Inhibition of FXa by the TFPI forms in the presence of protein S. (A) Progress curves for S-2222 hydrolysis by FXa in the presence of TFPI and various concentrations of PS. Final concentrations were S-2222 (500μM), TFPI (4nM), FXa (400pM), CaCl₂ (5mM), and cephalin (35μM). For each dataset, the concentrations of PS from top to bottom are 0, 5, 10, 40, 80, 160, and 320nM. The data were fitted by nonlinear regression using the program Grafit (Version 3.01) from Erithacus software as described.^21-23  (B) Plot of the final rates of S-2222 hydrolysis data from Figure 3A fitted into the IC₅₀ 4-parameter logistic equation of Halfman.²⁴  Note that, in the absence of PS (0nM PS), TFPI_WT, TFPI-_ΔK3, and TFPI_K3P1 inhibit FXa better, hence the lower A₄₀₅/minute values compared with TFPI_C1, TFPI_C2, and TFPI_C3.