IN FOCUS: A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant. I. In vitro study

Abstract

To cite this article: Szemraj J, Walkowiak B, Kawecka I, Janiszewska G, Buczko W, Bartkowiak J, Chabielska E. A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity -a staphylokinase variant. I. In vitro study. J Thromb Haemost 2005; 3: 2156-65.Summary. We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent -hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity.The plasminogen activation rate (determined by K M and K cat ) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125 I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.Keywords: antiplatelet activity, hirudin and K2 domain of t-PA, recombinant protein, staphylokinase, thrombolysis, thrombolytic and antithrombotic agents.

Locally activity-released bifunctional fusion protein enhances antithrombosis and alleviates bleeding risk

“…It has been widely used to remove tags of recombinant fusion protein with linker peptide IEGR in biochemistry. Recently, linker peptide IEGR also was introduced to construct FXadependent fusion proteins to improve anticoagulation [25,26] or thrombolysis [27,28]. Generally, the fibrinolytic or anticoagulant was linked with antibodies or fragment to improve the affinity to clot.…”

In VitroCharacterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt InducibleE. coliGJ1158

“…Fusion proteins SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul were constructed and produced in different eukaryotic expression host, Schizosaccharomyces pombe [17] and Pichia pastoris [18], respectively. Furthermore, a SAK variant (SAK-HV) with 12 amino acids thrombin-binding domain from hirudin was also constructed and expressed in E. coli BL21 (DE3) [19].…”

The evolution of recombinant thrombolytics: Current status and future directions

“…125 Szemraj and coworkers (2005) created a staphylokinase variant, SAK-RGD-K2-Hir by introducing the K2 domain of t-PA, the Arg-Gly-Asp tripeptide for preventing aggregation of platelets and the thrombin inhibitor hirudin to the C-terminus of staphylokinase. 126 Recently, another variant SAK-RGD-Hirulog has been developed with thrombin inhibition and antiplatelet aggregation abilities. 127 The recombinant fusion protein was more potent and faster-acting thrombolytic agent than rSAK (recombinant staphylokinase) with antithrombin and antiplatelet activities.…”