Affinity of phospholipid vesicles for C1 domain mutants of factor VIII. Wild-type factor VIII (●) and mutants 2159 (■), 2042/2043 (▾), 2090/2091 (◇), and 2092/2093 (♦) were incubated with various concentrations of sonicated phospholipid vesicles of PS/PE/PC/NBD-PC (4:20:71:5) for 30 minutes. Aliquots of 1-μL Superose beads were added and incubated for another 30 minutes. Phospholipid vesicle-factor VIII complexes bound to mAb GMA-8021-Superose beads were measured by flow cytometry. Values were corrected for the quantity of fluorescence from phospholipid vesicles associated with Superose beads lacking factor VIII. The quantity of bound phospholipid vesicles was normalized to the quantity of mAb BO2C11-fluorescein isothiocyanate that bound to aliquots of the same quantity of factor VIII incubated with Superose beads. Curves indicate the best fit values for wild-type factor VIII (solid line), mutants 2159 (solid line), 2042/2043 (dashed line), 2090/2091 (dotted line), and 2092/2093 (dash dotted line) obtained with the GraphPad Prism Version 5.0. Data are from a single experiment representative of 2 experiments from 2 separate COS cell transfections.