Identification of a factor VIII C1 domain epitope that participates in binding to phospholipid membranes. (A) Factor VIII residues K2092 to F2093 are critical for KM33 binding. KM33_scFv (5 nM) was passed over fVIII_YFP or fVIII_{YFP 2092/93} as described in detail in “Surface plasmon resonance.” Dissociation was initiated on replacement of ligand solution by buffer. (B) Space-filling display of the C1 and C2 domains of factor VIII crystal structure (PDB ID: 2r7e). The C2 domain is depicted in dark gray, whereas the C1 domain is medium gray. Mutated residues of the KM33_scFv epitope on the C1 domain and the 4 membrane-interactive hydrophobic residues of the C2 domain are highlighted by coloring according to the CPK scheme. (C) KM33_scFv inhibits membrane binding of factor VIII. Fluorescein-labeled factor VIII (3 nM) was incubated with KM33_scFv (0-12.5 nM) before the addition of 15% Ptd-L-Ser–containing lipospheres. After 15 minutes, bound factor VIII was measured by flow cytometry. The result displayed represents the average of 2 experiments. (D) fVIII_{YFP 2092/93} has reduced affinity for phospholipid membranes. fVIII_YFP (■) or fVIII_{YFP 2092/93} (▲) was incubated with lipospheres containing 15% Ptd-L-Ser. After 15 minutes, bound factor VIII was determined by flow cytometry. Data represent the mean ± SEM of 3 experiments. K_D values obtained from fitted curves were 31 nM ± 2 nM for fVIII_YFP and 91 nM ± 6 nM for factor fVIII_{YFP 2092/93}. The K_D value for fVIII_{YFP 2092/93} was obtained by assuming that the maximum phospholipid binding, at saturation, would be equivalent to wild-type factor VIII.