Interactions between Staphylokinase, Plasmin(ogen), and Fibrin

Abstract

Staphylokinase (STA), a protein of bacterial origin, induces highly fibrin-specific thrombolysis both in human plasma in vitro and in pilot clinical trials. Using fluorescence microscopy, we investigated the spatial distribution of fluorescein isothiocyanate (FITC)-labeled STA during lysis of a plasma clot and its binding to purified fibrin clots in the presence or in the absence of plasmin(ogen). STA highly accumulated in a thin superficial layer of the lysing plasma clot following the distribution of plasminogen (Pg) during lysis. Experiments with purified fibrin clots revealed that STA binds to Pg bound to partially degraded fibrin but not to Pg bound to intact fibrin. Binding of FITC-labeled STA to various forms of plasmin(ogen) in a buffer solution was studied by measuring fluorescence anisotropy. The binding constant for Glu-Pg was estimated as 7.4 M and for Lys-Pg as 0.28 M; for active-site blocked plasmin the binding constant was less than 0.05 M. The much lower affinity of STA for Glu-Pg compared with that for active siteblocked plasmin was mainly due to a lower association rate constant, as assessed by real time biospecific interaction analysis. Gel filtration of a mixture of STA with a molar excess of Glu-Pg demonstrated that STA migrated as an unbound 18-kDa protein when activation of Pg into plasmin was precluded by inhibitors of plasmin. When gel-filtered under the same conditions with plasmin, STA migrated in complex with plasmin with an apparent molecular mass of 100 kDa. Confocal fluorescence microscopy finally demonstrated that when FITClabeled STA was added to plasma before clotting, it did not bind to fibrin fibers during the first minutes (lag phase), although Pg bound to the fibers moderately. Then, both Pg and STA started to accumulate on the fibers progressively, followed by complete lysis of the clot. In conclusion, our results imply that, when STA is added to plasma, only a small percentage associates with Pg. In contrast, STA binds strongly to plasmin and to Pg, which is bound to partially degraded fibrin. These findings add a new mechanism to the known explanations for the inefficient Pg activation by STA in plasma and specify the mechanism for fibrin-dependent activation of Pg.

Staphylokinase Requires NH2-terminal Proteolysis for Plasminogen Activation

“…The K a values of all Sak42D variants studied were very similar to those of wild-type Sak42D. The affinity constants for binding to native Glu-Plg were approximately 1000-fold lower than those for binding to rPlg(S741A), as has previously been observed (20).…”

“…Determination of the association equilibrium constants for the binding of the Sak42D variants to plasminogen revealed no significant differences with Sak42D, with respect neither to the previously observed low affinity binding to native Glu-Plg in the presence of excess plasmin inhibitor, nor to the high affinity binding to rPlg(S741A) (20). In the absence of altered binding of Sak42D variants to plasminogen, the mutations that reduce the efficiency of Sak-plasmin complexes to activate plasminogen must affect the conversion of the active site of plasmin into the plasminogen activator configuration.…”

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

“…In addition, it has some desirable features that are superior to tPA (11). Notably, SAK mediates the lysis of platelet-rich and retracted clots efficiently (12,13) and shows exceptional fibrin specificity (9,10,14,15). These properties can help minimize reocclusion and bleeding complications.…”

“…The result is a preferential plasminogen activation by SAK at the fibrin surface that contributes to the fibrin specificity of SAK in a plasma milieu. This fibrin specificity is made even stronger by the preferential binding of SAK to plasmin(ogen) that is fibrinbound (14). The fibrin-specific property of SAK underlies an interesting observation in clinical trials.…”

Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

“…Consequently, the SAK-plasmin complex can activate plasminogen to generate plasmin locally on the surface of the clot. Furthermore, SAK is shown to bind preferentially to clot-bound plasmin(ogen) (15). SAK also has the ability to dissolve platelet-rich plasma clots and is much more efficient than tPA and streptokinase under both in vitro conditions and in animal models (16).…”