Effect of anti-fVIII mAbs on fVIII binding to fibrin. (A) fVIII-fluor, 4 nM, was incubated with 0.75 µg/mL ESH4 or 0.75 µg/mL ESH8 for 1 hour prior to mixing with fibrin–antifibrin–Superose or control beads lacking fibrin. ESH4 and ESH8 decreased binding to below control levels (*), observed when fVIII-fluor was incubated with control Superose beads. (B) fVIII was incubated with 10 μg/mL ESH4 or ESH8 for 1 hour at 23°C prior to the addition of factor IXa, factor X, thrombin, PLV, and Ca⁺⁺ as noted in Figure 4A description. In the absence of antibodies, the addition of 10 µg/mL fibrin increased Xase activity about twofold. Fibrin did not increase activity in the presence of ESH4 or ESH8 above the levels observed in the absence of fibrin. Results are from a single experiment representative of 4 experiments (A) and are mean ± SD for 4 experiments (B).