Z gly gly arg 7 amino 4 methylcoumarin

Thrombin generation in plasma was measured with the Calibrated Automated Thrombogram assay as previously described [23 (link)]. Briefly, 80 μl of platelet poor plasma was mixed with 20 μl of a mixture containing 6 pM tissue factor (TF, Dade-Behring) and 24 μM phospholipid vesicles (DOPS/DOPC/DOPE; 20/60/20 mol%/mol%/mol%; Avanti). After 5 minutes of incubation at 37°C, thrombin generation was started with 20 μl of the activator containing 100 mM CaCl₂ and 2.5 mM of the thrombin specific substrate, Z-Gly-Gly-Arg-7-amino-4-methylcoumarin (Bachem). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems). Samples were run in triplicate and each curve was calibrated to correct for inner-filter effects and substrate consumption. All procedures were performed at 37°C and data were analyzed with dedicated software (Thrombinoscope, Stago).

TG in plasma was measured with the calibrated automated thrombogram (CAT) assay as developed by Hemker and co-workers [18 (link)–20 (link)]. Briefly, 80 µl platelet poor plasma (PPP) was mixed with 20 µl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 µM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 µl of calibrator (α₂macroglobulin-thrombin complex, [19 (link)]) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z-Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 µM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) [20 (link)]. Thrombin generation was expressed based on endogenous thrombin potential (ETP); lagtime (LT); thrombin peak (TP), time-to-thrombin peak (TTP).