Although TXA2 and PGH2 are believed to be involved in the process of hemostasis and the genesis of certain forms of cardiovascular disease, little is known concerning the specific mechanisms by which TXA2/PGH2- receptor interaction leads to the modulation of platelet function. This lack of information is primarily due to two considerations: 1. the multiple feedback mechanisms which exist within platelets; and 2. the unavailability of purified signal transduction components, e.g., receptor protein, associated G-proteins, etc. Thus in a highly-coupled, positive feedback system, as apparently occurs in the TXA2/PGH2 pathway, pharmacological intervention at one site will undoubtedly effect multiple biochemical processes at additional sites. This, in turn, significantly complicates interpretation of the observed results. Based on this consideration, the most definitive approach to studying TXA2/PGH2 signal transduction would be to identify, isolate and characterize the primary components involved in this pathway. Once this has been accomplished one can begin to examine pharmacological and physiological modulation the reconstituted components in the absence of extraneous influences. The present application proposes to characterize the TXA2/PGH2 signal transduction pathway using this approach. Specifically, studies are designed to: 1. raise antipeptide and monoclonal antibodies libraries which will be used to probe TXA2/PGH2 receptors and their associated G- proteins; 2. purify TXA2/PGH2 receptors by affinity chromatography procedures; 3. purify, identify and characterize receptor-associated G- proteins; 4. define receptor/G-protein coupling domains; 4; identify cAMP/cGMP-mediated phosphorylation sites on the TXA2/PGH2 receptor/G- protein complex; 5. investigate the relationship between such phosphorylation and coupling of the receptor/G-protein complex in a reconstituted system; 6. apply specific photoaffinity ligand techniques to irreversibly label platelets. TXA2/PGH2 receptors, and identify the receptor ligand binding domains; and 7. utilize a newly synthesized, biotinylated TXA2/P6H2 receptor antagonist (in conjunction with antibodies) to label and define receptor location/distribution in resting and activated platelets. These studies represent a rigorous and in-depth characterization of human platelet TXA2/PGH2 receptors and the signal transduction mechanisms specifically involved in this pathway. The feasibility of these studies is great strengthened by the current availability of purified receptor/G- proteins, high affinity TXA2/PGH2 analogs receptor/G-protein-specific antibodies and substantial preliminary data. On this basis, it is anticipated that these studies will be of significant value in more clearly defining the underlying mechanisms of TXA2/PGH2-mediated platelet activation and, in turn, novel therapeutic approaches to the treatment of thromboembolic disorders.
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