Background: Although staphylokianse (SAK) is among the most promising blood dissolving agents, it is far from ideal. It is interesting to hypothesize that the clot lysis efficacy of SAK can be enhanced with direct active platelet binding ability, and at the same time the rethrombosis complication after successful recanalization can be minimized with an antiplatelet aggregation activity. The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK). Results: By using site-directed mutagenesis, an Arg-Gly-Asp (RGD) motif was engineered in the staphylokinase (SAK). This mutant of SAK designated RGD-SAK was expressed, purified and characterized. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis and the concentration required to obtain 50% clot lysis (C50) were reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation. Conclusion: RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.
ASJC Scopus subject areas
- Molecular Biology