Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme fromLachesis muta rhombeataSnake Venom

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).

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity

“…Every enzymatic assay performed was positive (Table 2 ), with high activity detected with azocasein substrate (566.53 ± 14.15 U/mg) when compared to others SVMPs 94 , 95 . The activity towards the BApNA substrate (0.17 ± 0,012 nmol/min) was lower compared to the Rhombeobin serine proteinase 96 . The lower activity found on BApNA substrate could be explained by the cleavage site specificity of the MMPs which require the presence of at least one hydrophobic amino acid, especially Leucine at the position P1′ 75 , 97 , 98 .…”

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase

“…In fact, this class is mainly reported in snakes from Crotalinae subfamily, which comprises the genus Lachesis among others [58]. TLEs have been already isolated from L. muta venoms [16, 59-61], and two are TLEs from LmrV [23, 60]. The first one was isolated in 1981 when L. m. rhombeata was designated as L. m. noctivaga [60, 62] and LMR-47 (sp|Q9PRP4) [23] was the only serine proteinase from LmrV whose sequence was available at UniProt so far.…”

“…SVSPs with plasminogen-activating or kallikrein-like activity from the genus Lachesis present molecular mass within the range of 27.9 to 33 kDa estimated by SDS-PAGE [14, 24, 67, 72]. On the other hand, Lachesis TLEs are usually larger proteins of more than 40 kDa [16, 23, 73]. However, after deglycosylation reaction, all of them present 27-28 kDa [67, 72, 73].…”

“…However, a more specific assay is needed to confirm this hypothesis. TLEs from Lachesis usually release fibrinopeptide A but some of them also slowly split off fibrinopeptide B from fibrinogen [16, 23, 61, 73]. During the envenoming process, thrombin-like serine proteinases act on fibrinogen converting that in a different form of fibrin which does not form a solid fibrin clot.…”

“…However, there is a certain difficulty in maintain Lachesis snakes in captivity due to the peculiar characteristics of their natural habitat [22] and this hinders the study of their venoms. Few components have been isolated from LmrV, including LAAO [21], PLA 2 [19], serine proteinases [23-25] and hyaluronidase [13]. Hyaluronidase was the last isolated component to be reported about 3 years ago.…”