Injury to coronary and cerebral arteries promotes platelet activation, recruitment and thrombotic occlusion. Prevention and reversal of the signals for platelet thrombus formation is a major therapeutic challenge with important implications for public health. In the presence of endothelial cells (EC), platelets are unresponsive to agonists, even when eicosanoid and nitric oxide production are blocked. This is due to EC CD39/ecto-ADPase, which rapidly metabolizes ADP released from activated platelets, thereby deleting the signal for platelet 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. Our CD39 mice exhibits exhibit a latent pro-thrombotic phenotype with increased susceptibility to injury-induced thrombosis, which is alleviated by infusion of solCD39. Thus CD39 plays a critical role in thromboregulation.
Specific aims i nclude: [1] Characterization of in vivo anti-thrombotic properties of CD39 using our backcrossed CD39 null mice, combined with studies of a novel, potentially even more pro-thrombotic CD39-annexin II double knockout. Expression of CD39 and other EC thromboregulators (prostacyclin and nitric oxide) will be examined during in vitro aging of normal EC, and EC immortalized by transfection with human telomerase reverse transcriptase (hTERT). [2] Characterization of the ecto- nucleotidase activity we identified in guinea-pig cardiac synapotosomes, and examination of the effects of ischemia/reperfusion on its expression. Does decreased nucleotidase activity lead to enhanced norepinephrine release? Does added solCD39 affect synaptosomal signaling in normal and ischemic guinea-pig heart? [3] Conformational analyses of solCD39, including effects of post-translational modifications on its stability and resistance to proteolysis, as well as CD and NMR studies to determine solCD39- nucleotide interactions, kinetics of binding as well as conformational information concerning the active site of solCD39. The ultimate health-related goal of our collaborative in vivo and in vitro experiments is to develop CD39 as a therapeutic agent that will block signals for platelet activation and recruitment in patients with coronary and cerebral vascular disorders induced by excessive platelet activation
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