Although changes in cytoplasmic Ca2+, cyclic nucleotides and the activation state of protein kinase C have been identified following platelet stimulation by single or multiple agonists, the manner in which these intracellular mediators interact to form a platelet aggregate has not yet been determined. The overall goal of the proposed studies is to establish which changes in intracellular mediators lead to the expression of fibrinogen receptors on the platelet surface. Based on our previous in vitro studies demonstrating the close link between intracellular Ca 2+ and platelet functional changes, my current working hypotheses are that (1) a Ca2+ rise produced by subthreshold concentrations of an initial agonist primes the platelets for synergistic functional response to additional agonists in similar concentrations and that (2) an initial required rise in cytoplasmic Ca2+ following a single agonist promotes activation of protein kinase C, which then causes fibrinogen binding and subsequent aggregation. Since aequorin and the fluorescent probes appear to measure different aspects of Ca2+ homeostasis, both will be used to record Ca2+ in platelets stimulated or inhibited by various agents. In washed human platelets, the individual and net effects of subthreshold concentrations of several agonists will be compared to assess the importance of Ca2+ in synergy. The mechanisms by which Ca2+ causes or potentiates platelet activation will be examined by measuring fibrinogen binding to surface receptors, pH change, formation of diacylglycerol, phosphorylation of platelet proteins, and translocation of protein kinase C in response to agonists alone or in subthreshold pairs. Antibodies to various epitopes of platelet receptors for adhesive proteins will be used to determine the importance of binding of such proteins to subsequent intracellular transduction. The possibility that Ca2+ entry associated with the platelet fibrinogen receptor causes protein phosphorylation and further platelet activation will be tested. Finally, the influence of agents that mimic endothelium-derived platelet inhibitors by elevating cyclic AMP (e.g.PGI-2, PGE-1) or cyclic GMP (e.g.,nitroprusside,nitric oxide) on agonist-induced changes in Ca2+, formation of diacylglycerol, and activation of protein kinase C will be tested. These studies should provide a greater understanding of platelet stimulus-response coupling, and may produce important findings both in general cell biology and in clinical approaches to disordered platelet function.
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