Alteration of the endogenous fII allele to limit activation to meizothrombin. (A) This map demonstrates the targeting vector used to alter the endogenous fII allele as well as a map of the endogenous allele. Incorporation of the targeting vector by homologous recombination targeting vector was confirmed in germline-competent mice by PCR. After transmission of the targeted allele by the chimeric animals, mice were crossed with CMV-Cre animals to remove the HPRT cassette, with the final targeted allele as illustrated. Loss of the HPRT cassette was also confirmed by PCR. (B) Liver fII mRNA levels in fII^WT and fII^MZ animals were assessed using qPCR. No significant difference in fII RNA levels was detected between the 2 genotypes. In contrast, significant decreases, as expected, were noted in fII^+/− and fII^lox/− animals (n = 6 in all cohorts). (C) Plasma from both fII^MZ and fII^WT mice was assayed for fII antigen levels via western blot. No difference in fII levels between fII^WT and fII^MZ animals was appreciable on the immunoblot. (D) Chromogenic fIIa activity from both fII^WT and fII^MZ plasma was assayed after activation with ecarin. There was no significant difference in plasma fIIa activity derived from either fII^MZ or fII^WT animals (n = 7 in both cohorts). The expected decrease in fIIa activity in both fII^+/− and fII^lox/− (n = 4 in both cohorts) was readily detectable.