Enhancement of TFPIα in the inhibition of FXa by increasing concentrations of FV-short in the presence or absence of WT protein S and protein S alanine composite variants. FXa activity (0.5 nM) was followed in real-time through cleavage of S-2765 (200 µM) at 405 mm in the presence of 25 μM phospholipids, TFPIα (0.5 nM) and the absence (A) or presence of 5 nM of WT protein S (B), LG1A (C), LG1B (D), or LG1C (E) at increasing concentrations of (F) FV-short (0-4 nM). Results from representative experiments are shown (n = 3). (E) The initial velocity (V₀) was calculated for each curve and plotted against FV-short concentration. Results are given as mean ± SD and are expressed as percentage of the V₀ for TFPIα either alone or together with WT protein S or its variants (n = 3). The EC₅₀ values of TFPIα enhancement by FV-short and WT protein S and its variants were derived and are presented as part of the figure. FV-s, FV-short; N.M., not measurable; PS, protein S.