Abstract
Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A2 inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja. As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins.
Highlights
Envenomation as a result of snakebites is an important public health issue, mainly in the tropical and subtropical countries of the developing world
Testing of LY315920 at the same concentration previously shown to be effective for other types of venom toxicity [20] was shown in Consistent with clinical observations of antivenom ineffectiveness [19], the antivenom efficacy testing in this study revealed an inability of the South African Institute for Medical Research antivenom to neutralise both Factor Xa (FXa) and thrombin inhibition activity by venoms (Figure 4)
This study revealed for the first time that thrombin inhibition and inhibition of prothrombinase complex formation by competitive binding to FXa, preventing Factor Va (FVa)’s binding to FXa, are shared features of the Hemachatus/Naja clade but are potent only in the African spitting Naja species, with
Summary
Envenomation as a result of snakebites is an important public health issue, mainly in the tropical and subtropical countries of the developing world. Inhibitors of the enzymatic activities of FXa and thrombin have been isolated from the non-spitting African cobra N. haje [10]. Our results demonstrate evolutionary patterns underlying anticoagulant activity within elapid species and have implications for the clinical treatment of elapid snakebites. 2. ResultsOur results demonstrate evolutionary patterns underlying anticoagulant activity within elapid species and have implications for the clinical treatment of elapid snakebites. A series of anticoagulant assays were performed on all venoms, including 8-point dose–response curves, to2.investigate the inhibition of thrombin’s ability to clot human fibrinogen (Figure 1) and on Results the impediment of Factor Xa’s ability to clot recalcified plasma by forming a prothrombinase complex. The four species within the clade of African spitting cobras
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