Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-af finity complex with its allosteric regulator, tissue factor (TF), and initi ates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVII a with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrop hobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupat ion of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the pr otease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in th is region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.