Laboratory assays

Thrombin generation (TG) was studied using Calibrated Automated Thrombography (CAT–Diagnostica Stago, Asnières-sur-Seine, France) with Stago reagents using Fluoroscan Ascent Fluorometer (version 5.0, Thermolab Systems, Helsinki, Finland). Runs were performed at 37°C after a 10 min pre-heating in the fluorometer of the round-bottom 96-well plate (Immulon 2HB) loaded with plasma samples and with procoagulant phospholipids and TF (see below). Fluo-Buffer together with FluCa was then automatically dispensed leading to recalcification of citrated plasma so that the reaction could start. Fluo-Buffer is a Hepes solution (pH 7.35) with calcium chloride, whereas Fluo-Substrate contains a thrombin-specific fluorogenic substrate (Z-GGR-AMC) solubilized in DMSO. To prepare FluCa, Fluo-Substrate was added to the warmed Fluo-Buffer shortly before the experiment. FluCa was freshly prepared for each run. Each TG run was calibrated according to the fluorescence curve obtained from a sample of the same plasma supplemented with a Thrombin Calibrator, with the specified amount of thrombin–α₂–macroglobulin complex, and FluCa. Inner-filter effect and substrate consumption were accounted for as well. Fluorescence was recorded every 15 s for 60 min. All samples were analysed in duplicate. Parameters of interest were derived from each TG curve (i.e., thrombogram) using the Thrombinoscope software, version 5.0 (Diagnostica Stago, Asnières-sur-Seine, France). All experiments were conducted with the same batches of reagents. TM (rabbit lung; BioMedica Diagnostics Inc., Stamford USA) was added to the TG mixture to assess the dynamic inhibitory protein C system. We used a TM concentration of 1.75 nM to induce a 50% decrease in ETP compared with values in the absence of TM with normal plasma.

First, we compared MidiCAT with the CAT method using frozen (after two centrifugations)–thawed plasma samples. Second, we compared the TG of plasma samples prepared with either a single-centrifugation scheme or a double-centrifugation scheme using MidiCAT.

For the CAT method acting as reference, experiments were performed at a total volume of 120 μL. To initiate TG, 20 μL of the reagent comprising procoagulant phospholipid vesicles only (MP-reagent) or recombinant TF and procoagulant phospholipid artificial vesicles (either PPP-low reagent 1 pM TF or PPP reagent 5 pM TF) were added to 80 μL of plasma into each well. TG was eventually triggered with automated dispensing of 20 μL FluCa reagent.

For the MidiCAT method (Bloemen et al. (16)), experiments were conducted in a reduced total volume of 60 μL. All volumes of the CAT method were halved. To modify the dispensing volume, we went to the folder: C: \Program Files (x86) \Thrombinoscope \Users \(username) \Setting, opened the file: default.set with notepad located the line “nDispenseVolume”, and entered the desired volume.