The long-term goal of the proposed studies is to determine the molecular mechanism by which streptokinase (SK) activates the human fibrinolytic system, which is the basis for the use of SK as a thrombolytic drug for the treatment of cardiovascular disease. The studies address significant gaps in the understanding of the unique mechanism of conformational activation of plasminogen (Pg) by SK and the coupled proteolytic activation pathway that converts Pg into the fibrinolytic proteinase, plasmin (Pm). In the hypotheses to be evaluated, conformational activation of Pg is triggered by rapid binding of SK and insertion of the sK aminoterminus into a binding pocket on Pg, in cooperation with stabilization of the activated conformation of the catalytic domain of the zymogen as a result of its preferentially higher affinity for SK. This initiates the proteolytic activation pathway by expression of a new binding site, first on SK-Pg and subsequently on SK-Pm complexes, that functions as an exosite to enable specific binding and cleavage of Pg as a substrate. Conformational and proteolytic activation of Pg by SK are modulated by intrinsic differences in the affinities of 5K for ys]Pg, and ysJPm, enhanced by involvement of lysine binding sites, and regulated in vivo by fibrinogen- and fibrin-promoted assembly of productive complexes. The hypotheses will be evaluated in quantitative equilibrium binding studies employing unique fluorescent derivatives of Pg, Pm, and SK, in combination with steady-state and rapid-reaction kinetics, and protein structural approaches.
Specific aims are: (1) To define the sequence of molecular events in the mechanism of conformational activation of Pg induced by SK binding. (2) To delineate the mechanism of the SK-initiated proteolytic activation pathway of Pm formation. (3) To determine the functional roles of the SK alpha, beta, and gamma-domains and individual lysine residues of SK in the mechanisms of conformational and proteolytic activation of Pg. (4) To elucidate the mechanism of fibrinogen and fibrin regulation of SK-initiated fibrinolysis and its role in the fibrin-specificity of SK therapy. The proposed studies of fundamental thermodynamic, kinetic, and structural aspects of the mechanism are expected to change the current conceptualization of the mechanism of action of SK as a thrombolytic drug. New information derived from these studies may enable more fibrin-specific thrombolytic agents to be developed.
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