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

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.

The Relative Efficacy of Chemically Diverse Small-Molecule Enzyme-Inhibitors Against Anticoagulant Activities of African Spitting Cobra (Naja Species) Venoms

“…This study is congruent with prior work, demonstrating the efficacy of varespladib against a wide range of snake venom PLA 2 -driven pathophysiological actions (20,26,30,(32)(33)(34)(43)(44)(45)(46)(47)(48), thereby underscoring the potential utility of this compound as a first-aid option. For instance, field treatment options such as varespladib could stabilize patients in remote locations while being transported to hospital, and or serve as the only treatment option for some snake species such as the potent FXa-inhibiting anticoagulant effect of the African spitting cobra venoms included in this study (18,24,26,(49)(50)(51).…”

“…We also ascertained the ability of prinomastat to cross-react with the anticoagulant PLA 2 of these medically important snakes. In addition to undertaking the first anticoagulant toxicity testing for N. ashei, N. katiensis, and N. nubiae venoms, this work builds upon increasingly popular research into non-immunoglobulin treatments for snakebites using temperature-stable small molecule inhibitors (SMIs) that may improve snakebite firstaid in remote areas (20,26,(30)(31)(32)(33)(34). Therefore, our results hopefully inform and streamline future platform testing while minimizing the need for in vivo animal testing.…”

“…Local effects include pain, blistering, swelling, and tissue death, while systemic effects include anticoagulant coagulopathy and flaccid paralysis (8,14,(16)(17)(18)(19). The diversity of effects are reflective of the complex composition of these venoms, which include threefinger toxins with cytotoxic and neurotoxic effects, anticoagulant phospholipase A 2 toxins that inhibit Factor Xa and thrombin, and metalloproteases that cause edema and degrade fibrinogen (20)(21)(22)(23)(24)(25)(26).…”

“…While the mechanism of action for inhibiting thrombin has not been elucidated, the ability to inhibit FXa is well-described. Rather than inhibiting FXa active site directly, the toxins compete with Factor Va for binding to FXa at a site distinct from the enzymatic pocket (20,(26)(27)(28)(29). These FXa-inhibiting toxins have been shown to unaffected by the SAVP polyvalent antivenom (which is made using the venoms of Bitis arietans, Bitis gabonica, Dendroaspis angusticeps, Dendroaspis jamesoni, Dendroaspis polylepis, Haemachatus haemachatus, Naja nivea, Naja melanoleuca, Naja annulifera, and Naja mossambica) but have been shown to be well neutralized by the PLA 2 inhibitor varespladib (26).…”

In vitro and in vivo venom-inhibition assays identify metalloproteinase-inhibiting drugs as potential treatments for snakebite envenoming by Dispholidus typus

“…A previous study observed a much clearer negative anticoagulant peak with D. typus venom, though the venom was applied at a five-fold higher concentration (5.0 mg/mL venom) than that used in this study (1.0 mg/mL venom) 29 . While varespladib has previously been demonstrated to be a potent inhibitor of anticoagulant venom activities induced by PLA2 toxins 33,[40][41][42] , in this study it produced no inhibitory effects on venom-induced coagulation, whether procoagulant or anticoagulant, at the maximal drug dose tested (20 μM) (Supplemental File S2). However, due to the weak anticoagulant venom activity observed in these experiments, the assay window for measuring such inhibition is limited.…”