ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. ADP causes platelets to change their shape, to aggregate, to release contents of granules, and to produce thromboxane A2, another potent agonist for platelets. The physiological effects and intracellular responses of ADP on platelets have been well characterized and the receptor mediating these effects has been cloned. However the molecular mechanism of these ADP-mediated physiological processes remains obscure. During the previous grant period, we have demonstrated 3 ADP receptor subtypes, P2Y1, P2Y12, and P2X1, on platelets and elucidated a number of signaling mechanisms in platelets. In this grant period, we propose to enhance our understanding by elucidating the molecular mechanisms of ADP-induced platelet activation and evaluate the relative contribution of P2Y receptors to thrombus formation. We hypothesize that different tyrosine kinases are activated downstream of different P2Y receptor subtypes. Our studies in the previous grant period led to the conclusions that Src family kinases contribute to fibrinogen receptor activation, thromboxane A2 generation, and phosphorylation of PKC-5 isoform and Akt downstream of the P2Y12 receptor activation. We propose to test this hypothesis by pharmacological, biochemical, and molecular genetic approaches. We hypothesize that charge interactions between negatively charged phosphates in ADP and some of the positively charged basic amino acid residues in the extracellular domains of the P2Y12 receptor are involved in ligand binding. We will test this hypothesis using chimeric receptors and site-directed mutagenesis. We will change the conserved basic amino acid residues in the P2Y12 receptor to alanine by site-directed mutagenesis, express the mutant receptors transiently in mammalian cells, and determine the effect on binding of labeled nucleotides and ADP-induced inhibition of adenylyl cyclase. In addition, we will pharmacologically characterize a constitutively active P2Y12 receptor generated in our lab. We hypothesize that the P2Y1 and P2Y12 receptors synergistically contribute to the thrombus growth and stability in vivo. We further propose to evaluate the relative contribution of the P2Y1 and the P2Y12 receptors to thrombus formation, using P2Y1 or P2Y12 deficient mice and treatment with antagonists and models of in vivo thrombosis and thromboembolism.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-HEME-D (02))
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Hasan, Ahmed AK
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Temple University
Schools of Medicine
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