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

Toxins

Mark C. Wilkinson¹  Nicholas R. Casewell¹  Taline D. Kazandjian¹  Arif Arrahman²  Govert W. Somsen²  Kristina B.M. Still²  Jeroen Kool²  Freek J. Vonk³ 

[1] Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK;Department of Chemistry and Pharmaceutical Sciences, Division of Bioanalytical Chemistry, Faculty of Sciences, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands;Naturalis Biodiversity Center, Darwinweg 2, 2333CR Leiden, The Netherlands;

关键词: snakebite;    Naja nigricollis;    coagulopathy;    varespladib;    marimastat;    nanofractionation;   

DOI  :  10.3390/toxins13050302

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown