Cloning, purification and characterization of a recombinant protease with novel thrombolytic activity in human plasma and rat thrombosis models. Issue 191 (July 2020)

• Record Type: Journal Article
• Authors: Lim, Do Sung; Park, Jung Eun; Park, Jong Woo; Cho, Yeong Hee; Park, Jong Kun; Lee, Jung Sup
• Is Part Of: Thrombosis research. Issue 191(2020)
• Page Start: 57
• Page End: 65
• Publication Date: 2020-07
• Subjects: Vibrio furnissii -- Recombinant metalloprotease rvFMP -- Fibrin(ogen)olytic enzyme -- Fibrin clots -- Thrombolytic enzyme -- Thrombosis rat models; Thrombosis -- Periodicals; 616.135
• DOI: 10.1016/j.thromres.2020.04.040
• Languages: English
• ISSNs: 0049-3848

Abstract

Abstract: Background: There is a need to identify and develop novel thrombolytic agents that can directly digest fibrin clots from various biological resources. Objective: To 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. Results: We 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 r ecombinant v ibrio f urnissii m etallop rotease). 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. Conclusion: rvFMP can be a candidate enzyme capable of being developed as a novel direct-acting thrombolytic agent. Highlights: A recombinant protease rvFMP can digest cross-linked fibrin even in human plasma milieu. rvFMP shows in vivo thrombolytic capability. rvFMP has no cytotoxicity and also is unlikely to induce a systemic bleeding, lethality, and inflammatory response. rvFMP can be a promising agent to be developed as a direct-acting enzyme that can dissolve vascular thrombi