Thrombin-catalyzed activation of D519V/E665V/K1813A, D519V/E665V, and K1813A FVIII mutants. WT-FVIII or FVIII mutants (10 nM) were incubated with thrombin (0.4 nM) before measuring FVIII activity at the indicated times in a 1-stage clotting assay. The symbols used are: open circles, WT; closed circles, D519V/E665V/K1813A; open squares, D519V/E665V; and closed squares, K1813A. The initial FVIII activities of the WT, and K1813A, D519V/E665V, and D519V/E665V/K1813A mutants were 6.1, and 12.8, 9.1, and 22.0 IU/mL, respectively, at t = 0. FVIII activity was expressed as a fold of initial (t = 0) and was plotted as a function of incubation time. Experiments were performed 3 times, and the average and standard deviation values are shown.