Function of KM33 epitope in activity of factor VIII. KM33_scFv inhibited activity of factor VIII on vesicles with 15% Ptd-L-Ser (A) and on vesicles with 4% Ptd-L-Ser (B). Factor VIII (1 U/mL) in the absence (■) or presence (▲) of excess KM33_scFv was added to various concentrations of phospholipid vesicles. Factor VIII was preincubated with KM33_scFv for 30 minutes at room temperature. Displayed values represent the mean ± SEM for 3 separate experiments, each performed in duplicate. (C-D) FVIII_{YFP K2092A/F2093A} had diminished apparent affinity for phospholipid vesicles. fVIII_YFP (■) or fVIII_{YFP 2092/93} (▲) (1 U/mL) was added to various concentrations of phospholipid vesicles containing 15% Ptd-L-Ser (C) or 4% Ptd-L-Ser (D), and activity was measured as described for panels A and B. Each point represents the mean value ± SEM of 3 experiments. Curves were fitted to a single-site binding model, assuming that vesicles offered a single class of binding sites, and fitted with nonlinear least squares curve fitting.