title: A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning.
abstract: A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.
authors.affiliation: Departamento do Biofísica, Universidade Federal de Pernambuco, Recife, PE, Brazil.
authors.forename: Márcia B
authors.initials: MB
authors.lastname: Silva
authors.forename: Mirta
authors.initials: M
authors.lastname: Schattner
authors.forename: Celso R R
authors.initials: CR
authors.lastname: Ramos
authors.forename: Inácio L M
authors.initials: IL
authors.lastname: Junqueira-de-Azevedo
authors.forename: Míriam C
authors.initials: MC
authors.lastname: Guarnieri
authors.forename: María A
authors.initials: MA
authors.lastname: Lazzari
authors.forename: Claudio A M
authors.initials: CA
authors.lastname: Sampaio
authors.forename: Roberto G
authors.initials: RG
authors.lastname: Pozner
authors.forename: Janaina S
authors.initials: JS
authors.lastname: Ventura
authors.forename: Paulo L
authors.initials: PL
authors.lastname: Ho
authors.forename: Ana M
authors.initials: AM
authors.lastname: Chudzinski-Tavassi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017837:Bothrops; D015815:Cell Adhesion Molecules / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry*; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020536:Enzyme Activators / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D005337:Fibrin / Q000187:drug effects; D005340:Fibrinogen / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D003435:Crotalid Venoms; D018076:DNA, Complementary; D020536:Enzyme Activators; D014841:von Willebrand Factor; D011516:Prothrombin; D005337:Fibrin; D005340:Fibrinogen