Apparent membrane-independent activity of FV mutants resulting from copurification of phospholipid. (A) The potential presence of phospholipid was evaluated by testing the capacity of the factor FV_WT and mutants to support the FXase complex. FV preparations were mixed with FVIIIa, FIXa, and FX in the absence of phospholipid vesicles. FV mutants supported FXase activity, whereas FV_WT supported little or no activity. Data represent the mean ± SEM for 2 experiments for FV_LL and FV_WT and one experiment for the other mutants each performed in duplicate. (B) Lactadherin inhibition of FV_MF-supported FXase activity was evaluated using increasing concentrations of lactadherin. Activity was inhibited with half-maximal inhibition at approximately 4nM. Data represent the mean ± SEM for a single experiment performed in duplicate. (C) Prothrombinase activity supported by CHAPS-washed FV_WT and FV mutants in the absence of phospholipid vesicles was evaluated in the presence of various concentrations of lactadherin. Lactadherin inhibited activity supported by FV_MF (▵) with half-maximal inhibition at approximately 16nM lactadherin. Half-maximal inhibition of activity supported by FV_LL (▿) occurred at approximately 1nM, whereas half-maximal inhibition of FV_MF/LL (♢) occurred at an intermediate concentration. Data represent the mean ± SEM for 2 experiments. (D) Inhibition of prothrombinase activity by phospholipase A₂ was evaluated for FV_LL (▿) in the absence of phospholipid vesicles and for FV_WT (□) in the presence of phospholipid vesicles (inset). Phospholipase A₂ inhibited prothrombinase activity under both conditions. Data represent the mean ± SEM for 3 experiments performed in duplicate.