Fibrinolysis is the enzyme system that eliminates existing fibrin blood clots and inhibits the deposition of new fibrin in normal blood vessels. Plasmin is the primary proteinase responsible for fibrin digestion in vivo. The reaction of plasmin with proteinase inhibitors in plasma is well characterized; a2AP is the primary plasmin inhibitor; however, in vivo, fibrinolysis probably occurs on the surfaces of fibrin, endothelial cells, and extracellular matrix proteins. Little is known about the function of fibrinolysis proteinase inhibitors in these environments. The major goal of this research program is to study the regulation of plasmin and plasminogen activators in experimental systems that model important """"""""non-plasma"""""""" sites of fibrinolytic activity. The function of a2AP, a2- macmacroglobulin (a2M), and antithrombin III (AT) will be evaluated. The regulation of plasmin will be studied in the presence of soluble fibrin and other preparations that mimic the polymerizing or degrading clot. Results obtained in experiments with purified proteins will be confirmed in whole plasma systems and in vivo in mice. Human umbilical vein endothelial cells and rat hepatocyte will be used to study the reaction of proteinase inhibitors with plasmin that is bound to cell surface receptors. Plasmin that is bound to endothelial cells reacts less readily with a2AP. Therefore, other plasmin inhibitors such as a2M or AT play critical roles in the regulation of plasmin at this site. The reaction of plasmin with proteinase inhibitors will be studied in the presence of an endothelial cell extracellular matrix protein preparation, immobilized laminin, immobilized fibronectin and a basement membrane preparation secreted by EHS sarcoma cells. Many of these studies will be extended to consider thr regulation of plasminogen activators such as streptokinase- plasmin (SkP1) and tissue plasminogen activator in the same environments. For example, experiments are planned to evaluate preliminary data which suggest that SkP1 binds to cell surface receptors analogously to plasmin. The applicants speculate that similar interactions may be important with new acylated-SkP1 derivatives such as APSAC (anistreplase). Finally, since heparin is frequently administered concomitantly with thrombolytic agents, complexes formed between AT, fibrin and heparin will be studied with regard to antifibrinolytic activity. Once again, plasma """"""""non plasma"""""""" environments will be considered. The goal of this research program is to provide basic research data that will aid in the design of thrombolysis protocols and in the management of patients with hemostasis imbalances.
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