A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning.

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

Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil

The Caatinga lancehead, Bothrops erythromelas, is a medically relevant species, responsible for most of the snakebite accidents in most parts of its distribution range in northeastern Brazil. The spectrum and geographic variability of its venom toxins were investigated applying a venomics approach to venom pools from five geographic areas within the Caatinga ecoregion. Despite its wide habitat, populations of B. erythromelas from Ceará, Pernambuco, Juazeiro, Paraiba, and Ilha de Itaparica exhibit highly conserved venom proteomes. Mirroring their compositional conservation, the five geographic venom pools also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated in Vital Brazil (BR) and Clodomiro Picado (CR) Institutes, using different venoms in the immunization mixtures. The paraspecificity exhibited by the Brazilian SAB and the Costa Rican BCL antivenoms against venom toxins from B. erythromelas indicates large immunoreactive epitope conservation across genus Bothrops during the last ~ 14 million years, thus offering promise for the possibility of generating a broad-spectrum bothropic antivenom. Biological Significance Accidental snakebite envenomings represent an important public health hazard in Brazil. Ninety per cent of the yearly estimated 20-30,000 snakebite accidents are caused by species of the Bothrops genus. Bothrops erythromelas, a small, moderately stocky terrestrial venomous snake, is responsible for most of the snakebite accidents in its broad distribution range in the Caatinga, a large ecoregion in northeastern Brazil. To gain a deeper insight into the spectrum of medically important toxins present in the venom of the Caatinga lancehead, we applied a venomics approach to define the proteome and geographic variability of adult B. erythromelas venoms from five geographic regions. Although intraspecific compositional variation between venoms among specimens from different geographic regions has long been appreciated by herpetologists and toxinologists as a general feature of highly adaptable and widely distributed snake species, the five B. erythromelas populations investigated exhibit highly conserved venom proteomes. The overall toxin profile of the Caatinga lancehead's venom explains the local and systemic effects observed in envenomations by B. erythromelas. The five geographic venom pools sampled also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated using different bothropic venoms in the immunization mixtures. The large immunoreactive epitope conservation across genus Bothrops offers promise for the generation of a broad-spectrum bothropic antivenom.Ministerio de Ciencia é Innovación/[BFU2010-17373]//EspañaMinisterio de Economía y Competitividad/[BFU2013-42833-P]//EspañaGeneralitat Valenciana/[PROMETEO/2010/005]//EspañaPrograma Iberoamericano de Ciencia y Tecnología para el Desarrollo/[BIOTOX P211RT0412]/CYTED/EspañaUniversidad de Costa Rica/[741-B2-652]/UCR/Costa RicaConsejo Nacional de Rectores/[741-B2-652]/FEES-CONARE/Costa RicaGenoprot/[560931/2010-7]//BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP