Abstract
BackgroundThere is a need to identify and develop novel thrombolytic agents that can directly digest fibrin clots from various biological resources. ObjectiveTo clone, express, purify, and characterize a recombinant protease named rvFMP capable of cleaving fibrinogen, fibrin polymer, and cross-linked fibrin in human plasma milieu and rat thrombosis model systems. ResultsWe cloned a vFMP-encoding gene from the genomic DNA of V. furnissii KCCM41679 using polymerase chain reaction (PCR), expressed in Escherichia coli, and purified rvFMP (stands for recombinant vibrio furnissii metalloprotease). The proteolytic activity of purified rvFMP enzyme could be clearly inhibited by 1,10-phenanthroline and ethylene glycol tetraacetic acid, but not by diisopropyl fluorophosphate, suggesting that it can be a typical metalloprotease. rvFMP showed an effective proteolytic activity in cleaving cross-linked fibrins in human plasma milieu. Remarkably, rvFMP exhibited a clear thrombolytic activity in rat thrombosis models such as ferric chloride-exposed rat carotid artery and carrageenan-treated rat tail. However, rvFMP (1.5 mg/kg) evoked no internal bleeding and also showed no lethal effect in mice. The recombinant enzyme also showed no cytotoxicity and had an inability to induce tumour necrosis factor-α (TNF-α) in Raw264.7 cells. ConclusionrvFMP can be a candidate enzyme capable of being developed as a novel direct-acting thrombolytic agent.
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