This Program Project is directed toward elucidating the pathophysiology of thrombosis through both basic and clinical studies. This goal has been and is being pursued by the use of radioimmunoassays for peptides derived from fibrinogen by proteolysis by thrombin (fibrinopeptide A and des-arginine fibrinopeptide B) and by plasmin (BBeta1-42) and for proteins secreted from platelets (platelet factor 4 and Beta-thromboglobulin). These assays are being applied in Unit VI to characterize the effects of thrombolytic therapy on the hemostatic system and they will be applied in a study of renal transplantation to determine whether activation of coagulation is associated with transplant rejection. ELISA's have been developed in Unit I for two fibrinogen AAlpha-chain peptides for the study of cross-linking of fibrin Alpha-chains and eventually for detection of cross-linking in vivo. Factor XIII is being purified in Unit I to study the interactions between its subunits prior to activation of zymogen. In Unit VII, these radioimmunoassays and ELISA's for fibrinopeptides have been employed to characterize the fibrinogen/fibrin content of thrombi and atherosclerotic plaques. Immunohistochemical studies have been initiated to identify fibrin, tissue plasminogen activator, and platelet proteins within thrombi and plaques. Work in Unit V is focused on leukocyte elastase cleavage of fibrinogen. Cleavage occurs at a unique site, releasing a specific peptide. An indirect assay has been developed for this peptide, and development of a specific radioimmunoassay is planned. Studies in Unit III have concentrated on defining the molecular defect in fibrinogen NY-1 and are moving toward defining the mechanism by which the molecular defect leads to a functional defect. Work in this Unit has also focused on the interaction of tissue plasminogen activator with fibrin and with endothelial cells. Studies in Unit II concentrated on the interaction of coagulation factors and inhibitors with endothelial cells and demonstrated that activation of coagulation could proceed on the endothelial cell surface, but that inhibitors (antithrombin III, protein C, and protein S) could also interact with endothelial cells and modulate the coagulation process. Studies in Unit IV have begun to examine the cellular mechanisms involved in the generation of endothelial cell pro- and anticoagulant activities, and studies are also underway to characterize the interaction of secreted platelet proteins with endothelial cells and with endothelial cell extracellular matrix.
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