Figure 3. Factor Xa (A, and B) and thrombin (C and D) generation tests in normal (A and C) and serpin-free (B and D) plasmas.

Defibrinated plasmas (50 μL) were incubated in the absence (■) or in the presence of 0.2 μg/mL of heparin (▲) or sulfated galactan (●) with 50 μL of TS/PEG buffer and 10 μL of Cephalin reagent. After incubation for 2 min at room temperature, the protease generation reaction was started by addition of 100 μL 12.5 mM CaCl₂, and aliquots of 10 μL were removed each 15 s into microplate wells containing 40 μL TS/PEG buffer + 50 mM EDTA. The amounts of factor Xa or thrombin generated were determined using the chromogenic substrates S-2765 or S-2238, respectively. Substrate hydrolysis was detected using a Thermomax Microplate Reader. Reactions were recorded continuously at 405 nm for 15 min at 37°C. The panels show mean±SD, n=3.