Thrombin-catalyzed activation of FVIII mutants of K1813A, K1818A, and K1813A/K1818A. FVIII WT or mutants (10 nM) were incubated with thrombin (0.4 nM) before measuring FVIII activity at the indicated times in a 1-stage clotting assay, as described in the Methods section. The symbols used are defined in Figure 1. The initial FVIII activities of WT, K1813A, K1818A, and K1813A/K1818A mutants were 7.8, 16.1, 14.3, and 15.1 IU/mL at t=0, respectively. FVIII activity was expressed as a fold of initial (t=0) and was plotted as a function of incubation time. Experiments were performed 3 separate times, and the average and standard deviation values are shown.