Inhibition of FVIIa/TF-induced FX activation by Ixolaris.

(A) Progress curves: FVIIa (1 nM)/TF (0.2 pM) was added to a mixture containing FX (200 nM), previously incubated with increasing concentrations of Ixolaris (a, 0 nM; b, 0.1 nM;c, 0.25 nM; d, 0.5 nM; e, 1 nM;f, 2.5 nM; g, 5 nM; h, 10 nM), or full-length clone TFPI (inset: a, 0 nM; b, 0.05 nM; c, 0.125 nM; d, 0.25 nM; e, 0.5 nM; f, 1.25 nM; g, 2.5 nM; h, 5 nM). Progress curves are representative experiments (n = 3). (B) Data generated in panel A were transformed as described in “Materials and methods” to estimate FXa production for Ixolaris (a, 0 nM;b, 0.1 nM; c, 0.25 nM; d, 0.5 nM;e, 1 nM; f, 2.5 nM; g, 5 nM;h, 10 nM), or TFPI (inset: a, 0 nM; b, 0.05 nM; c, 0.125 nM; d, 0.25 nM; e, 0.5 nM). (C) For determination of the IC₅₀, Vs and Vo were plotted against Ixolaris concentration at different FX concentrations: (●) 3 nM; (▪) 15 nM; (▴) 40 nM; (▾) 75 nM; (○) 100 nM; (■) 140 nM); (▵) 175 nM; (▿) 200 nM (n = 3). Inset: Vs and Vo were plotted against TFPI concentration at different FX concentrations: (●) 15 nM; (▪) 40 nM; (▴) 75 nM; (▾) 200 nM (n = 3). Vs, inhibited velocity; Vo, control (uninhibited velocity). (D) IC₅₀ for Ixolaris or TFPI (inset) at different concentrations of FX (n = 3).