Relative activities, membrane interaction, and affinities for FVIII mutants. (A) Membrane interaction for FVIII_WT (■) or FVIII_LS (▾) was evaluated by mixing FIXa, FX, and various concentrations of sonicated vesicles. The quantity of FXa formed was measured with chromogenic substrate S-2765. The membrane reactivity of FVIII_LS was less than FVIII_WT. The vesicles had a composition of PS:PE:PC 4:20:76; concentration of FVIII or mutants was 0.2nM; FIXa, 4nM; and FX, 130nM. Results represent the mean ± SEM for at least 3 experiments, each performed in duplicate and fitted to a single-site binding model (smooth curves). (B) Apparent K_M for the FVIIIa-FIXa complex was determined as in panel A, except that the phospholipid concentration was constant (100μM) and the FX concentration varied. (A-B) Fitted results were normalized to the B_max value for each curve to optimize comparison of the relative affinities. (C) The specific activities, apparent phospholipid interactivity, and apparent FX affinities for each mutant are displayed with reference to control experiments performed with wild-type FVIII (as in panels A and B for FVIII_LS). The displayed values represent the mean ± SEM. Each fit was performed on a minimum of 3 separate experiments, each performed in duplicate. (D) Apparent VWF affinity was determined by incubating FVIII_WT and mutants with various concentrations of VWF for 60 minutes before addition to a microtiter well coated with a monoclonal antibody that competes for the VWF-binding epitope. Bound FVIII was detected with a second antibody. The fraction of VWF-bound FVIII was calculated as the difference between the maximum signal in the absence of VWF and the signal at each concentration of VWF. Smooth curves represent best-fit curves corresponding to K_D values of 0.7 ± 0.1nM, 2.2 ± 0.7nM, and 4.8 ± 0.8nM for FVIII_WT (■), FVIII_WW (▴), and FVIII_LS (▾), respectively. Displayed results are the mean ± SEM of at least 2 experiments, each performed in duplicate.