Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors

Abstract

Palearctic vipers are medically significant snakes in the genera Daboia, Macrovipera, Montivipera, and Vipera which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of in vitro coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes (Daboia genus, Macrovipera genus, and Vipera ammodytes uniquely within the Vipera genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the Montivipera genus and Vipera latastei uniquely within the Vipera genus) were each accompanied by a shift away from procoagulant action, with the Montivipera species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of Vipera species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing V. berus but, reflective of this being a monovalent antivenom, it was not effective against other Vipera species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation.

Keywords: antivenom; coagulopathy; enzyme inhibition; snakebite; venom.

Figure 1

Phylogenetic relationships of Palearctic vipers (14). Green indicates species included in this study.

Figure 2

Sequence alignment of representative Factor X activating P-IIId SVMP characteristic of the Palearctic viper clade. Highlighted is the diagnostic cysteine that forms the interchain disulfide bond to a covalently linked lectin dimer. Uniprot (Daboia and Macrovipera) and Genbank (Vipera) accession codes are given for each sequence.

Figure 3

Clotting times on human plasma produced by Vipera venoms (20 µg/ml).

Figure 4

8-point concentration curves, x-axis showing concentrations of venom in μg/ml and y-axis showing clotting times in seconds of human plasma with venom and relative antivenom efficacy. For each species, linear graphs are presented on the left and logarithmic views on the right. Shown are: venom-induced clotting times (red curves); venom-induced clotting times after preincubation with Inoserp AV (final concentration 0.5%; spontaneous control- 420.2 +/27.7) (blue curves), ViperaTAb AV (final concentration 0.4%; spontaneous control- 478.4 +/33.3) (purple curves), or VIPERFAV AV (final concentration 0.2%; spontaneous control- 509.7 +/13.5) (green curves). Values are mean ± SD of N = 3 and shown as dots with error bars. Some error bars are too small to see.

Figure 5

X-fold magnitude of shift of plasma clotting time due to incubation of antivenoms indicated by: Inoserp in blue bars, ViperaTAb in purple bars; and VIPERFAV in green bars. X-fold magnitude of shift was calculated by the formula [(AUC of antivenom + venom/AUC of venom) -1]. A value of 0 indicates no shift (no neutralization by antivenom), while a value above 0 indicates neutralization by antivenom. Values are mean ± SD of N = 3.

Figure 6

(A) 8-point concentration curves, x-axis showing concentrations of venom in μg/ml and y-axis showing clotting times in seconds of human plasma with venom and relative inhibitor efficacy. For each species, linear graphs are presented on the left and logarithmic views on the right. Shown are venom-induced clotting times (red curves), effect of venoms after preincubation with prinomastat (final concentration 0.2 mM%; spontaneous control- 484.8 +/- 11.0) (blue curves), and effect of venoms after preincubation with DMPS (final concentration 0.2 mM %; spontaneous control- 425.8 +/3.3) (green curves). Values are mean ± SD of N = 3 and shown as dots with error bars. Some error bars are too small to see and the failure of DMPS to shift the curves results in an identical line to the red (venom only) curves. (B) Bar graphs of X-fold magnitude of shift of plasma clotting time due to induction of prinomastat. X-fold magnitude of shift was calculated by the formula [(AUC of inhibitor + venom/AUC of venom) -1]. A value of 0 is no shift (no neutralization by inhibitor), while a value above 0 indicates neutralization by inhibitor. Values are mean ± SD of N = 3.

Figure 7

Overlaid thromboelastography traces of spontaneous clotting negative control (blue), Factor X and thrombin positive controls (green) and venom-induced clotting experimental condition (red). Parameters: SP = the split point (time till clot formation begins) (sec); R = time until detectable clot (2 mm +) is formed (sec); MA = maximum amplitude of clot (mm); MRTG = maximum rate of thrombus generation (dynes/cm2/s); TMRTG = time to maximum rate of thrombus generation (min); and TGG = total thrombus generated (dynes/cm2). Values are mean ± SD of N = 3.

Figure 8

Ability of venoms to activate Factor X (left) compared to prothrombin (right). Results are presented relative to the control of the same amount of the corresponding activated enzyme form (note difference in y-axis scales between the two graph sets). Data points are N = 3 mean ± SD.

Figure 9

Shift of clotting time relative to control due to inhibition of Factor Xa (no effect relative to control would be a value of 0). Data points are N = 3 mean ± SD.