2.4. PPP‐TG measurement with the CAT assay

TG in PPP was measured with the CAT technique on a Fluoroskan Microplate Fluorometer (Thermolabsystems OY) as originally described.9 The final concentration of TF was 5 pm with 4 µmol/L PL in the absence or presence of 10 nmol/L TM. The concentration of TM was chosen to inhibit TG by 50% in normal pooled plasma (NPP). TG parameters were calculated with Thrombinoscope software version 5.0, and TG parameters including the lagtime, TTP, peak, and ETP were chosen for further analyses.

In each run of TG measurement, NPP was also measured on the same plate. The ETP and peak values of the study subjects were normalized as the percentage of the ETP and peak of the NPP tested without TM in the same run, respectively. For better standardization and comparison, the PPP‐TG parameters of samples measured in the presence of TM, including peak™⁺ and ETP™⁺, were also normalized as the percentage of the ETP and peak of the NPP tested without TM in the same run. The preparation of the NPP has been described previously.20 Blood from 116 healthy adult volunteers who gave written consent and did not take any anticoagulant or antiplatelet drugs for at least 2 weeks before blood draw was collected at Maastricht University Medical Center. After an initial centrifugation step (2500 g, 5 minutes) plasma was pooled, followed by ultracentrifugation (100 000 g, 10 minutes). Aliquots of 500 µL were snap‐frozen in liquid nitrogen and stored at −80°C until analysis.