Amino-Terminal Fusion of Epidermal Growth Factor 4,5,6 Domains of Human Thrombomodulin on Streptokinase Confers Anti-Reocclusion Characteristics along with Plasmin-Mediated Clot Specificity

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.

EGF‐like and Other Disulfide‐rich Microdomains as Therapeutic Scaffolds

“…In agreement with functional data, the thrombomodulin:thrombin crystal structure suggests excised EGF‐based fragments of thrombomodulin might have therapeutic applications. Indeed, the EGF4‐6 fragment has been used to block the thrombomodulin:thrombin interaction and hence prevent clot formation by inhibiting protein C activation . In that study, it was attached to streptokinase, a potent clot dissolver, to overcome the unwanted downstream reformation of clots, a common issue associated with thrombolytics.…”

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“…CRME was observed to manifest moderate central analgesic activity in comparison to morphine sulfate whose effect was reduced and increased gradually with the time span at 200 mg/kg and 400 mg/kg, respectively. Commercially available thrombolytic drugs like SK activates other plasminogen to plasmin by complexing with circulatory plasminogen, which in turn dissolves fibrinogen and fibrin, the insoluble matrix of the clot that results in lysis of blood clot 27 .…”

EGF‐artige und andere disulfidreiche Mikrodomänen als therapeutische Molekülgerüste

“…In Übereinstimmung mit funktionellen Daten, lässt die Thrombomodulin:Thrombin‐Kristallstruktur vermuten, dass ausgeschnittene Fragmente von Thrombomodulin basierend auf EGF therapeutische Anwendungen haben könnten. Tatsächlich wurde das EGF4‐6‐Fragment verwendet, um die Thrombomodulin:Thrombin‐Wechselwirkung zu blockieren und so die Gerinnselbildung durch Hemmung der Protein‐C‐Aktivierung zu verhindern . In dieser Studie wurde es an Streptokinase, einem wirksamen Gerinnsellöser, gebunden, um die unerwünschte Neubildung von Gerinnseln zu überwinden, welches ein häufiges Problem im Zusammenhang mit Thrombolytika darstellt.…”