Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara

Senis, Y. A., Kim, P. Y., Fuller, G. L. J., Garcia, A., Prabhakar, S., Wilkinson, M. C., Brittan, H., Zitzmann, N., Wait, R., Warrell, David A., Watson, S. P., Kamiguti, A. S., Theakston, R.David G., Nesheim, M. E. and Laing, Gavin (2006) 'Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara'. Biochimica Et Biophysica Acta-Proteins and Proteomics, Vol 1764, Issue 5, pp. 863-871.

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Abstract

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active a-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex, The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloprotemase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloprotemases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloprotemase family of proteins. (c) 2006 Elsevier B.V All rights reserved.

Item Type:	Article
Uncontrolled Keywords:	snake venom toxin cotiaractivase prothrombin activator bothrops cotiara snake-venom platelet-adhesion cloning metalloproteinases receptors proteins thrombin collagen family
Subjects:	QY Clinical Pathology > Blood. Blood Chemistry > QY 410 Coagulation WD Disorders of Systemic, Metabolic or Environmental Origin, etc > Animal Poisons > WD 410 Reptiles WH Hemic and Lymphatic Systems > Hematologic Diseases. Immunologic Factors. Blood Banks > WH 310 Mechanism of blood coagulation. Hemostatis
Faculty: Department:	Groups (2002 - 2012) > Molecular & Biochemical Parasitology Group
Digital Object Identifer (DOI):	https://doi.org/10.1016/j.bbapap.2006.03.004
Depositing User:	Martin Chapman
Date Deposited:	31 May 2011 17:46
Last Modified:	06 Feb 2018 13:02
URI:	https://archive.lstmed.ac.uk/id/eprint/1589

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