2.1. Thrombin generation experiments

Thrombin generation assays were performed using calibrated automated thrombography as reported previously with a Fluoroskan Ascent plate reader (Thermo Labsystems, Helsinki, Finland) and the Thrombinoscope software (Thrombinoscope BV, Maastricht, the Netherlands). 20 Twenty microliters of activating reagent (5 pM or 1 pM tissue factor, 4 μM phospholipids; final concentration) or calibrator (both Thrombinoscope BV, Maastricht, the Netherlands) were placed into the respective wells of a 96‐well‐plate (Immulon 2 HB, Thermo Scientific), and then 80 μL of PPP was added. The measurement was started by the automatic dispension of 20 μL fluobuffer‐CaCl₂, containing a fluorogenic substrate (Z‐Gly‐Gly‐Arg‐amino‐methyl‐coumarin, Bachem, Bubendorf). The thrombin generation profiles were recorded in triplicates. Parameters derived from the thrombin generation traces were lag time, time to peak, endogenous thrombin potential (ETP), and peak height (Figure ​(Figure2A).2A). The velocity index was calculated with the formula: (peak height/[time to peak−lag time]).

Thrombin generation parameters with 5 pM exogenous tissue factor. A, Representative thrombin traces of one patient at onset and during follow‐up. B‐F, Absolute values of thrombin generation parameters at onset and follow‐up depicted in boxplots (left) and relative changes for each patient with median and IQR (right). ETP, endogenous thrombin potential. *P < .05