Figure 4.
FVII activation by FXa determined by western blotting and FVIIa amidolytic activity. (A) Equivalent molar concentrations (0.5 nM) of WT, R147A, K192A, and R147A/K192A were incubated with 60-nM FXa for 0 and 30 minutes at 37°C. At each time point, the enzymatic reactions were quenched with the reducing sample buffer, run on 12% SDS-PAGE, transferred to PVDF membrane, and probed with the rabbit anti–FVII monoclonal antibody as described in the Methods section. FVIIa LC, the light chain of protease FVIIa; rFVIIa, recombinant FVIIa (NovoSeven, 10 ng). (B) FVII (15 nM) was activated by FXa in reaction buffer at 37°C in the presence of 60-nM FXa for 30 minutes. Rivaroxaban (120 nM) was then added to the inhibit activity of FXa at 37°C for 30 minutes. FVIIa substrate (0.8 mM) was added to determine FVIIa amidolytic activity. Data were presented in percentage of activity compared with WT FVII.

FVII activation by FXa determined by western blotting and FVIIa amidolytic activity. (A) Equivalent molar concentrations (0.5 nM) of WT, R147A, K192A, and R147A/K192A were incubated with 60-nM FXa for 0 and 30 minutes at 37°C. At each time point, the enzymatic reactions were quenched with the reducing sample buffer, run on 12% SDS-PAGE, transferred to PVDF membrane, and probed with the rabbit anti–FVII monoclonal antibody as described in the Methods section. FVIIa LC, the light chain of protease FVIIa; rFVIIa, recombinant FVIIa (NovoSeven, 10 ng). (B) FVII (15 nM) was activated by FXa in reaction buffer at 37°C in the presence of 60-nM FXa for 30 minutes. Rivaroxaban (120 nM) was then added to the inhibit activity of FXa at 37°C for 30 minutes. FVIIa substrate (0.8 mM) was added to determine FVIIa amidolytic activity. Data were presented in percentage of activity compared with WT FVII.

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