Calibrated automated thrombogram
Thrombin generation kinetics was measured with the Calibrated Automated Thrombogram (CAT) (Thrombinoscope BV, Maastricht, the Netherlands) according to the manufacturer’s instructions in the 96-well plate fluorometer (Ascent Reader, Thermolabsystems OY, Helsinki, Finland) equipped with the 390/460 filter set at a temperature of 37°C14. Briefly, 80 μl of platelet-poor plasma was diluted with 20 μl of the reagent containing 5 pmol/l recombinant tissue factor (TF), 4 μmol/l phosphatidylserine/phosphatidylcholine/
phosphatidylethanolamine vesicles, and 20 μl of FluCa solution (Hepes, pH 7.35, 100 mmol/l CaCl2, 60 mg/ml bovine albumin, and 2.5 mmol/l Z-Gly-Gly-Arg-amido methyl coumarin). Each plasma sample was analyzed in duplicate, and the intraassay variability was 7%. The maximum concentration of thrombin formed during the recording time is described as the ‘thrombin peak’ and the area under the curve represents ETP.