Conformation of Factor VIIa Stabilized by a Labile Disulfide Bond (Cys-310–Cys-329) in the Protease Domain Is Essential for Interaction with Tissue Factor

Abstract

Unlike other trypsin-type serine proteases, zymogento-enzyme transition of conformation of factor VII apparently requires not only conversion of the zymogen to active form factor VIIa (VIIa) but also interaction of VIIa with tissue factor (TF). To determine the region of interaction that correlates with maturation of the VIIa active site, we modified intramolecular disulfide bonds in VIIa and examined the interaction of the modified VIIa with soluble TF (sTF). We found that partial reduction and S-carboxamidomethylation of disulfide bonds in VIIa led to losses of amidolytic activity and the binding ability to sTF. To determine the sites of modification that associate with the loss of functions, partially S-carboxamidomethylated VIIa was separated on a column of immobilized sTF. Each of the sTF-bound and sTF-unbound fractions and native VIIa was then digested by trypsin, and the digest was analyzed by reversed-phase high performance liquid chromatography. We found that reduction and S-carboxamidomethylation of a disulfide bond between Cys-310 and Cys-329 in the protease domain of VIIa led to loss of the binding ability with sTF, and the modification of a disulfide bond between Cys-340 and Cys-368 of VIIa led to loss of the amidolytic activity. In the three-dimensional structures of trypsinogen and trypsin, the disulfide bonds corresponding to Cys-340-Cys-368 and Cys-310-Cys-329 of VIIa are, respectively, in and adjacent to the activation domain, which has flexible conformation in trypsinogen but not in trypsin. Furthermore, the crystal structure of human VIIa.TF complex indicates that the region next to Cys-310-Cys-329 is in contact with sTF. We speculate that a regional flexibility, reflected by the labile nature of disulfide bonds of Cys-310-Cys-329 and Cys-340-Cys-368 in the protease domain, contributes to the inability of VIIa to attain the active conformation. Interaction of TF with this flexible region may stabilize the structure in a conformation similar to that of the active state of VIIa.