Rapid purification and procoagulant and platelet aggregating activities of Rhombeobin: a thrombin-like/gyroxin-like enzyme from Lachesis muta rhombeata snake venom.

Frank Denis Torres-Huaco,Sérgio Marangoni,Ana Cláudia Coelho Nery-Diez,Cristina Pontes Vicente,Edson Antunes,Cláudio C Werneck,Talita Vassequi-Silva,Camila B Mendes,Daniela C S Damico

doi:10.1155/2013/903292

Frank Denis Torres-Huaco, Sérgio Marangoni + Show 7 more

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https://doi.org/10.1155/2013/903292

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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).

Highlights

Snakes of the genus Lachesis, commonly known as bushmasters, or surucucu in Brazil, can be found in the tropical forests of Central and South Americas
Lachesis m. rhombeata crude venom shows lower lethal and hemorrhagic activities when compared with L. m. muta venom, while both venoms induce similar edema-forming and myotoxic activities
Rechromatography of F-16 fraction in an RP-HPLC, with a linear gradient, results in a single symmetric peak eluted with 57% of buffer B and a retention time of 36.23 ± 2.2 min (Figure 1(b)); SDS-PAGE showed that this fraction in nonreduced and reduced conditions is a single-chain protein, with a relative molecular mass of 45 kDa (Figure 1(b): insert)

Summary

Introduction

Snakes of the genus Lachesis, commonly known as bushmasters, or surucucu in Brazil, can be found in the tropical forests of Central and South Americas. There are three different Lachesis species: L. stenophrys, L. melanocephala (both found in Central America), and L. muta. Poisoning by Lachesis is characterized by local damage, pain, edema, hemorrhage, and myonecrosis, as well as systemic complications, such as coagulation disorders, hemolysis, neurotoxicity, renal failure, diarrhea, hypotension, and bradycardia, among others [2,3,4,5]. Rhombeata crude venom shows lower lethal and hemorrhagic activities when compared with L. m. Muta venom, while both venoms induce similar edema-forming and myotoxic activities. Rhombeata venom exhibits higher coagulant and defibrinogenating effects [6] L. m. rhombeata venom exhibits higher coagulant and defibrinogenating effects [6]

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