Rapid Purification of a New P-I Class Metalloproteinase fromBothrops moojeniVenom with Antiplatelet Activity

Abstract

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMPα-II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMPα-II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMPα-II cleaves Aα-chain of fibrinogen followed by Bβ-chain, and does not show any effect on the γ-chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30–50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMPα-II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMPα-II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMPα-II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.

Systemic alterations induced by phospholipase A2, BmooTX‐I, isolated from Bothrops moojeni snake venom

“…The enzyme CK is an important marker of muscle injury because it is at high plasma levels under these conditions, in view of its extravasation from the intracellular environment (muscle cells) to the extracellular (blood) environment. Thus, our results showed high plasma concentrations of CK in the first hours (3 hours; Figure A) after BmooTX‐I consistent with the results of Santos‐Filho et al and with previous data on muscle damage caused by B. moojeni venom when injected to animals . According to Santos‐Filho et al, optical and electron microscopy analysis revealed that BmooTX‐I was able to cause severe myonecrosis accompanied by inflammatory infiltrate.…”

“…Thus, our results showed high plasma concentrations of CK in the first hours (3 hours; Figure 5A) after BmooTX-I consistent with the results of Santos-Filho et al 22 and with previous data on muscle damage caused by B. moojeni venom when injected to animals. 60 According to Santos-Filho et al, 22 optical and electron microscopy analysis revealed that BmooTX-I was able to cause severe myonecrosis accompanied by inflammatory infiltrate. The gastrocnemius muscle fibres were extensively destroyed presenting different stages of degeneration and oedema.…”

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

“…Platelet aggregation assays were performed in PRP and measured using the automated Aggregometer 4 channels (AggRAMTM version 1.1, Helena Laboratories, USA) as described by Queiroz et al [ 16 ]. Human blood, collected in the presence of sodium citrate (3.2%), was centrifuged at 100× g for 12 min at room temperature to obtain PRP.…”

The role of platelets in hemostasis and the effects of snake venom toxins on platelet function

“…The imbalance of the hemostatic system is the most studied biological effect caused by these toxins (Markland Jr and Swenson, 2013;Ramos and Selistre-De-Araujo, 2006;Takeda et al, 2012). They can interfere in platelet aggregation and in the coagulation cascade, causing pro-or anti-coagulant effects (Camacho et al, 2014;Gutiérrez and Rucavado, 2000;Queiroz et al, 2014;Sajevic et al, 2011;Takeda et al, 2012).…”

“…jararacussu (Marcussi et al, 2007), atroxlysin-I from B. atrox (Sanchez et al, 2010), BmooMPα-II from B. moojeni (Queiroz et al, 2014), and barnettlysin-I (Bar-I) from B. barnetti (Sanchez et al, 2016). The enzymatic activity of these PI SVMPs is critical for significant inhibition of platelet function.…”