolongs porcine and murine bleeding times in vivo. CD39 null mice exhibit a latent prothrombotic phenotype with increased susceptibility to ischemic cerebral and pulmonary injury and thrombosis, which is alleviated by infusion of solCD39. This demonstrates a critical role for CD39 in thromboregulation. Broad, long-term objectives and specific aims include: 1) Molecular and biochemical characterization of CD39/ecto-ADPase: Using site-directed mutagenesis we will examine primary structural features focusing on specific amino acids affecting CD39 enzyme and biological activity, and determine the importance of cysteines for protein folding. In addition, the investigator will study regulation of CD39/ecto-ADPase in an immortalized endothelial cell line; 2) Biochemical and functional studies on CD39 null mice: In vitro and in vivo platelet and endothelial function will be assessed in myocardial and lung model systems, in comparison with wild-type mice; 3) Clone and characterize the chick cDNA homolog of human CD39 (E-NTPDase-1), and study expression pattern(s) of ecto-nucleotidases during embryonic development in the chick; and 4) Metabolism of extracellular AMP, formed by CD39, will be studied. Preliminary data indicate that this pathway amplifies CD39-mediated thromboregulation. The role of adenosine formation, involvement of cAMP generation, and function of plasma and cell-bound 5' nucleotidase(s) and phosphodiesterase(s) will be characterized. The ultimate health-related goal of these in vivo and in vitro experiments is to develop a therapeutic agent that will block platelet activation and recruitment in the circulation of patients with vascular disorders due to excessive platelet activation.
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