Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two

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... active variants, FVIIa DVQ and FVIIa VEAY , imply that enhanced catalytic efficiency was attained by two different mechanisms. Regions protected from exchange in FVIIa DVQ include the N-terminal tail and the activation pocket, which is a subset of the regions of FVIIa protected from exchange upon TF binding. FVIIa DVQ appeared to adopt an intermediate conformation between the free (zymogen-like) and TF-bound (active) form of FVIIa and to attain enhanced activity by partial mimicry of TF-induced activation. In contrast, exchange-protected regions in FVIIa VEAY were confined to the vicinity of the active site of FVIIa. Thus, the changes in FVIIa VEAY appeared to optimize the active site region rather than imitate the TF-induced effect. Hydrogen exchange analysis of the FVIIa M306D variant, which was unresponsive to stimulation by TF, correlated widespread reductions in exchange to the single mutation in the TFbinding region. These results reveal the delicate interplay between key allosteric sites necessary to achieve the transition of FVIIa into the active form.