Figure 4.

Thrombin generation profile of wild-type prothrombin and PT6 (1 μM) activation by (A) FXa/FVa, (B) FXa/PCPS, or (C) FXa alone, measured by FRET analysis using DAPA. Reaction mixtures for each set of conditions were as follows: (A) FXa (5 nM) and FVa (20 nM) (FXa/FVa), (B) FXa (5 nM) and PCPS (50 μM) (FXa/PCPS), and (C) FXa alone (5 nM). Under all conditions, experiments were initiated with the addition of FXa in HBST buffer with CaCl₂ (5 mM) and DAPA (10 μM). Fluorescence was measured by a SpectraMax M2 fluorescent plate reader, with an excitation wavelength of 280 nm, emission wavelength of 545 nm and an emission cutoff at 530 nm at 20 s intervals.