CHARACTERIZATION, MOLECULAR CLONING AND SEQUENCE ANALYSIS OF cDNA CODING FOR LOPAP, A PROTHROMBIN ACTIVATOR FROM LONOMIA OBLIQUA CATERPILLAR.

REIS, CLEISON V.; HO, PAULO, L; POZNER, ROBERTO G.; LAZZARI, MARIA A.; 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)

The Lonomia obliqua venom causes a severe consumptive coagulopathy, which can lead to an hemorrhagic syndrome. The crude bristles extract displays a procoagulant activity due to Factor X, and a prothrombin activating activity. Lopap, a 69 kDa prothrombin activator protease isolated from L. obliqua bristle, In vivo, Lopap was able to evoke thrombus formation in microcapillar vessels of rats, fibrinogen depletion, 30% reduction on platelet number and inhibition of platelet aggregation induced by colagen. Histologycal analysis of isolated tissue, after 1h Lopap e.v. injection  revealed glomerular infiltration and tubular necrosis. In vitro, treatment of HUVECs with Lopap significantly increased ICAM-1 in a concentration dependent manner. VWillebrand factor synthesis or release was observed after incubation of endothelial cells with different concentrations of  Lopap . In vitro  The titration of the putative reactive serines of Lopap by NPGB indicated the stoichiometry of 1.2 serine residue per molecule of NPGB. The kinetic hydrolysis parameters of the internally quenched fluorescent peptide substrate, based on prothrombin sequence, Abz-YQTFFNPRTFGSQ-EDDnp, obtained by Lopap activity were Kmapp 4.5 mM; kcat 5.32 sec-1; kcat/Kmapp 1.2 x 106 M-1.sec-1, indicating good affinity and a high catalytic efficiency. Total RNA was extracted from the bristles of L. obliqua and a cDNA library was constructed in the pGEM11zf(+) plasmid (Promega). Lopap cDNA (900 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, CONICET and Fundación R Barón, Argentina).