The involvement of platelets in diverse physiological processes as well as in hypersensitivity and inflammatory reactions is mediated by structurally distinct platelet activating factors. Of these, one of the most potent is the phospholipid 1-0-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). AGEPC activates platelets by binding to specific, high-affinity receptors and recently, a 180,000 dalton platelet membrane protein which binds AGEPC was purified to homogeneity by ligand specific affinity chromatography. The goal of the proposed research program is to delineate the subcellular mechanisms by which AGEPC contributes to immunological reactions by exploring in detail the interaction of AGEPC with its specific receptor and by delineating the biochemical consequences of this interatin which mediate platelet activation. The relationship between the binding protein and the AGEPC receptor will be determined by producing antisera to the binding protein and monoclonal antibodies which specifically modulate AGEPC binding. New methods for quantitating AGEPC binding by the solubilized receptor and the purified binding protein will be developed in order to analyze in depth their binding constants and affinities. If these antigenic and functional studies indicate that the binding protein is not the AGEPC receptor then monoclonal antibodies to the receptor will be utilized to isolate the receptor for a detailed analysis of its functional properties. Specific antibody will be used also to define the location of the receptor in platelets, to track the receptor during platelet activation, to compare the receptor to other platelet membrane proteins and to compare the receptor in platelets and other cell types. While the new methods and antibodies are being developed the mechanisms of activating signal transduction will be examined by analyzing the initiation of protein kinase and phospholipase activity in platelet membranes and solubilized receptor systems. The information derived from these binding and functional studies of the receptor in unpurified systems will then be utilized to guide studies which characterize the functional properties of the isolated, purified receptor.
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