Factor VIII C1 domain mutations and the effects on apparent phospholipid binding affinity. (A) Space-filling display of the C1 and C2 domains of factor VIII (PDB ID: 3CDZ). Dark gray represents the C2 domain; and medium gray, the C1 domain. Mutated residues encompassing the lower surface of the C1 domain are highlighted in pink (Gln 2042/Tyr 2043), red (Arg 2159), blue (Arg 2090/Gln 2091), and green (Lys 2092/Phe 2093). The C2 domain epitopes of mAb ESH4 (magenta) and BO2C11 (cyan) are also shown. (B) Factor VIII mutants had diminished apparent affinity for phospholipid vesicles compared with wild-type factor VIII. Factor VIII wild-type or mutants (0.15 U/mL) were added to various concentrations of vesicles containing 4% or 15% PS, and factor Xase activity was measured as described in “Factor Xase assay.” Data are mean ± SEM for 3 experiments, each performed in duplicate.