A PROTHROMBIN ACTIVATOR FROM BOTHROPS ERYTHOMELAS VENOM: CHARACTERIZATION, MOLECULAR CLONING AND SEQUENCE ANALYSIS OF CODING cDNA.

SILVA, MARCIA B.; HO, PAULO, L; LAZZARI, MARIA A.; POZNER, ROBERTO G.; LITWAK, SARA; SCHATTNER, MIRTA; CHUDZINSKI-TAVASSI, ANA M.

Paris, Francia.

Conferencia; International Conference on Exogenous Factors affecting Thrombosis and Haemostasis (ICEFATH).; 2001

Registry of Exogenous Hemostatic Factors (subcommittee of the ISTH Scientific and Standardization Committee)

Bothrops erythromelas snakes are widely distributed in the Northeastern of Brazil. This specie is particularly interesting since its venom presents a more potent procoagulant activity than other species in this genus.  It has been demonstrated that one of the components that contributes for this high procoagulant activity is a Factor II activator protein, which was found after a screening of the crude venom. We have isolated and characterized  the Factor II activator protein in B. erythromelas venom. Aliquots of the crude venom (2.5 mg) were submitted to purification steps in FPLC and HPLC systems. Prothrombin activator was purified in a Resource-S column (Pharmacia) and its homogeneity was confirmed by SDS-PAGE. It is a 78 kDa  single chain metalloproteinase with no hemorrhagic activity in vivo. The activator produced thrombin by cleavage of the prothrombin in the first five minutes (SDS-PAGE). A Kcat/Km of 1,5 X 104 (M s)-1 was obtained from kinetic parameters of the amidolytic activity of the prothrombin activator towards chromogenic substrate S-2238. In vitro treatment of HUVECs with the purified protein induced marked morphological changes, ICAM-1 expression and vWF release in a concentration dependent manner.Total RNA was extracted from the venom gland of B. erythromelas and a cDNA library was constructed in the pGEM11zf(+) plasmid (Promega). The Berythrom activador cDNA (1,700 bp) was amplified from this library by PCR using degenerate primers corresponding to N-terminal sequence of protein. This fragment was sequenced and sub-cloned in E. coli expression vector for recombinant protein production. (Supported by CNPq and FAPESP, Brazil and CONICET and Fundación R Barón, Argentina).