Profiling elapid venoms for coagulation and paralysis modulators by liquid chromatography with post-column in vitro/in vivo bioassays and mass spectrometry

Abstract This doctoral research presents an integrated analytical framework that combines chemical profiling with both in vitro and in vivo biological assays to advance the characterization of toxins in elapid snake venoms. The work focuses on two major pathological outcomes of snakebite envenoming—coagulopathy and neurotoxicity—by identifying the specific venom components responsible for these effects and assessing the potential of small-molecule inhibitors as therapeutic candidates. Central to this thesis is the application of nanofractionation analytics, which couples high-resolution chromatographic separation with parallel mass spectrometric analysis and post-column bioassays. The first part of the research investigates coagulopathic toxins in medically important elapid venoms. Using coagulation bioassays in combination with nanofractionation, toxins modulating blood clotting were identified, and their susceptibility to inhibition by varespladib, a phospholipase A₂ (PLA₂) inhibitor, and marimastat, a metalloproteinase inhibitor, was systematically evaluated. This approach enabled detailed mapping of venom-induced coagulopathy and provided evidence supporting the potential of varespladib as a supplementary treatment for elapid snakebite. The second part integrates in vitro targeted ion-channel assays with in vivo zebrafish embryo assays to examine venom-induced paralysis and lethality. Fractionated toxins were screened for α7 nicotinic acetylcholine receptor modulation and correlated with behavioural toxicity profiles in zebrafish. Parallel mass spectrometric and proteomics analyses allowed direct linkage between toxin identity, ion-channel activity, and in vivo effects. Collectively, this research demonstrates the effectiveness of nanofractionation analytics for resolving complex venom mixtures and correlating chemical identity with biological function. The findings improve understanding of elapid venom pathophysiology and highlight the promise of small-molecule inhibitors, particularly varespladib, as candidates for early-stage snakebite intervention. Future directions include refinement of toxin separation strategies, increased automation of proteomics workflows, and broader adoption of complementary mammalian models to support translational venom research.