title: Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara.
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 alpha-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 metalloproteinase. 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 metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.
authors.affiliation: Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, Wolfson Drive, Edgbaston, Birmingham B15 2TT, UK. y.senis@bham.ac.uk
authors.forename: Yotis A
authors.initials: YA
authors.lastname: Senis
authors.forename: Paul Y
authors.initials: PY
authors.lastname: Kim
authors.forename: Gemma L J
authors.initials: GL
authors.lastname: Fuller
authors.forename: Angel
authors.initials: A
authors.lastname: García
authors.forename: Sripadi
authors.initials: S
authors.lastname: Prabhakar
authors.forename: Mark C
authors.initials: MC
authors.lastname: Wilkinson
authors.forename: Helen
authors.initials: H
authors.lastname: Brittan
authors.forename: Nicole
authors.initials: N
authors.lastname: Zitzmann
authors.forename: Robin
authors.initials: R
authors.lastname: Wait
authors.forename: David A
authors.initials: DA
authors.lastname: Warrell
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
authors.forename: Aura S
authors.initials: AS
authors.lastname: Kamiguti
authors.forename: R David G
authors.initials: RD
authors.lastname: Theakston
authors.forename: Michael E
authors.initials: ME
authors.lastname: Nesheim
authors.forename: Gavin D
authors.initials: GD
authors.lastname: Laing
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology* / Q000302:isolation & purification; D045726:Metalloproteases / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011516:Prothrombin / Q000378:metabolism*
chemical_list: D003435:Crotalid Venoms; C511889:cotiaractivase, Bothrops cotiara; D011516:Prothrombin; D045726:Metalloproteases