EFFECT OF STAPHYLOKINASE CONCENTRATION ON PLASMINOGEN ACTIVATION

Activation of Glu- and Lys-plasminogen by various concentrations of re combinant staphylokinase (SAK) were studied by the generation of amido lytic activity from the chromogenic substrate S-2251(H-D-Val-Leu-Lys-p NA) and by SDS-PAGE analysis. Surprisingly, excess SAK decreased and f ixed the rate of S-2251 hydrolysis in a mixture of Lys-plasminogen and SAK. Since the effect of SAK on S-2251 hydrolysis by plasmin was simi lar, the hydrolysis kinetics by free plasmin and plasmin-SAK complex w ere studied. Hydrolysis by either enzyme form followed Michaelis-Mente n kinetics with a Km of 0.38 mM for plasmin and 3.74 mM for SAK-plasmi n complex. The catalytic rate constant was 22.7 s(-1) for plasmin and 21.0 s(-1) for the SAK-plasmin complex. With excess SAK and vigorous r emoval of plasmin activity from plasminogen, the pre-activation lag pe riod differed greatly between Glu- and Lys-plasminogen. Based on the d ifferent substrate specificity of plasmin and plasmin-SAK complex, we analyzed the Glu-plasminogen activation with either catalytic or exces s SAK. With excess SAK, almost no Lys-plasminogen was detectable and w hole Glu-plasminogen was converted directly to Glu-plasmin, then gradu ally to Lys-pIasmin. In contrast, Lys-plasminogen appeared rapidly wit h catalytic amount of SAK. These results suggest that inhibition of Gl u-plasminogen to Lys-plasminogen conversion in the plasminogen-SAK com plex in the presence of excess SAK prolonged the initial lag phase of activation.