256 On the mechanism of the fibrin-specificity of staphylokinase

Abstract

Staphylokinase (STA), a protein of bacterial origin, was recently demonstrated to induce highly fibrin-specific thrombolysis both in human plasma in vitro and in limited clinical trials. Using fluorescence microscopy, we investigated the spatial distribution of FITC-labelled STA during lysis of a plasma clot and its binding to purified fibrin clots in the presence or in the absence of plasminogen (Pg). When added to the outer plasma, STA accumulated highly in a thin superficial layer of a lysing plasma clot following the distribution of Pg during lysis. Experiments with fibrin clots revealed that STA binds to Pg which is bound to partially degraded fibrin, but not to Pg which is bound to intact fibrin. Binding of FITC-STA to various forms of plasmin(ogen) in solution was studied by measuring fluorescence anisotropy. The binding constant for Glu-Pg was estimated as 8 μM, for Lys-Pg as 300 nM, for active-site blocked plasmin (PI) it was less than 50 nM. Gel filtration of a mixture of STA (0.5 /iM) with a two-fold molar excess of Glu-Pg demonstrated that STA migrated as an unbound 18 KD protein when activation of Pg into PI was precluded hy inhibitors of PI. When gel-filtered under the same conditions with active-site blocked PI, STA migrated in a complex with PI with an apparent molecular mass of 90-100 KD. Confocal fluorescense microscopy demonstrated that when FITC-STA was added to plasma before clotting, it did not hind to fibrin fibers during the first minutes (lag-phase), whereas Pg bound to the fibers moderately. Then, both Pg and STA started to accumulate on the fibers progressively, followed by lysis of the clot. Our results suggest that in plasma, STA is not significantly bound to Pg. This adds a new mechanism to the known explanations for the remarkable stability of plasminogen in plasma in the presence of STA. STA binds strongly to PI and to Pg which is bound to partially degraded fibrin, providing a basis for fibrin-dependent activation of Pg.