Erythrocyte haemotoxicity profiling of snake venom toxins after nanofractionation

Chunfang Xie, Matyas A. Bittenbinder, Julien Slagboom, Arif Arrahman, Sven Bruijns, Govert W. Somsen, Freek J. Vonk, Nicholas R. Casewell, Juan J. García-Vallejo, Jeroen Kool*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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 haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput haemolytic 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 haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic 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 haemolytic venom toxins identified were mainly phospholipase A2s 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 neutralisation of venom cytotoxins.

Original languageEnglish
Article number122586
Pages (from-to)1-11
Number of pages11
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Early online date16 Feb 2021
Publication statusPublished - 30 Jun 2021

Bibliographical note

Funding Information:
C.X. was funded by a China Scholarship Council (CSC) fellowship (201706250035). N.R.C. acknowledges support from a UK Medical Research Council (MRC) Research Grant (MR/S00016X/1) and Confidence in Concept Award (CiC19017), and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and Royal Society. The APC was funded by the Wellcome Trust.

Publisher Copyright:
© 2021 The Author(s)

Copyright 2021 Elsevier B.V., All rights reserved.


  • Erythrocytes haemolysis assay
  • Haemolytic toxins
  • Nanofractionation analytics
  • Proteomics analysis
  • Snakebite
  • Venom


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