Biochemical characterization of TF/FVIIa/XK1/10H10 complexes. (A) Schematic of the TF/FVIIa/XK1/10H10/nanodisc complex. (B) TF exosite mutations K165A and K166A reduce the rates of FX activation by TF/FVIIa, with higher PS content moderating this effect. TF was incorporated into small unilamellar liposomes, after which FVIIa and FX were added, and initial rates of FX activation were quantified. Bar graphs show FX activation rates (normalized to WT TF) observed when TF/liposomes contained 5% PS (left) or 20% PS (right). Fold reductions are listed in supplemental Table 1. (C) Elevated XK1 concentrations are required to inhibit TF/FVIIa when TF exosite residues are mutated, with higher PS content moderating this effect. TF/FVIIa was assembled on TF/liposomes with 5% PS (left) or 20% PS (right), then preincubated with varying XK1 concentrations. The residual TF/FVIIa enzymatic activity was quantified (normalized to the FX activation rate without XK1) and plotted vs XK1 concentration, from which IC₅₀ values were derived (listed in supplemental Table 1). Individual data points are graphed in panels B-C (n ≥ 3). ∗∗∗P < .001. WT, wild-type.