Inhibition of FXa by the TFPI variants in the presence or absence of protein S and binding of protein S to TFPI studied by SPR. (A-D) Cleavage of 200μM S-2765 by 0.5nM FXa was monitored at 405nm in the presence of phospholipids (25μM), 2nM WT TFPI (A), TFPI R199Q (B), and TFPI E226Q (C) and in the presence or absence of 100nM protein S (PS). Results from a representative experiment are shown. Increasing concentrations of protein S (0-320nM; Enzyme Research Laboratories) were added to 2nM WT TFPI, TFPI R199Q, and TFPI E226Q. The v0 (initial velocity; expressed as percentage of the v0 in the absence of TFPI) was calculated and plotted against the protein S concentration. Values are given as means ± SD (n = 3) in panel D. (E-H) A CM5 chip was coupled with WT TFPI (E), TFPI R199Q (F), or TFPI E226Q (G) to 2500 resonance units (RU). The flow cells were perfused with increasing concentrations (0-4000nM) of protein S. Results from a representative experiment are shown (n = 3). The maximum RU (RU at 550 seconds of association) was plotted against the protein S concentration. Data are given as means ± SEM (n = 3) in panel H.
