Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib.

Taline D Kazandjian,Jeroen Kool,Freek J Vonk,Mark C Wilkinson,Nicholas R Casewell,Kristina B M Still,Govert W Somsen,Arif Arrahman

doi:10.3390/toxins13050302

Abstract

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

Highlights

Snakebite has been defined by the World Health Organization (WHO) as a neglected tropical disease since June 2017
To analyse the effects of N. nigricollis venom on blood coagulation, crude venom was applied to citrated bovine plasma in a 384-well plate bioassay
Our findings demonstrate that Naja nigricollis venom displays considerable anticoagulant activity, as shown by coagulation assays utilising bovine plasma, and that despite 3FTxs being the dominant constituents of this venom, this anticoagulant activity observed is caused by PLA2 toxins, as exhibited through testing of N. nigricollis toxins isolated by nanofractionation or standard chromatographic means

Summary

Introduction

Snakebite has been defined by the World Health Organization (WHO) as a neglected tropical disease since June 2017. Elapids (Elapidae: including cobras, mambas, and related species) are a venomous, front-fanged family of snakes responsible for many snakebite cases in Africa. The black-necked spitting cobra, Naja nigricollis, is a medically-important species that inhabits a wide range across the central belt of Africa [14], and is thought to be responsible for the majority of elapid bites in many of these areas [15,16,17]. Bites from this species have been shown to result in local swelling and tissue necrosis, bleeding, and blistering [11,13]. In vivo studies show that, when injected intradermally, the venom causes oedema, blistering and necrotic lesions in a mouse model [18], and exhibits strong cytotoxic effects on the myogenic cell line C2C12 [19]

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