Tiny but Mighty: Vipera ammodytes meridionalis (Eastern Long-Nosed Viper) Ontogenetic Venom Variations in Procoagulant Potency and the Impact on Antivenom Efficacies

Abstract

The Eastern Long-Nosed Viper (Vipera ammodytes meridionalis) is considered one of the most venomous snakes in Europe. However, it is unknown whether ontogenetic variation in venom effects occurs in this subspecies and how this may impact antivenom efficacy. In this study, we compared the procoagulant activities of V. a. meridionalis venom on human plasma between neonate and adult venom phenotypes. We also examined the efficacy of three antivenoms-Viperfav, ViperaTAb, and Inoserp Europe-across our neonate and adult venom samples. While both neonate and adult V. a. meridionalis venoms produced procoagulant effects, the effects produced by neonate venom were more potent. Consistent with this, neonate venom was a stronger activator of blood-clotting zymogens, converting them into their active forms, with a rank order of Factor X >> Factor VII > Factor XII. Conversely, the less potent adult venom had a rank order of FXII marginally more activated than Factor VII, and both much more so than Factor X. This adds to the growing body of evidence that activation of factors besides FII (prothrombin) and FX are significant variables in reptile venom-induced coagulopathy. Although all three examined antivenoms displayed effective neutralization of both neonate and adult V. a. meridionalis venoms, they generally showed higher efficacy on adult venom than on neonate venom. The ranking of antivenom efficacy against neonate venom, from the most effective to the least effective, were Viperfav, Inoserp Europe, ViperaTAb; for adult venom, the ranking was Inoserp Europe, Viperfav, ViperaTAb. Our data reveal ontogenetic variation in V. a meridionalis, but this difference may not be of clinical concern as antivenom was effective at neutralizing both adult and neonate venom phenotypes. Regardless, our results highlight a previously undocumented ontogenetic shift, likely driven by the documented difference in prey preference observed for this species across age classes.

Keywords: Echis; antivenom; coagulopathy; factor activation; small-molecule enzyme inhibitor.

Figure 1

Thromboelastography using human plasma (1800 s total run time). Blue traces = spontaneous clot control (negative control), green traces = thrombin control, and red traces = venom samples. All traces are overlaid with the spontaneous clot control. SP = the split point, the time in seconds until clot formation begins. R = reaction time, the time in seconds until a detectable clot (>2 mm) is formed. A = amplitude, the width of tracing at the latest time point, representing clot strength (mm). Data are n = 4 mean ± standard deviation. Thrombin control is at a concentration of 1.94 NIH units/mL. Venom samples are at a concentration of 19.44 μg/mL.

Figure 2

Logarithmic views of (A) adult and (B) neonate venom and antivenom plasma clotting dose-response curves (0.05, 0.125, 0.25, 0.66, 1.66, 4, 10, and 20 μg/mL). (C) Relative shifts in the area under the curve (AUC) for the venom and antivenom plasma clotting dose-response curves. No antivenom effect = 0%. p-values are comparisons between neonate and adult venoms within the same antivenom type, comparisons between antivenom types for neonate venom, and comparisons between antivenom types for adults. p-values classifications are as follows: ns = not significant (0.62 in this case). Statistics are Brown–Forsythe and Welch ANOVA tests with post-hoc Dunnett’s T3 multiple comparisons. All data are n = 3 ± standard deviation.

Figure 3

Adult and neonate relative ability to convert clotting factor zymogens into their corresponding activated enzyme. p-values are comparisons between neonate and adult venoms within the same factor type, comparisons between factor types for neonate venom, and comparisons between factor types for adults. p-values classifications are as follows: **** = p ≤ 0.0001. Statistics are Brown–Forsythe and Welch ANOVA tests with post-hoc Dunnett’s T3 multiple comparisons. Data are n = 3 mean ± standard deviation.