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BioMed Research International
Volume 2013 (2013), Article ID 903292, 12 pages
http://dx.doi.org/10.1155/2013/903292
Research Article

Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme from Lachesis muta rhombeata Snake Venom

1Department of Biochemistry, Institute of Biology, University of Campinas (UNICAMP), P.O. Box 6109, 13083-970 Campinas, SP, Brazil
2Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), P.O. Box 6109, 13083-865 Campinas, SP, Brazil
3Department of Pharmacology, State University of Campinas (UNICAMP), P.O. Box 6109, 13083-887 Campinas, SP, Brazil

Received 4 June 2013; Accepted 29 June 2013

Academic Editor: Saulo Luís da Silva

Copyright © 2013 Frank Denis Torres-Huaco et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).