Inhibition of FXa by ATIII and TFPI. The rate of inactivation of FXa by ATIII was measured under pseudo-first order conditions. Antithrombin III (A, ●, 0; ▴, 0.1; ▾, 0.25; ■, 0.50; and ♦, 1.0μM; B, ○, 0; ▵, 1.0; ▿, 2.5; □, 5.0; and ◇, 10μM) was incubated with FXa (A, 2.0 nM wt-FXa; B, 10.0nM FXa^I16L) in assay buffer for up to 90 minutes. At various time points, residual enzyme activity was measured using the chromogenic substrate SpecXa. For the inhibition of FXa by TFPI, wt-FXa (C, 0.2 or 0.4nM,) was incubated with increasing concentrations of TFPI (0.1-2.0nM), whereas FXa^I16L (D, 2.0 or 4.0nM) was incubated with higher concentration of TFPI (1.0-40nM) for 3 hours at 25°C. Residual enzyme activity was determined after the addition of SpecXa. The lines are drawn following analysis to equations detailed in “Methods,” and the fitted values are given in Table 1. The data are representative of 2 to 3 similar experiments.