5.3.2. Procoagulant Activity

The procoagulant activity was evaluated in recalcified chicken (White leghorn) plasma, obtained under license CEUAIB n◦ 13,710-14, Instituto Butantan, by thromboelastometry using a four-channel ROTEM® system (Pentapharm, Munich, Germany), as previously described [26]. The plasma was prepared as 3.2% citrated stock and stored at −80 ◦C until needed in 1 mL aliquots. For the experiments, an aliquot was defrosted by placing it into a 37 ◦C water bath for 10 min. Venom samples and controls were dissolved in PBS (60 μL) and added in the specific cups with 20 μL of CaCl2 (0.02 M) and 260 μL of citrated plasma (final volume = 340 μL), pipette-mixed, and the clotting time was immediately recorded. PBS and ellagic acid were used as negative and positive controls, respectively. Under such conditions, recalcified chicken plasma treated with PBS only is unclottable for at least 30 min (CT value = 1800 s). The results are shown as Coagulant Dose 50% (DC50), defined as the amount of venom (μg) that reduces the CT parameter to 900 s.
