Recent reports have suggested that one important endothelial- derived relaxing factor (EDRF) is nitric oxide or a nitric oxide- like substance. Given the known ability of nitrates and nitroxides to inhibit platelet aggregation and the role of the endothelium in maintaining vessel patency, we plan to study the physiologic and biochemical effects of the NO-related EDRF on the formation and stability of platelet aggregates in normal human platelets. We propose to study: (1) the inhibitory effects of EDRF on platelet aggregation and the promotion of disaggregation by EDRF in normal platelets, (2) the potential additive or synergistic effects between EDRF and known endothelial-derived inhibitors of platelet function, (3) the role of S-nitrosothiols and the cyclic GMP pathway in the inhibition of platelet aggregation by EDRF, and (4) interactions of fibrinogen with its platelet receptor, glycoprotein IIb-IIIa, in the presence of EDRF. The experimental design and methods in this proposal begin with a systematic examination of EDRF inhibition of platelet aggregation, the role of EDRF in disaggregation, and the potential synergistic effects of EDRF with known inhibitors of platelet function, including prostacyclin and tissue plasminogen activator. Sources of EDRF will include the perfused vessel of the standard bioassay system, and endothelial cells cultured on microcarrier beads used in columns or directly coincubated with platelets. Intraplatelet events involving S-nitrosothiols and the guanylate cyclase pathway in the presence of EDRF and nitrovasodilators will be examined by biochemical analyses of S-nitrosothiol accumulation, cytosolic free calcium, and cyclic GMP production within intact platelets, as previously performed in the sponsor's laboratory. Alterations in the platelet glycoprotein IIb-IIIa complex in the presence of EDRF will next be studied by performing equilibrium and kinetic analyses of fibrinogen-platelet interactions in the presence of EDRF using radiolabeled fibrinogen and monoclonal antibodies against glycoprotein IIb-IIIa. Given the participation of platelets in the pathophysiology of ischemic cardiovascular disorders, it is believed these studies will improve our understanding of a complex and important dynamic that exists between the endothelial surface and circulating platelets.
