Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom.
Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombin's activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators.
Berger
Markus
M
Laboratório de Bioquímica Farmacológica, Centro de Biotecnologia Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15005, 91501-970 Porto Alegre, RS, Brazil.
Pinto
Antônio F M
AF
Guimarães
Jorge A
JA
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002413:Cations, Divalent; D003435:Crotalid Venoms / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000378:metabolism*; D030162:Reptilian Proteins / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D003435:Crotalid Venoms; D030162:Reptilian Proteins; D011516:Prothrombin; D045726:Metalloproteases