Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. in this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrino-genolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate D-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca2+ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators. (C) 2013 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Inst Butantan, CAT Cepid, Lab Especial Toxinol Aplicada, BR-05503000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, São Paulo, BrazilFAPESP: 98/14307-9FAPESP: 11/08514-8FAPESP: 2011/11308-0Web of Scienc

Activation of leukocyte rolling by the cysteine-rich domain and the hyper-variable region of HF3, a snake venom hemorrhagic metalloproteinase

AbstractThe functionality of the disintegrin-like/cysteine-rich domains of snake venom metalloproteinases (SVMPs) has been shown to reside in the cysteine-rich region, which can interact with VWA-containing proteins. Recently, the hyper-variable region (HVR) of the cysteine-rich domain was suggested to constitute a potential protein–protein adhesive interface. Here we show that recombinant proteins of HF3, a hemorrhagic P-III SVMP, containing the cysteine-rich domain (disintegrin-like/cysteine-rich and cysteine-rich proteins) but not the disintegrin-like protein were able to significantly increase leukocyte rolling in the microcirculation. Peptides from the HVR also promoted leukocyte rolling and this activity was inhibited by anti-alphaM/beta2 antibodies. These results show, for the first time, that the cysteine-rich domain and its HVR play a role in triggering pro-inflammatory effects mediated by integrins

Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

International audienceSnake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues

Interaction of Bothrops jararaca venom metalloproteinases with protein inhibitors

Made available in DSpace on 2015-06-08T14:01:51Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) ana_ferreiraetal_IOC_2014.pdf: 1325949 bytes, checksum: 8a1af05b523f11b0f45b9059129701ab (MD5) Previous issue date: 2014Instituto Butantan. Laboratório Especial de Toxinologia Aplicada-CeTICS. São Paulo, SP, Brasil.Instituto Butantan. Laboratório Especial de Toxinologia Aplicada-CeTICS. São Paulo, SP, Brasil.Instituto Butantan. Laboratório Especial de Toxinologia Aplicada-CeTICS. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxonologia. Rio de Janeiro, RJ, Brasil.Instituto Butantan. Laboratório Especial de Toxinologia Aplicada-CeTICS. São Paulo, SP, Brasil.Snake venom metalloproteinases (SVMPs) play important roles in the local and systemic hemorrhage observed upon envenomation. In a previous study on the structural elements important for the activities of HF3 (highly hemorrhagic, P-III-SVMP), bothropasin (hemorrhagic, P-III-SVMP) and BJ-PI (non-hemorrhagic, P-I-SVMP), from Bothrops jararaca, it was demonstrated that they differ in their proteolysis profile of plasma and extracellular matrix proteins. In this study, we evaluated the ability of proteins DM43 and a2- macroglobulin to interfere with the proteolytic activity of these SVMPs on fibrinogen and collagen VI and with their ability to induce hemorrhage. DM43 inhibited the proteolytic activity of bothropasin and BJ-PI but not that of HF3, and was not cleaved the three proteinases. On the other hand, a2-macroglobulin did not inhibit any of the proteinases and was rather cleaved by them. In agreement with these findings, binding analysis showed interaction of bothropasin and BJ-PI but not HF3 to DM43 while none of the proteinases bound to a2-macroglobulin. Moreover, DM43 promoted partial inhibition of the hemorrhagic activity of bothropasin but not that of HF3. Our results demonstrate that metalloproteinases of B. jararaca venom showing different domain composition, glycosylation level and hemorrhagic potency show variable susceptibilities to protein inhibitors

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process