The importance in the understanding of the basic mechanism of human plasminogen activation is underscored by the wide use of fibrinolysis in the therapy of some blood clotting and cardiovescular disorders. Staphylokinase is a plasminogen activator protein secreted by strains of Staphylococcus aureas. Limited data from ours and other laboratories suggest that this protein activates plasminogen by strong binding of the two proteins, similar to the mechanism of streptokinase. The lack of information on staphylokinase has been due to the difficulty to obtain sufficient amounts of pure protein. This project proposes (1) to obtain pure staphylokinase form cloned gene in E. coli, (2) to examine themechanism of human plasminogen activated by kinetic and binding studies, (3) to determine the functional groups involved in plasminogen binding and activation by differential chemical methods, and (4) to change the functional groups of staphylokinase using site-directed mutagenesis of cloned gene. These changes involve lysines, tyrosines, tryptophan, N-terminal regiona and C-terminal region. The changes on the N- and C-terminal regions will test the functional roles of these residues. The changes in Tyr and Trp will be done in conjunction with 19F-nuclear magnetic resonance studies of the role of these residues. The studies described above will permit a greater understanding of mode of plasminogen activation by staphylokinase in specific and of contact activation of serine protease zymogens in general.
