Activation of the fibrinolytic system is required to restore blood vessel patency after coagulation and occurs in wound healing and inflammation in which proteolysis may regulate cell migration. Activation of this system results in conversion of plasminogen to plasmin, a broad spectrum serine protease. Perturbation of this system can have pathological results ranging from thrombosis to hemorrhagic diatheses. Known mechanisms for regulating fibrinolysis include the availability of plasminogen activators, protease inhibitors and fibrin. The overall objective of this proposal is to test the hypothesis that cell surface receptors for plasminogen and its activator, urokinase, constitute a unique and previously unrecognized mechanism for regulating the fibrinolytic system. This proposal is expanded from a N.l.R.A proposal. Specifically, as initially proposed, the functions of plasminogen receptors on platelets and monocytes will be evaluated.
The first aim i s to test the hypothesis that plasminogen binding to cells enhances the catalytic efficiency of plasminogen activation.
The second aim i s to test the hypothesis that the plasminogen binding site on the monocyte is functionally similar to that of unstimulated platelets. For both cell types, the hypothesis than cell-bound plasmin is protected from inhibition and than plasmin affects cellular functions will be tested.
The aims of the expanded proposal are to characterize and compare the binding of urokinase and prourokinase to monocytes to elucidate the mechanisms of regulation of urokinase and plasminogen receptors, and to test the hypothesis that concomitant binding of plasminogen and urokinase promotes formation of plasmin on the cells. The experimental objectives include: (1) comparison of kinetic parameters for activation of cell-bound and soluble plasminogen; (2) comparison of rates of inactivation of cell-bound vs soluble plasmin (3) identification of cell-binding region(s) of plasminogen: and (4) assessment of consequences of plasmin formation on the cells. Additional objectives for monocytes are: (1) characterization of their interactions with plasminogen and urokinase. (2) comparison of inactivation of cell-bound vs soluble urokinase; (3) identification of both the plasminogen and urokinase receptors; (4) assessing regulation of both receptors by monocyte agonists; (5) determining the mechanism by which urokinase enhances plasminogen binding: (6) assessment of plasminogen synthesis by the cells. The results obtained may suggest mechanisms for therapeutic regulation of the fibrinolytic system.
