Spontaneous decay on the stability of D519V/E665V/K1813A, D519V/E665V, and K1813A FVIIIa mutants. (A) WT-FVIII or FVIII mutants (1 nM) were incubated with thrombin (30 nM) before measuring FVIII activity at the indicated times in a 1-stage clotting assay, as described in “Methods.” As for the A2 dissociation in the intrinsic Xase assay (B), FVIII (1 nM) in buffer containing PL vesicles (20 μM) was activated by the addition of thrombin (30 nM) and thereafter terminated after 30 seconds by the addition of hirudin (10 U/mL). At various time intervals (0-15 minutes), FXa generation was initiated by the addition of FIXa (40 nM) and various concentrations of FX (300 nM). The symbols used are: open circles, WT; closed circles, D519V/E665V/K1813A; open squares, D519V/E665V; and closed squares, K1813A. FVIIIa activity was expressed as the fold-change of the initial value and was plotted as a function of incubation time. The experiments were performed 3 times, and the average and standard deviation calculations are shown. The data were fitted to the formula given in Equation 2.