The overall objective of this proposal is to define the molecular basis of plateletfibrin interactions, which play a major role in both the normal hemostatic process and the development of thrombosis. In response to vascular injury, platelets adhere to the exposed subendothelium, and release their granule contents which in turn activate other platelets, causing aggregate formation in a fibrinogen-dependent process. Activated platelets are critical for the catalysis of the coagulation cascade, especially the conversion of prothrombin to thrombin. Next, thrombin removes fibrinopeptides from fibrinogen, triggering rapid protofibril growth, followed by the formation of a platelet-fibrin clot. Subsequent platelet-fibrin interactions, which involve the actin-myosin system of the activated platelet, lead to a dramatic contraction of the fibrin gel. Data obtained in the first project period have raised the following questions, which are addressed in this proposal: Does removal of the fibrinopeptides from fibrinogen unmask a new binding site on the central E-domain of fibrin, which is important for platelet-fibrin binding? Do protofibrils bind to platelets through the same contacts which promote their lateral association into a network of thick fibrin fibers? Does fibrin bind to platelets through surface receptor proteins other than the glycoprotein IIb:IIIa complex involved in fibrinogen binding? What is the relationship between fibrin binding, platelet aggregation, and clot retraction? A multifaceted approach will be required to answer those questions. Measurements of the rate and equilibrium constants for binding protofibrils to stimulated platelets will be carried out by radiolabelled ligand binding and flow cytometric techniques, and the development of quantitative fluorescence microscopic techniques to study binding of platelets to the fibrin network will be undertaken. Specific inhibitory fragments derived from the D- and E-domains of fibrin(ogen) will be used to define which regions of the multinodular fibrin molecule interact with platelets. Experiments with Glanzmann's Thrombasthenia platelets and monoclonal antibodies specific for the glycoprotein IIb:IIIa complex are planned, to ascertain if fibrin binds to platelets solely through the fibrinogen receptor. In addition, experiments will determine if a platelet protein which has been isolated by its affinity for immobilized fibrin monomer is involved in fibrin binding. Characterization of the interactions of this protein with fibrinogen, protofibrils, and fibrin by light scattering and electron microscopic techniques will be carried out.
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