Ppp reagent

TG was measured by Calibrated Automated Thrombinography (CAT) using PPP reagent, calibrator and FluCa from Diagnostica Stago (France). TG was measured in the presence or absence of thrombomodulin (TM; the concentration causing 50% inhibition of the peak height in pooled normal plasma; Synapse Research Institute, the Netherlands) to test the sensitivity of the activated protein C (APC) system. In the cohort of ICU patients, who were all treated with low molecular weight heparin, heparin was neutralized with 0.045 mg/mL polybrene added to the plasma prior to the measurement of TG. In addition to the classical TG parameters ETP, peak height, time-to-peak and lag time, novel time-to-tail and curve width were quantified. The time-to-tail was quantified as the time it takes until the thrombin concentration stays below 1 nM at the end of thrombin generation. The curve width was quantified as the difference between the lag time and time-to-tail, providing a measure for the broadness of the curve.

The cover and capillary chips used in the flow chamber system (Fig. S1A) were manufactured by Richell Corp. (Toyama, Japan). The following materials were obtained from commercial sources: porcine type I collagen (Nitta Gelatin, Inc., Osaka, Japan), tissue thromboplastin (Sysmex, Hyogo, Japan), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD41 immunoglobulin G (IgG), and FITC-conjugated mouse IgG (Beckman Coulter, Miami, FL, USA), rabbit anti-human fibrinogen IgG (Dako, Tokyo, Japan), normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Alexa594 (Invitrogen, Carlsbad, CA, USA).
Dabigatran and rivaroxaban were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). AR-C66096, a specific P₂Y₁₂-receptor antagonist, was obtained from Tocris Bioscience (Bristol, UK). For the TG assay, PPP-Reagent (with phospholipids), PRP-Reagent (without phospholipids), and FluCa-reagent, a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) dissolved in HEPES buffer and calcium chloride, were purchased from Diagnostica Stago (Parsippany, NJ). Recombinant TF (r-TF) was purchased from Mitsubishi Chemical Medience (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan). Corn trypsin inhibitor (CTI) was prepared as reported previously [13] (link).

Calibrated automated thrombogram (CAT; Thrombinoscope BV, Maastricht, Netherlands) was performed 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 °C. Briefly, to 80 µL platelet-poor plasma 20 µL of tissue factor (TF)-based activator (PPP Reagent; final TF concentration, 5 pM) and FluCa solution (both Diagnostica Stago) were added. Each plasma sample was analyzed in duplicate. The maximum concentration of thrombin formed during the recording time is described as the peak thrombin generated and the area under the curve represents endogenous thrombin potential (ETP) [24 (link)]. The intra-assay variability was 8%.