Effect of ESH4 and ESH8 on fVIII binding to thrombin-stimulated platelets. (A) fVIII-fluor was incubated with ESH8 for 1 hour on ice prior to mixing with platelets stimulated by thrombin as noted in Figure 1C description. The fVIII-fluor/antibody mix was added at a final fVIII concentration of 8 nM and allowed to bind for 10 minutes prior to dilution and reading. ESH8 decreased bound fVIII by ∼50%. (B) The same procedure was followed using ESH4. ESH4 inhibited ∼80% of fVIII binding. (C) ESH8 or ESH4, 10 µg/mL, was mixed with fVIII for 60 minutes prior to mixing with platelets (1 × 10⁸/mL), factor IXa, factor X, Ca⁺⁺, and thrombin. ESH8 inhibited 84% and ESH4 inhibited 78% of activity, respectively. To obtain average aggregate values, factor Xase activity was normalized to the value in the absence of ESH4 or ESH8 for each experiment. All data are corrected for the signal obtained from unstimulated platelets and normalized to facilitate comparison. Results are mean ± SEM for 3 experiments (A) and 4 experiments (B). (C) Mean ± SEM from 4 experiments, each performed in duplicate.