Abstract
Phospholipase A2 (PLA2) enzymes are important toxins found in many snake venoms, and they can exhibit a variety of toxic activities including causing hemolysis and/or anticoagulation. In this study, the inhibiting effects of the small molecule PLA2 inhibitor varespladib on snake venom PLA2s was investigated by nanofractionation analytics, which combined chromatography, mass spectrometry (MS), and bioassays. The venoms of the medically important snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus, Echis ocellatus, and Oxyuranus scutellatus were separated by liquid chromatography (LC) followed by nanofractionation and interrogation of the fractions by a coagulation assay and a PLA2 assay. Next, we assessed the ability of varespladib to inhibit the activity of enzymatic PLA2s and the coagulopathic toxicities induced by fractionated snake venom toxins, and identified these bioactive venom toxins and those inhibited by varespladib by using parallel recorded LC-MS data and proteomics analysis. We demonstrated here that varespladib was not only capable of inhibiting the PLA2 activities of hemotoxic snake venoms, but can also effectively neutralize the coagulopathic toxicities (most profoundly anticoagulation) induced by venom toxins. While varespladib effectively inhibited PLA2 toxins responsible for anticoagulant effects, we also found some evidence that this inhibitory molecule can partially abrogate procoagulant venom effects caused by different toxin families. These findings further emphasize the potential clinical utility of varespladib in mitigating the toxic effects of certain snakebites.
Highlights
Phospholipases A2 (PLA2 s) are key enzymes involved in many events in cellular signaling and act by cleaving ester bonds in phospholipids to generate fatty acids [1,2,3].They are pervasive in the mammalian pancreas and are highly abundant in many animal venoms [4,5].Venom Phospholipase A2 (PLA2) enzymes show a wide variety of functional activities, and can contribute to several distinct pathologies in envenomed prey/people, as well as potentially helping with prey digestion [4,5].They are recognized as the most thoroughly investigated venom toxins both in hemotoxic and neurotoxic snake venoms [6,7]
A recently developed analytical platform combining liquid chromatography (LC), mass spectrometry (MS), and PLA2 and coagulation activity bioassays was applied to evaluate the inhibitory properties of varespladib against the enzymatic PLA2 and coagulopathic activities of toxins found in the venoms of several medically important snake species
All venoms analyzed in this study showed constituents with clear coagulopathic toxicities, while only the venoms of B. asper, D. russelii, E. carinatus, and O. scutellatus displayed components with a clear enzymatic PLA2 activity
Summary
Phospholipases A2 (PLA2 s) are key enzymes involved in many events in cellular signaling and act by cleaving ester bonds in phospholipids to generate fatty acids (hydrolysis reactions) [1,2,3].They are pervasive in the mammalian pancreas and are highly abundant in many animal venoms [4,5].Venom PLA2 enzymes show a wide variety of functional activities, and can contribute to several distinct pathologies in envenomed prey/people, as well as potentially helping with prey digestion [4,5].They are recognized as the most thoroughly investigated venom toxins both in hemotoxic and neurotoxic snake venoms [6,7]. Snake venom PLA2 s are capable of contributing to presynaptic and/or postsynaptic neurotoxicity, myotoxicity, and cardiotoxicity, which can induce platelet aggregation disorders, hemolysis, anticoagulation, convulsions, hypotension, edema, and necrosis [4,5,8]. They play an important role in contributing to the morbidity and mortality of snakebite victims, via paralysis and destruction of respiratory muscle tissues, and/or due to their effect on homeostatic mechanisms involved in coagulation and oxygen transport [9]. Snakebite envenoming is a severe medical problem that was recently added to the World Health Organization (WHO) list of Neglected Tropical
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