Parameters of hemostasis in vivo following tail-clipping assay of C57Bl/6 hemophilia B/CD4 knock-out mice 8 weeks following intramuscular injection of AAV vectors encoding F.IX-WT or variants. (A) Tail-bleeding times were determined by visual inspection of blood flow into a saline solution, and time to cessation was recorded. At the time of assay, mean circulating F.IX levels for F.IX-WT- and K5A/V10K-treated mice were 439 ng/mL and 487 ng/mL, respectively. Whereas for R338A, F.IX levels were 248 ng/mL. Control groups consisted of hemostatically normal C57Bl/6 or untreated hemophilia B mice matched by age and sex. The experiment in untreated hemophilia B mice was terminated at 10 minutes. (B) Blood loss (mean ± SD) was determined by measuring the absorbance at A₅₇₅ of hemoglobin content in the saline solution in which the tail was placed. The number of animals for each group is indicated at the top of each column. ^*ANOVA was used for statistical analysis with Tukey-Kramer comparison test for all groups; the results are indicated. ^**WT represents hemophilia B mice injected with AAV-F.IX-WT. NS indicates P value not significant.