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.