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

Abstract

Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.

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.…”

Staphylokinase-Annexin XI Chimera Exhibited Efficientin VitroThrombolytic Activities

“…The resistance of secondary clots, which are rich in platelets, to the effect of plasminogen activators is another concern. 1,6) This has led to many efforts to improve the potency and safety of thrombolytic agents. 5,7,8) Following injury to the endothelium and vessel wall, platelets adhere rapidly to the sub-endothelial matrix, then become activated and form the primary hemostatic plug.…”

“…1,35,36) Hence, SAK has been chosen as the clot dissolving component in several chimeras. 6,37,38) Although a chimera composed of SAK and an ANX might exert both fibrin selective plasminogen activation and platelet targeting anticoagulation, no such chimera lacking a linker in-between has been investigated so far.…”

“…[26][27][28][29] Many efforts have been made to construct ANXV based chimeras for evaluation of their targeting effect at a potential thrombus. 6,10,30,31) Staphylokinase (SAK) is one of the promising blood clot dissolving agents. 1,6,[32][33][34] Although the thrombolytic potency of SAK has been found to be comparable to that of tPA, clinical trials have indicated that SAK has a superior fibrin specificity.…”

“…6,10,30,31) Staphylokinase (SAK) is one of the promising blood clot dissolving agents. 1,6,[32][33][34] Although the thrombolytic potency of SAK has been found to be comparable to that of tPA, clinical trials have indicated that SAK has a superior fibrin specificity. 1,35,36) Hence, SAK has been chosen as the clot dissolving component in several chimeras.…”