Kinetics of the Inhibitory Effect of Factor IX_Bm in the Activation of Factor X by Factor VII and Bovine Tissue Factor

F.IX_Bm is giving a prolonged prothrombin time with bovine tissue factor (T.F.). In the present study the inhibitory effect of purified F.IX_Bm was tested in a F.VII assay that measures the ability of F.VII to activate F.X in the presence of bovine T.F. and Ca⁺⁺, followed by the quantitation of formed F.Xa by the synthetic substrate benzoyl -L-Ile-Glu-Gly-L-Arg-p-nitroanilide. Experiments were carried out with different concentrations of F.IX_Bm and F.X, keeping the concentrations of F.VII and bovine T.F. constant. Lineweaver-Burk plot of the data showed that all the curves crossed the l/V axis in one point - no inhibition by F.IX_Bm at infinite concentratlons of F.X. Thus, F.IX_Bm acts as competitive in inhibitor to F.X in the activation of F.X by F.VII and bovine T.F. By plotting the 1/V against the inhibitor concentrations (F.IX_Bm conc), a Dixon plot, the K_i for F.IX_Bm was determined to be 0.017 μM.

Normal F.IX was also found to be a competitive inhibitor to F.X but 4-5 times higher concentrations of F.IX were needed to give the same inhibition as F.IX_Bm. Utilizing human T.F. instead of bovine T.F. in this assay resulted in no significant inhibition by either F.IX of F.IX_Bm.