ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Although the physiological effects and intracellular responses of ADP on platelets have been well characterized, the receptor(s) mediating these effects have not been identified and the molecular mechanism of these processes remains obscure. The cell surface receptors mediating effects of nucleotides are referred to as P2 receptors and the platelet ADP receptor(s) has been designated P2T receptor. The applicants have proposed a three receptor model to explain the effects of ADP on platelets: a receptor coupled to phospholipase C, designated P2TPLC, a second receptor coupled to inhibition of adenylyl cyclase, P2TAC, and the third receptor of the intrinsic ion channel family, P2X receptor, coupled to rapid calcium influx. They have cloned the P2Y1 receptor from a platelet cDNA library and have identified it as the P2TPLC receptor on platelets. They hypothesize that the P2Y1 receptor plays an important role in ADP-induced physiological responses in platelets. Preliminary studies indicate that the P2Y1 receptor plays an important role in the ADP-induced platelet shape change and aggregation. The P2Y1 receptor specific antagonists and inhibitory P2Y1 receptor antibodies will be used to selectively block the responses mediated by the P2Y1 receptor. On the other hand, the P2Y1 receptor will be selectively activated in buffers containing nominal extracellular calcium, to block effects of the P2X receptor, and in the presence of ARL 66096, a P2TAC specific antagonist. They will generate P2Y1 receptor gene knockout mice and assess the platelets for ADP-induced physiological responses. The intracellular response cascade mediated by the P2Y1 receptor which leads to phosphorylation of myosin light chain and pleckstrin will be elucidated. Amino acid residues essential for binding of ligands will be identified and the mechanism of the homologous desensitization of the platelet P2Y1 receptor will be characterized. The studies proposed in this application will provide evidence for a role for the P2Y1 receptor in ADP-induced activation and will form the basis for the design of anti-platelet drugs acting through this mechanism.
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