Abstract
Streptokinase (SK) is a potent clot dissolver but lacks fibrin clot specificity as it activates human plasminogen (HPG) into human plasmin (HPN) throughout the system leading to increased risk of bleeding. Another major drawback associated with all thrombolytics, including tissue plasminogen activator, is the generation of transient thrombin and release of clot-bound thrombin that promotes reformation of clots. In order to obtain anti-thrombotic as well as clot-specificity properties in SK, cDNAs encoding the EGF 4,5,6 domains of human thrombomodulin were fused with that of streptokinase, either at its N- or C-termini, and expressed these in Pichia pastoris followed by purification and structural-functional characterization, including plasminogen activation, thrombin inhibition, and Protein C activation characteristics. Interestingly, the N-terminal EGF fusion construct (EGF-SK) showed plasmin-mediated plasminogen activation, whereas the C-terminal (SK-EGF) fusion construct exhibited ‘spontaneous’ plasminogen activation which is quite similar to SK i.e. direct activation of systemic HPG in absence of free HPN. Since HPN is normally absent in free circulation due to rapid serpin-based inactivation (such as alpha-2-antiplasmin and alpha-2-Macroglobin), but selectively present in clots, a plasmin-dependent mode of HPG activation is expected to lead to a desirable fibrin clot-specific response by the thrombolytic. Both the N- and C-terminal fusion constructs showed strong thrombin inhibition and Protein C activation properties as well, and significantly prevented re-occlusion in a specially designed assay. The EGF-SK construct exhibited fibrin clot dissolution properties with much-lowered levels of fibrinogenolysis, suggesting unmistakable promise in clot dissolver therapy with reduced hemorrhage and re-occlusion risks.
Highlights
Blood clot or thrombus formation within the vascular system is a life-saving process only when it occurs subsequent to a natural hemorrhage
As mentioned in literature Epidermal growth factor (EGF) 4,5,6 domains contain 9 disulfide bonds and their proper oxidative refolding is necessary for full functional activity that is not possible in reduced cytoplasmic environment of bacteria whereas mammalian eukaryotic expression systems often provide low yield and are expensive for production; Pichia pastoris seemed the best system for these constructs
Streptokinase and other plasminogen activators activate plasminogen to plasmin, which dissolves pathological blood clots, but hemorrhage and reformation of clot/s are major clinically challenging problems as the free human plasminogen (HPG) circulates within the system unless HPG activation is limited to the locale of the clot [46,47,48]
Summary
Blood clot or thrombus formation within the vascular system is a life-saving process only when it occurs subsequent to a natural hemorrhage. Thrombus formation, or its subsequent migration, can be life-threatening when it occurs either inside the blood vessels such as that of the brain (causing thrombotic strokes), or the coronary artery- causing heart attacks. Most preferred and available thrombolytics such as streptokinase (SK), Urokinase (UK) and tissue-type Plasminogen Activator (tPA) or its engineered derivatives [1,2,3], have been widely used against various circulatory disorders. All essentially operate through a broadly similar mechanism wherein they catalyse the scission of the scissile peptide bond between residues Arg561-Val562 of plasminogen, and convert it into its proteolytically activated form, plasmin. A detailed description of these constructs has been summarized in a recent review article [4]
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