ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. We have proposed a three receptor model to explain the effects ADP on platelets: a receptor coupled to phospholipase C, designated P2Y1, a second receptor coupled to inhibition of adenyl cyclase, P2T/AC, and the third receptor of the intrinsic ion channel family, P2X1 receptor, coupled to rapid calcium influx. We have shown that the P2Y1 receptor plays an essential role in ADP-induced platelet shape change. Furthermore, e have demonstrated that fibrinogen receptor activation requires co-activation of both the P2Y1 and P2T/AC receptors and concomitant signaling from Gq and Gi is sufficient and necessary for platelet aggregation. We hypothesize that platelet aggregation by any agonist requires concomitant signaling from Gq and Gi pathways. We propose that all platelet aggregating agents, with the exception of thrombin and ADP, activate only either Gq or Gi, but not both, signaling cascades. We further hypothesize that these platelet agonists cause fibrinogen receptor activation by supplementary signaling events from either P2YQ or P2T/AC, through Gq and Gi, respectively. We will elucidate the role of ADP, the P2Y1, and P2T/AC receptors, in other platelet agonist-induced aggregation. We will elucidate the role of ADP, the P2Y1, and P2T/AC receptors, in other platelet agonist-induced aggregation by selective P2 receptor antagonists. We hypothesize that activation of novel and atypical protein kinase C isoforms is essential for fibrinogen receptor activation. We propose to test our hypothesis by developing a cell model for fibrinogen receptor activation and by transfection of constitutively active or dominant negative PKC isoform constructs. We propose to clone the P2T/AC receptor by homology screening under low stringency hybridization conditions. Alternative strategies include RT-PCR approach with degenerate primers, expression cloning, and screening with oligonucleotide probes. Finally, we will elucidate the molecular defect in the P2Y1 or P2T/AC receptors in the patients with abnormal ADP-induced platelet activation. These studies will shed light on the mechanisms of fibrinogen receptor activation and role of ADP receptor subtypes in platelet activation.
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