CHARACTERIZATION & ISOLATION OF PLATELET ADP RECEPTORS

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The mechanism by which ADP activates platelets is not understood. A major question is whether it acts via a single type of receptor or through two receptors, one causing platelet activation and the other inhibition of stimulated adenyl cyclase: a corollary to the two receptor hypothesis is that C2-substituted ADP analogues should act only a: the receptor modulating adenyl cyclase. The proposed studies are a major commitment by this laboratory intended to resolve these questions as well as to define the ADP receptor, or receptors, of the platelet surface in terms of number, size, specificity and structural configuration and function. In preliminary studies, formaldehyde-fixed platelets were used to avoid complications due to metabolism and secretion and high (Kd 0.35 uM) and low (Kd 7.9 uM) affinity binding sites for ADP and C2- substituted ADPs have been identified. A new C2-substituted ADP photoaffinity probe has been synthesized that specifically labels three binding sites with molecular weights of 188,000, 93,000 and 50,000 in isolated platelet membranes. In order to identify the ADP receptor, or receptors, of intact platelets the following specific aims are proposed: (i) characterize the binding sites for ADP and 2-methylthioADP in situ by equilibrium binding and radiation inactivation to determine their number and affinity and utilize radiation inactivation to differentiate them on the basis of functional sizes; (ii) utilize the C2-substituted photoaffinity label and a new affinity probe directed against a possible active thiol at the receptor site to identify putative receptors; (iii) isolate the products of photoaffinity and affinity labeling and their parent polypeptides by a variety of techniques including affinity chromatography on C2-ADP-Sepharose; (iv) measure the ability of these polypeptides, and antibodies prepared against them, to block ADP-induced platelet activation and to reverse the inhibition of stimulated adenyl cyclase; (v) use a fluid phase assay to differentiate receptors accessible to C2-ADP analogues and those accessible only to ADP; (vi) characterize the receptor, or receptors, so defined by structural analysis, including sequencing through the ADP-binding domains and by evaluation of functional activities; (vii) evaluate endothelial cells and other cells and tissues for proteins related to the ADP receptor(s) of platelets.