In vivo hemostatic effect assessed by tail clip assay in HA mice after infusion of D519V/E665V/K1813A and D519V/E665V FVIII mutants. The HA mice were infused with FVIII-WT (2 and 4 μg/kg) or mutant; D519V/E665V/K1813A (0.25 and 0.5 μg/kg) or D519V/E665V (1 μg/kg), or PBS. At 5 minutes after infusion, the terminal 5 mm of the tail was amputated, and shed blood was collected for 40 minutes, as described in “Methods.” The blood loss in (A) WT-FVIII at 2 μg/kg and (B) 4 μg/kg were compared with that in WT mice and HA mice infused with FVIII mutants. Statistical analysis between WT-FVIII and FVIII mutants or no FVIII administration was performed using the Dunnett multiple comparison test. Each point represents a mouse. The straight lines in each graph indicate the average values. Significant differences are defined as P < .05 (∗P < .05 and ∗∗P < .01). ns, not significant.