Abstract
The mechanism of plasminogen activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with plasminogen that activates plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface.
Highlights
Istration is associated with extensive systemic fibrinogenolysis [2]
0.12-ml '251-fibrin-labeled humanplasmaclotsubcently, it was shown that recombinant staphylokinase is a mersed in 0.5 ml of citrated human plasma;50%lysis more potent and more fibrin-specific fibrinolytic agent than in 2 h is obtained with 17 nM staphylokinase and is streptokinase in human plasmian vitro [14]
A plasminogen activator secreted by S. aureus has been cloned by recombinant DNA technology and expressed in E. coli [5,6] or B. subtilis [7]
Summary
Complex Formation with Plasminogen-Fig. 1 shows that, in mixtures of plasminogen witha &fold molar excess of either staphylokinase or streptokinase, the active site,as monitored with the chromogenic substrate S-2251, is rapidly exposed. 5 nM) in the absence or the presence of a2-antiplasmin (final concentration, 3 p ~ )A., activation of plasminogen (in percent) by staphylokinase (0,m)or streptokinase (0,Oa)s a function of time, whereas in the mixture with plasminogen-streptokinase, no complex formation was observed (lane 5). SDS-PAGE under reducing conditions (Fig. 5, panel 11) shows that the single-chain plasminogen moiety is converted to a two-chain plasmin derivative in both the complex with staphylokinase (lane 4 ) and that with streptokinase (lane 3). The data represent the concentration of plasminogen activator (nM) required to obtain 50% lysis within 1 h (Cso) of purified fibrin clots (preparedin the absence or the presence of plasminogen) submersed in buffer containing 1.5 PM Glu-plasminogen in the absence or the presence of 1.0 p~ az-antiplasmin.The data are mean f S.E. of three to five independent determinations
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