Purification of a prothrombin activator from Bothrops insularis venom and it activity on melanoma cells.

Modesto, JCA¹; Maria, DA¹; Neves-Ferreira, AGC²; Fritzen, M¹; Chudzinski-Tavassi, AM¹.

1 Laboratório de Bioquímica, Instituto Butantan, São Paulo; 2 Departamento de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro.

Snake venoms contain proteins whose interfere on numerous biological processes of their prey, and some of them affect directly the haemostatic system. In contrast to other bothropic snakes, the diet of Bothrops insularis consists of birds and some invertebrates, consequently its venom may present specific toxins which are selected to better capture that kind of animals. In this study we described the first group-I metalloprotease prothrombin activator purified from B. insularis venom and its action on different cell lines. The P141 was purified by affinity chromatography (Benzamidine Sepharose 6B), followed by a cation-exchange (Resource S) and molecular exclusion chromatography (Superdex 200). Analyzed by MALDI-TOF (Voyager DE-PRO Applied Biosystems) the molecular masses of P141 were 22,639 and 22,645 (respectively in the absence and presence of reducing agent), similar to the MW found by SDS-PAGE. The peptide sequences of P141, obtained by digestion with endoproteinase Lys-C, were analyzed by BLASTQ3 program, and show high similarity to known metalloprotenases. Prothrombin activator activities were measured using specific chromogenic substrates, the P141 was able to activate prothrombin in a dose-dependent manner, independently of phospholipids and exogenous calcium. The P141 is devoid of hemorragic activity evaluated by Kondo et al. (1960) and by intravital microscopy. The P141 was tested on human umbilical vein endothelial cells (HUVECs), vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta, chinese hamster ovary cells (CHO-K1 cells) and murine B16F10 melanoma cell line, and it was capable to induce detachment in a concentration-dependent manner only of B16F10 cell line, and the viability of the detached cells was not significantly reduced as evaluated by exclusion trypan blue staining 24h after treatment with P141. In summary, these results indicate that P141 is an effective prothrombin activator capable to act specifically on adhesion of B16F10 melanoma cells without affecting its viability, moreover the mechanism of action of P141 on B16F10 cells has been investigated.

Supported by Fapesp.

Correspondence to: clainealbuquerque@hotmail.com