Prothrombin^WE limits wild-type thrombin generation in plasma. (A) Representative TGA tracings using plasma isolated from individual wild-type, fII^WT/Null, and fII^WT/WE mice. As expected based on rate-limiting prothrombin concentration, fII^WT/Null plasma supported the generation of approximately half of the thrombin activity observed using wild-type plasma. Thrombin generation tended to be incrementally less in TGA studies using plasma collected from fII^WT/WE mice relative to samples collected from fII^WT/Null mice, but this was not statistically different over multiple specimens (see panel C). (B) Representative TGA tracings using plasma from fII^flox/WT mice (carrying wild-type prothrombin at a concentration ∼ 60% of normal), fII^flox/Null mice (carrying wild-type prothrombin at a concentration ∼ 10% of normal), and fII^flox/WE mice (carrying wild-type prothrombin at a concentration ∼ 10% of normal and an ∼ 5-fold molar excess of prothrombin^WE over wild-type prothrombin). Note that thrombin generation with plasma from fII^flox/Null mice was limited (consistent with wild-type prothrombin being present at approximately 10% of normal levels), but was barely detectable with plasma from fII^flox/WE, with a similar concentration of wild-type prothrombin and appreciably more prothrombin^WE. (C) Comparison of peak thrombin concentration. The TGA assay was performed in duplicate on plasma collected from 3 mice per genotype. Note that the mean peak thrombin concentration in fII^flox/WE plasma, in which the ratio of fII^WE thrombin to wild-type thrombin was approximately 5:1, was significantly lower than that observed for fII^flox/null plasma. Data are presented as the means ± SD and were analyzed using the Student t test.