An accurate and reproducible method to measure snake venom anticoagulant activity

Anticoagulant activity is an important clinical effect of human envenoming, seen in Australian black snake (Pseudechis) and cobra (Naja) envenoming. Anticoagulant toxins are not well characterised and anticoagulant assays not standardised. We aimed to develop and validate an accurate and standardised anticoagulant assay for snake venoms.

We used a turbidimetric assay previously developed to measure procoagulant activity and modified for anticoagulant activity. Recalcified fresh frozen plasma was mixed with varying concentrations of venom in a buffer for 5 s in a microplate well, and optical density was recorded for 340 nm every 10 s in a plate reader. Clotting time was defined as the time until a sharp increase occurred in optical density, which was normalised by dividing the venom clotting time by the plasma control clotting time (CT/CT_Pl). We investigated the effect of changing calcium concentration, the presence of a recombinant tissue factor (Innovin®), venom reconstitution, three different buffers, microplate type, and experimental timing.

Optimal conditions were a calcium concentration of 0.4 M, physiological buffer solution (PBS) and a regular microplate. Freshly reconstituted venom and immediately thawed plasma modestly improved the assay. Assay sensitivity improved without a recombinant tissue factor (Innovin®) trigger, particularly for weakly anticoagulant venoms. To reduce assay variability the assays were best done by selecting 4-6 venoms, and doing these on 4-6 days, with each venom at a different time each day; median coefficient of variation for all venoms <10%.

The anticoagulant assay had significantly reduced variability and improved sensitivity when conditions were standardised and a triggering agent was not used.