Fibrinogen binding to specific receptors on stimulated platelets is a prerequisite for platelet aggregation. In previous work we determined the conditions and the kinetics of the fibrinogen-platelet interaction. In the present proposal we intend to extend our observations into five areas. First, we intend to identify and isolate the platelet-fibrinogen receptor using crosslinking reagents, affinity chromatography, and receptor-specific monoclonal antibodies. identification and isolation of the fibrinogen receptor will aid study of the membrane changes that follow platelet stimulation and the organization of membrane structures. Second, the effect of alterations in the fibrinogen molecule on fibrinogen binding will be studied. Fibrinogen with degraded Aalpha chains will be isolated from human plasma. Fibrinogen fragments will also be produced by plasmin cleavage of the molecule. The altered fibrinogen will then be used in studies of fibrinogen binding and platelet aggregation. These experiments will definethe structural features of fibrinogen that permit platelet binding. Third, the role of ADP in the fibrinogen binding stimulated by other platelet agonists will be evaluated by examining the effect of various ADP antagonists on several aspects of the fibrinogen binding reation. Fourth, the requirement for divalent cations in fibrinogen binding will be investigated. Because both the platelet surface and fibrinogen avidly bind divalent cations, calcium binding to each will be investigated separately. Lastly, the contribution of fibrinogen binding to the growth of a hemostatic platelet plug will be examined by measuring the binding of radiolabeled fibrinogen to platelets stimulated by ristocetin and von Willebrand factor and by using fluoroscein-conjugated anti-fibrinogen to detect fibrinogen on platelets adherent to segments of denuded aorta. Platelet aggregations plays a critical role in normal hemostosis and is likely to be involved in the evolution of atherosclerotic lesions and arterial thrombosis. The demonstration of fibrinogen receptors on stimulated platelets was a significant step in understanding platelet aggregation. Further examination of the platelet-fibrinogen interaction will extend this understanding toward the ultimate goals of successful prevention and treatment of vascular disease.
