Introduction
Crotalidae (pit viper) envenomation produces a consumptive coagulopathy, most commonly treated with Crotalidae polyvalent immune antibody fragments (CroFab, BTG International). Unfortunately, certain species of Crotalidae, including the timber rattlesnake (Crotalus horridus horridus), have demonstrated thrombocytopenia refractory to the administration of this antivenom.
Objective
No current data have sufficiently explained the refractory thrombocytopenia produced by C horridus horridus (CH) venom. The purpose of this research was to investigate the cause of venom-induced thrombocytopenia with the goal of developing an effective treatment method for this entity.
Methods
Whole blood from a healthy man was collected in 3.2% sodium citrate. CH venom (Type A neurotoxin; Kentucky Reptile Zoo, Slade, KY) was serially diluted and mixed with 0.5 mL whole blood ± CroFab antivenom. Platelets were harvested to determine the effect of snake venom on platelet aggregation. Platelet aggregation studies were performed using a Chronolog Aggregometer. Prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR), and fibrinogen also were monitored.
Results
Ristocetin-induced platelet aggregation was exacerbated in the presence of increasing concentrations of venom. CH venom alone induced aggregation, and this effect was inhibited when venom was incubated with CroFab antivenom. PT, PTT, INR, and fibrinogen levels were within normal range when incubated with <200 mcg/mL CH venom. Venom concentrations >200 µg/mL caused significant clotting, excluding these samples for analysis. Antivenom alone did not affect platelet aggregation.
Conclusion
Platelet aggregation induced by CH Type A neurotoxin venom appears to be reversible by increasing concentrations of Crotalidae polyvalent immune antibody fragments in vitro. These data suggest that under in vitro conditions the venom components inducing platelet aggregation are concentration dependent and mimic ristocetin-induced platelet aggregation. Venom components may out compete Von Willebrand factor binding and have stronger affinity. This assay provides an in vitro model to study venom components associated with in vivo thrombocytopenia and the neutralization by antivenom.
Funding
WMS Research-in-Training Grant (2010).