Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom.

Pamella G Gutierres,Edward G Rowan,Rafael S Floriano,Lilian F Arantes,Rosa M B Nogueira,Nelson J Silva,Kristian A Torres-Bonilla,Diego R Pereira,Karen Morais-Zani,Stephen Hyslop,Nataly L Vieira

doi:10.3389/fphar.2021.812295

Abstract

Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2 in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001–1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 ‘v/w’) in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

Highlights

Snakes of Lachesis genus are represented by three species found in Central America (L. stenophrys, L. melanocephala, and L. acrochorda) and one in South America (L. muta), with the latter being recognized as two subspecies distributed in the Amazon river basin ( L. muta muta) and Atlantic rainforest ( L. muta rhombeata) in Brazil (Costa and Bérnils, 2018; Nogueira et al, 2019; Diniz-Sousa et al, 2020)
MO, United States) and antiBothrops/Lachesis serum was from Butantan Institute (São Paulo, SP, Brazil); varespladib was dissolved in DMSO prior to use, whereas the antivenom was provided ready for injection and maintained under refrigeration
L. m. rhombeata (1 mg/ml) exhibited moderate enzymatic activity for phospholipases A2 (PLA2) which was promptly abolished from 0.01 mM of varespladib; the lowest concentration of varespladib (0.001 mM) did not produce inhibitory effect on the PLA2 activity of this venom (Figure 1A)

Summary

Introduction

Snakes of Lachesis genus are represented by three species found in Central America (L. stenophrys, L. melanocephala, and L. acrochorda) and one in South America (L. muta), with the latter being recognized as two subspecies distributed in the Amazon river basin ( L. muta muta) and Atlantic rainforest ( L. muta rhombeata) in Brazil (Costa and Bérnils, 2018; Nogueira et al, 2019; Diniz-Sousa et al, 2020). Envenomation by Lachesis spp. is characterized by intense local pain, oedema and necrosis (Damico et al, 2006; Ferreira et al, 2009; Damico et al, 2012), systemic myotoxicity (Fuly et al, 2000; Fuly et al, 2003; Damico et al, 2006), renal failure (Damico et al, 2007), haemorrhage and coagulopathy (Sánchez et al, 1987; Sánchez et al, 1991; Sánchez et al, 1995; Fuly et al, 1997; Rucavado et al, 1999; Estêvão-Costa et al, 2000; Torres-Huaco et al, 2013), including severe cardiovascular disorders (Diniz and Oliveira, 1992; Giovanni-De-Simone et al, 1997; Dias et al, 2016a; Dias et al, 2016b) Such effects have been associated predominantly with the presence of phospholipases A2 (PLA2) (Cordeiro et al, 2015; DinizSousa et al, 2018), metalloproteases (Cordeiro et al, 2018) and serine proteases (Wiezel et al, 2019), including biologically active peptides (Graham et al, 2005; Soares et al, 2005; Sanz et al, 2008; Pla et al, 2013; Pinheiro-Júnior et al, 2018), in these venoms.

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