Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

Abstract

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C₁₆₄ I₁₆₅M₁₆₆ motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 μg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and β-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and β-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.

Clotting and fibrinogenolysis inhibition by essential oils from species of the Asteraceae family

“…The fibrinogenolytic activity of B. atrox venom was not inhibited by the essential oils from B. dracunculifolia, C. bonariensis, T. diversifolia or A. polystachya. Cintra and coworkers 4 described that Batroxase, a metalloproteinase with high fibrinogenolytic and thrombolytic activities present in the same venom, is probably one of the enzymes responsible for the proteolysis observed, which was not inhibited in the present study 4 . Tests using different volumes of oils, incubation times, run times, concentrations of polyacrylamide in the gel and variations of the staining times in the presence of Coomassie Brilliant Blue (results not shown) resulted in the hypothesis that the fibrinogen molecules could be interacting with constituents of the oils, making it impossible to bind the dye with fibrinogen molecules.…”

“…Annually, about 40,000 people die of snakebites worldwide, including about 25,000 in India, 10,000 in the United States and the rest in other countries 3 . In Brazil, accidents with snakes of the Bothrops genus (Viperidae), widely distributed throughout the national territory, stand out in number and severity, being of great relevance because of permanent sequels characterized mainly by the loss of the affected limb 4 . The snakes of the Lachesis genus (Viperidae) are also relevant because of the severity of the local effects induced by their venoms, although they have a geographical distribution restricted to the Amazon basin (Lachesis muta muta L.) and the Atlantic forest, from north of Rio de Janeiro to Paraiba (Lachesis muta rhombeata L.) 5 .…”

Comparison of venoms from wild and long-term captive Bothrops atrox snakes and characterization of Batroxrhagin, the predominant class PIII metalloproteinase from the venom of this species

“…However, in these studies PIIISVMPs were characterized at the protein family level and the proteoform expressed in each sample was not characterized. The literature describes the isolation of several PI-SVMPs from B. atrox venom as atroxlysin-I [51], batroxase [52] and Batx-I [53]. Only one PIII-SVMP has been isolated [54], however, its structural characterization was partial and the results did not address the major functional activities of this class of toxin.…”

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

“…Enquanto isso, as metaloproteases P-I, apesar da pouca ou nenhuma atividade hemorrágica, têm revelado alta atividade proteolítica sobre os componentes da cascata de coagulação sanguínea, especialmente sobre o fibrinogênio (Fator I). Muitas das SVMPs da classe P-I apresentam este caráter, das quais temos BJ-P12 de B. jararaca (SILVA et al, 2012); Batroxase de B. atrox (CINTRA et al, 2012); BleucMP de B. leucurus (GOMES et al, 2011); dentre outras. Estas metaloproteases são chamadas de enzimas fibrinogenolíticas, porque provocam o consumo do fibrinogênio, causando assim uma desfibrin (ogen)ação e, por consequência, tornando o sangue mais fluido e aumentando o tempo de coagulação.…”