Analysis of tissue factor–stimulated thrombin generation by Western blot in factor IX–deficient plasma supplemented with 5% recombinant factor IX wild-type, R170A, and R233A. Thrombin generation was initiated with 0.2 pM of human tissue factor, 8.3 μM of PC:PS vesicles, and 40 μg/mL of CTI (plasma concentrations) in factor IX-deficient plasma supplemented with 5% recombinant factor IX wild-type (A), R170A (B), and R233A (C). Individual reactions were quenched over time with loading buffer containing 5 M of urea and analyzed by 10% SDS-PAGE under nonreducing conditions as described in “The time course of plasma thrombin generation by Western blotting.” Proteins transferred to Immobilon-P were detected with a polyclonal sheep anti–human thrombin primary antibody, followed by a peroxidase-conjugated affinity-purified donkey anti–sheep IgG, and subsequent development of signal with chemiluminescent substrate. Prothrombin indicates prothrombin/meizothrombin band; TAT, thrombin-antithrombin complex.