Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus)

Abstract

Background: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear.

Objectives: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom.

Methods: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured.

Results: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation.

Conclusions: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.

Fig 1. Examples of clinical findings in envenomated dogs.

Facial swelling at presentation (T1) (A), extensive facial swelling 36 hours after bite (T4) (B), subcutaneous extravasation of blood 24 hours after bite (T3) (C) and continued bleeding from the bite wound in the same individual, 24 hours after bite (T3) (D).

Fig 2. Thrombin generation parameters in envenomated dogs and controls.

Lag time, peak thrombin and endogenous thrombin potential (ETP) measured by calibrated automated thrombography with three reagents, PPP low, PRP and no exogenous reagent, in envenomated dogs at five timepoints after bite (black dots) and healthy controls (green dots). T1 = presentation, T2 = 12 hours, T3 = 24 hours, T4 = 36 hours and T5 = 15 days after bite. * indicates P < 0.05 for comparisons between envenomated dogs at each time point and the control group. indicates a significant change between timepoints for envenomated dogs (P < 0.05).

Fig 3. Lag time in dogs treated with and without antivenom.

Box and Whisker plots for lag time with three different reagents (PPP low (A), PRP (B) and no exogenous reagent (C)), in dogs treated with (red) and without (blue) antivenom, and healthy controls (green). T1 = presentation, T2 = 12 hours, T3 = 24 hours, T4 = 36 hours and T5 = 15 days after bite.* indicates a significant difference (P < 0 .05) between treatment groups at a given timepoint. Statistical analysis was not performed at T1 due to a low number of antivenom treated dogs (n = 2). N/A = not analysed.

Fig 4. Phosphatidylserine equivalents in envenomated dogs and controls.

Phosphatidylserine (PS) equivalents in envenomated dogs at five timepoints after bite (black dots) and controls (green dots). T1 = presentation, T2 = 12 hours, T3 = 24 hours, T4 = 36 hours and T5 = 15 days after bite.* indicates P < 0.05 for comparisons between envenomated dogs at each time point and the control group. indicates a significant change between timepoints for envenomated dogs (P < 0.05).

Fig 5. Thrombin-antithrombin complexes in envenomated dogs and controls.

Concentrations of thrombin-antithrombin (TAT) complexes in envenomated dogs at five timepoints after bite (black dots) and controls (green dots). T1 = presentation, T2 = 12 hours, T3 = 24 hours, T4 = 36 hours and T5 = 15 days after bite.* indicates P < 0.05 for comparisons between envenomated dogs at each time point and the control group. indicates a significant change between timepoints for envenomated dogs (P < 0.05).