| Summary: | 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 (<i>Naja nigricollis</i>) 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 <i>N. nigricollis</i> venom toxins as well as PLA<sub>2</sub>s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A<sub>2</sub> 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 <i>N. nigricollis</i> venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA<sub>2</sub> proteins. We, therefore, conclude that PLA<sub>2</sub>s are the most likely candidates responsible for anticoagulant effects stimulated by <i>N. nigricollis</i> venom.
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