Simultaneous measurement of thrombin and plasmin generation potential of plasma samples were performed with modifications to previous methods (25 (link), 26 (link)). Briefly, plasma samples were mixed with 512 μM of either thrombin specific substrate, Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland) or plasmin specific substrate, Boc-Glu-Lys-Lys-AMC (Bachem) and 16 nM of thrombomodulin (PeproTech, Rocky Hill, NJ, USA) similar to a previous method designed to measure thrombin and plasmin in parallel (26 (link)).
The reaction was initiated by adding an activator solution that yielded a final concentration of 1 pM tissue factor (Diagnostica Stago, Parsippany, NJ, USA), 0.7 μg/mL of tissue plasminogen activator (Sigma-Aldrich, St. Louis, MO, USA) and 16 mM CaCl2. Sample wells were supplemented with buffer (150 mM NaCl, 20 mM HEPES and pH 7.5) and AMC fluorophore instead of activator solution for background and calibrator measurements respectively. Calculation of thrombin and plasmin concentration was performed as described previously (25 (link)).
The reaction was initiated by adding an activator solution that yielded a final concentration of 1 pM tissue factor (Diagnostica Stago, Parsippany, NJ, USA), 0.7 μg/mL of tissue plasminogen activator (Sigma-Aldrich, St. Louis, MO, USA) and 16 mM CaCl2. Sample wells were supplemented with buffer (150 mM NaCl, 20 mM HEPES and pH 7.5) and AMC fluorophore instead of activator solution for background and calibrator measurements respectively. Calculation of thrombin and plasmin concentration was performed as described previously (25 (link)).
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