Abstract
Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.
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This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP—No 2011/23236-4) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—No 142499/2010-2).
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Supplementary Figure I. Obtaining factor Xa. Purified factor Xa was obtained from MooA activation of human coagulation factor X. Initially, moojenactivase was coupled to CNBr-activated Sepharose resin according to the manufacturer’s instructions (GE Healthcare). The MooA-Sepharose complex was incubated with human factor X (250 µg/mL) in 50 mM Tris–HCl buffer, pH 8.0, containing 10 mM of benzamidine. After a range of time periods (1 to 60 min), the reaction mixture was centrifuged at 3000 g for MooA-Sepharose sedimentation, and aliquots of supernatant were removed and (A) analyzed on 12 % SDS-PAGE. The material obtained after 60 min of incubation, composed mostly of activated factor X (FXa), was desalted using an ultracentrifugation system (cutoff pore size 9 kDa) for benzamidine removal and applied to a benzamidine-Sepharose resin. (B) The chromatographic procedure was performed using a FPLC system. The non-bonded material was washed with 50 mM Tris–HCl buffer, pH 8.0, containing 300 mM NaCl with a flow rate of 1 mL/min and collected (fraction F I). The material that bonded to the column (fraction F II) was eluted with 50 mM glycine buffer, pH 3.5, and immediately buffer exchanged for 50 mM Tris–HCl buffer, pH 8.0. Both fractions (FI and FII) were assessed for amidolytic activity toward chromogenic substrate S-2222 and specific activity calculated as shown in parenthesis. (C) Human coagulation factor X, fractions F I and F II were analyzed by 12 % SDS-PAGE. Fraction F II corresponds to isolated enzymatically active factor Xa and was used as described in section Determination of the rate of thrombin and factor Xa generation. (PPTX 725 kb)
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Sartim, M.A., Costa, T.R., Laure, H.J. et al. Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes. Arch Toxicol 90, 1261–1278 (2016). https://doi.org/10.1007/s00204-015-1533-6
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DOI: https://doi.org/10.1007/s00204-015-1533-6