The mechanism(s) of thrombin-platelet interaction(s) will be studied with three long range objectives: i) to characterize and establish the function of the thrombin-induced inhibition of platelet adenylate cyclase (AC), ii) to use inhibition of AC as an assay of a thrombin receptor in soluble preparations, and iii) to isolate thrombin-binding proteins and to identify components of the putative thrombin """"""""receptor"""""""". Comparisons of thrombin-induced activation of control vs chymotrypsin-treated platelets and of activation of platelets by Alpha-thrombin vs Gamma-thrombin reveal that when the ability of thrombin to activate platelets quickly is impaired (pretreatment with chymotrypsin; activation by Gamma-thrombin), the ability to inhibit AC is blocked. We will test the hypotheses that i) inhibition of AC is necessary for normal platelet activation by thrombin and other agonists, and ii) thrombin regulates AC activity by either a receptor or an effector that is distinct from that for platelet activation, with only the AC system highly sensitive to proteolysis. Thrombin-induced inhibition of AC will be analyzed in membrane and soluble preparations, and the effects of proteases on regulation of cyclase by thrombin and other agonists will be correlated with the effects of protease pretreatment on the polypeptide composition of the cyclase preparation. The solubilized system will be used as an assay for receptor activity to establish an apparent mass for the receptor, to study dissociation and stability of the receptor and for following the course of purification. With similar experiments, the effect of collagen on the regulation of AC will be analyzed. To identify platelet thrombin-binding proteins, photoactivatable crosslinking reagents will be used in two ways: i) as a means for enrichment of the binding proteins, and ii) as a means for specifically attaching a radioactive label for screening for monoclonal antibodies against the binding proteins. For isolation of the binding proteins, a cleavable crosslinking reagent will be used. The derivatized thrombin will be crosslinked to the surface of labeled platelets, the complex will be solubilized and adsorbed to an immobilized antibody against thrombin, and the platelet protein will be released by cleavage of the crosslinking reagent. The product will be used as an immunogen for the preparation of monoclonal antibodies, using the immunogen and crosslinked thrombin-platelet complexes for clone selection. Antibody affinity columns will be used to isolate the binding proteins in high yield.
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