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Rapid purification, pro-coagulant and platelet aggregating activities of Rhombeobin (LMR-47), a Thrombin-like/gyroxin enzyme from Lachesis muta rhombeata snake venom

Grant number: 13/14706-2
Support type:Regular Research Grants - Publications - Scientific article
Duration: August 01, 2013 - January 31, 2014
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Sergio Marangoni
Grantee:Sergio Marangoni
Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

In this article we report a rapid purification method using one step chromatography of SVSP Rombeobin (LMR-47) from Lachesis muta rhombeata venom and its pro-coagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C-8 Discovery® Bio Wide column, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BApNA, with a broad optimum pH (7-10) and was stable in solution up to 60 oC. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents; but not for chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with with other SPSV, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terificus). Rhombeobin acts, in vitro, as a strong pro-coagulant enzyme on mice citrated plasma, shortening the APTT and PT test in dose-dependent manner. The protein showed, "ex vivo", a strong defibrigenating effect with 1¼g/animal. At lower doses activated the intrinsic and extrinsic coagulation pathway and impair the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that producing a Venom-induced consumptive coagulopathy (VICC). (AU)