Coagulotoxic Cobras: Clinical Implications of Strong Anticoagulant Actions of African Spitting Naja Venoms That Are Not Neutralised by Antivenom but Are by LY315920 (Varespladib)

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.

Keywords: LY315920; antivenom; coagulopathy; coagulotoxicity; cobra; snakebite; tissue damage; varespladib; venom.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Dose–response curves for thrombin inhibition. A comparison of clotting curves showing the relative inhibitory effects of the venom of 30 different species of elapid snakes on thrombin. x axis: venom concentration (µg/mL); y axis: clotting time in seconds. Negative control values were 43.4 ± 0.6 s. Data points are mean and standard deviations for N = 3. Note that for most data points, the error bars are smaller than the line icon. Note also that the y axis begins at 35 to improve viewability.

Figure 2

Dose–response curves for Factor Xa (FXa) inhibition. A comparison of clotting curves showing the relative inhibitory effects of the venom of 30 different species of elapid snakes on FXa. x axis: venom concentration (µg/mL); y axis: clotting time in seconds, with a machine maximum reading time of 999 s. Negative control values were 11.9 ± 0.1 s. Data points are mean and standard deviations for N = 3. Note that for most data points, the error bars are smaller than the line icon.

Figure 3

Ancestral state reconstruction of thrombin and FXa inhibition. A reconstruction of the ancestral state of thrombin and FXa inhibition, based on AUC (area under the curve) values derived from dose–response curves for thrombin and FXa inhibition by venoms. Warmer colours represent larger AUCs (greater anticoagulant venom potency). The two clades which have independently evolved hooding defensive displays are indicated with light brown vertical bards, while the three lineages which have evolved defensive spitting are indicated with dark brown vertical bars [16]. Phylogeny based upon [17,18].

Figure 4

Dose–response curves for LY315920 (varespladib) and SAIMR antivenom efficacy. Comparison of clotting curves showing the relative inhibitory effects of venom of the four most potent anticoagulant African spitting Naja species, with and without the addition of antivenom or LY315920. x axis: venom concentration (µg/mL); y axis: clotting time in seconds. Thrombin negative control values were 43.4 ± 0.6 s. FXa negative control values were 11.9 ± 0.1 s. Data points are mean and standard deviations for N = 3. Note that for most data points, the error bars are smaller than the line icon. Also, the venom + antivenom blue line is not visible in some doses/species due to the lack of effect resulting in an identical line to that of the venom-only red line. To improve viewability, the y axis of thrombin inhibition begins at 35.