The modulation of FXa and thrombin activity was evaluated by colorimetric assay.
Briefly, CTX (0.5-13.5 µg/mL) or PBS (control) was incubated with 43 mU/mL of
human FXa or thrombin (Sigma-Aldrich, St. Louis, USA) for 10 minutes.
Afterwards, specific chromogenic substrate S-2222 or S-2238 (400 µM,
Chromogenix, Milan, Italy), were added and the hydrolysis of chromogenic
substrate by FXa or thrombin was measured at 405 nm using a Spectramax 190
microplate reader (Molecular Devices, San Jose, USA). The reaction was carried
out in a 96-well plate at 25ºC in PBS, pH 7.4. The enzymatic activity was
calculated based on the slope of the activity curve, obtained from sequential
readings at 405 nm, and considered the values generated by the incubation of the
factors and PBS (control) to be 100% activity.
Briefly, CTX (0.5-13.5 µg/mL) or PBS (control) was incubated with 43 mU/mL of
human FXa or thrombin (Sigma-Aldrich, St. Louis, USA) for 10 minutes.
Afterwards, specific chromogenic substrate S-2222 or S-2238 (400 µM,
Chromogenix, Milan, Italy), were added and the hydrolysis of chromogenic
substrate by FXa or thrombin was measured at 405 nm using a Spectramax 190
microplate reader (Molecular Devices, San Jose, USA). The reaction was carried
out in a 96-well plate at 25ºC in PBS, pH 7.4. The enzymatic activity was
calculated based on the slope of the activity curve, obtained from sequential
readings at 405 nm, and considered the values generated by the incubation of the
factors and PBS (control) to be 100% activity.