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Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro
Habibollah Faraji1, Fatemeh Soltani2, Mohammad Ramezani2
1Department of Laboratory Sciences, Faculty of Para-medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
Objective:
Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris.
Result:
Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420 μg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay.
Conclusion:
Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.
