Inhibition of FIXa by AT. (A) Inhibition of FIXa-WT and its variants by AT in the purified system. FIXa (500 nM) was incubated with AT (2 μM) for different periods of time, and the residual amidolytic activity was measured by the addition of SpecIXa. The residual activity is shown as a percentage of the activity corresponding to time 0, which was fitted to an exponential decay function. (B) Inhibition kinetics of FIXa-WT and its variants by AT were measured using an ELISA assay that detects FIXa-AT formation after the addition of FIXa-WT or FIXa variants (25 nM) to human HB (hHB) plasma. The solid lines represent the fit (R2 ≥ 0.97) of the points for a single exponential increase. (C) Residual activity of FIXa was determined after 1 nM FIXa-WT, -V17I, -V16L, or 30 nM -V16T were incubated in human HB plasma for different periods of time. The residual activity is shown as a percentage of the activity corresponding to time 0, which was fitted to an exponential decay function. Each curve is representative of 3 independent experiments. The fitted parameters are listed in Table 2.
