Characterization of Klkb1^−/− mice. (A, top) An agarose gel from a polymerase chain reaction showing genotype of WT control (Klkb1^+/+) and Klkb1^−/−; (bottom) immunoblots for prekallikrein antigen in murine Klkb1^+/+ and Klkb1^−/− plasmas and normal human plasma (NHP). Both murine and human plasmas were added at 2 different concentrations (see Materials and methods). (B) Plasma kallikrein activity from Klkb1^+/+ and Klkb1^−/− mice after activation of PK as determined by cleavage of chromogenic substrate HD-Pro-Phe-Arg-pNA. The data are presented as percentage of pooled normal murine plasma KK activity. Three individual mice plasmas were examined in each genotype. (C) aPTT (left 3 columns) and PT (right 3 columns) were determined in WT, Klkb1^−/−, and F12^−/− plasmas (n = 4 in each group). The absence of the line on the top of 2 aPTT bar graphs indicates that they were >200 seconds. (D) Carotid artery occlusion times on the rose bengal thrombosis model. Klkb1^+/+, Klkb1^+/−, Klkb1^−/−, and Klkb1^−/− mice reconstituted with purified human plasma prekallikrein to make the blood 450 nM were examined in this assay. Five to 12 animals were examined in each group. (E) Carotid artery occlusion times on the ferric chloride thrombosis model. (F-G) Contact activation–induced thrombin generation times (TGT) in WT (Klkb1^+/+), Klkb1^−/−, Klkb1^−/− + 450 nM human prekallikrein, or F12^−/− plasma without incubation (F) or after incubation for 3 hours at room temperature (G) (n = 4 in each group). The slopes of the TGT are relative values normalized to WT. (H) The AUCs were calculated and quantified from the TGT curves in (F). Data were normalized to the WT value for each set of experiments.