Plasma-Based Thrombin Generation Assays

Thrombin generation assays were performed in 96-well microtiter plates at 37°C as previously described ^{22 24 28} using final concentrations of 3 pM tissue factor (HemosIL HS PLUS), 4 µM PCPS, 0.5 mM Z-Gly-Gly-Arg-AMC, and 7.5 mM CaCl ₂ , without added corn trypsin inhibitor. Studies were done in the absence or presence of rivaroxaban or apixaban at concentrations up to 2,300 and 2,120 µM (1,000 ng/mL), respectively. Assays were calibrated using Technothrombin TGA thrombin calibrators (Technoclone) according to the manufacturer's instructions with high and low thrombin concentration controls included in each run. The reactions were monitored over time using SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, California, United States) at excitation and emission at 360 and 460 nm, respectively, with the cutoff filter at 455 nm. All samples and controls were tested in quadruplicate.