Mechanisms contributing to the cofactor activity of Mim8. (A) Concentration-dependent stimulation of FX (25 nM) activation by monovalent anti-FIXa OA antibodies (as indicated) in the presence of 0.02 to 2 nM FIXa and 500 µM PS:PC vesicles at pH 7.4. FIXa stimulation (mean ± standard deviation, n = 3) was calculated as the ratio of FX activation in the presence and absence of antibody and fitted to a 1:1 binding model (solid lines) to obtain stimulation indices at saturation of (23 ± 0.4) × 103 and 3.3 ± 0.16 for Mim8 and biAb46376, respectively. In comparison, emicizumab SIA stimulated FX activation 729 ± 11.3–fold relative to free FIXa. (B) FX (25 nM) activation by FIXa (varying concentrations) in the presence of a limiting concentration of Mim8 or emicizumab SIA (0.1 nM) and PS:PC vesicles as indicated (mean ± standard deviation, n = 3). Curve fitting was performed as described in the supplemental Methods to derive a functional KD value of 16.0 ± 2.9 nM for the Mim8-FIXa-FX complex assembly in the presence of membrane. A functional KD value of 0.4 ± 0.1 nM was observed for emicizumab SIA.