Erythrocyte haemotoxicity profiling of snake venom toxins after nanofractionation

Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including hemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, hemolytic activity can greatly debilitate victims and contributes to systemic hemotoxicity. Current assays designed for studying hemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput hemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving hemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for hemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The hemolytic venom toxins identified were mainly phospholipases A2 and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralization of venom cytotoxins.