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 78kDa. 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 Aα-chain was slowly digested only after longer incubation with berythractivase, and no effect on the β- or γ-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 2330bp 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.
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January 2003
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Research Article|
January 01 2003
A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning
Márcia B. SILVA;
Márcia B. SILVA
∗Departamento do Biofísica, Universidade Federal de Pernambuco, Recife, PE, Brazil
¶Laboratório do Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brazil, 1500 São Paulo, SP, Brazil
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Mirta SCHATTNER;
Mirta SCHATTNER
†Departamento de Trombosis y Hemostasia, Academia Nacional de Medicina, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
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Celso R.R. RAMOS;
Celso R.R. RAMOS
‡Centro Do Biotecnolgia, Instituto Butantan, Av. Vital Brazil, 1500 São Paulo, SP, Brazil
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Inácio L.M. JUNQUEIRA-de-AZEVEDO;
Inácio L.M. JUNQUEIRA-de-AZEVEDO
‡Centro Do Biotecnolgia, Instituto Butantan, Av. Vital Brazil, 1500 São Paulo, SP, Brazil
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Míriam C. GUARNIERI;
Míriam C. GUARNIERI
§Departamento do Zoologia, Universidade Federal de Pernambuco, Recife, PE, Brazil
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María A. LAZZARI;
María A. LAZZARI
†Departamento de Trombosis y Hemostasia, Academia Nacional de Medicina, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
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Claudio A.M. SAMPAIO;
Claudio A.M. SAMPAIO
∥Departamento do Bioquímica, Universidade Federal de São Paulo-Escola Paulista de Medicina (UNIFESP-EPM), SP, Brazil,
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Roberto G. POZNER;
Roberto G. POZNER
†Departamento de Trombosis y Hemostasia, Academia Nacional de Medicina, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
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Janaina S. VENTURA;
Janaina S. VENTURA
¶Laboratório do Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brazil, 1500 São Paulo, SP, Brazil
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Paulo L. HO;
Paulo L. HO
‡Centro Do Biotecnolgia, Instituto Butantan, Av. Vital Brazil, 1500 São Paulo, SP, Brazil
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Ana M. CHUDZINSKI-TAVASSI
Ana M. CHUDZINSKI-TAVASSI
1
¶Laboratório do Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brazil, 1500 São Paulo, SP, Brazil
1To whom correspondence should be addressed (e-mail amchudzinski@hotmail.com).
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Publisher: Portland Press Ltd
Received:
March 22 2002
Revision Received:
July 26 2002
Accepted:
September 11 2002
Accepted Manuscript online:
September 11 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 369 (1): 129–139.
Article history
Received:
March 22 2002
Revision Received:
July 26 2002
Accepted:
September 11 2002
Accepted Manuscript online:
September 11 2002
Citation
Márcia B. SILVA, Mirta SCHATTNER, Celso R.R. RAMOS, Inácio L.M. JUNQUEIRA-de-AZEVEDO, Míriam C. GUARNIERI, María A. LAZZARI, Claudio A.M. SAMPAIO, Roberto G. POZNER, Janaina S. VENTURA, Paulo L. HO, Ana M. CHUDZINSKI-TAVASSI; A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning. Biochem J 1 January 2003; 369 (1): 129–139. doi: https://doi.org/10.1042/bj20020449
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