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

Unlike other trypsin-type serine proteases, zymogen-to-enzyme transiti on of conformation of factor VII apparently requires not only conversi on of the zymogen to active form factor VIIa (VIIa) but also interacti on of VIIa with tissue factor (TF), To determine the region of interac tion that correlates with maturation of the Wa active site, we modifie d intramolecular disulfide bonds in VIIa and examined the interaction of the modified VIIa with soluble TF (sTF). We found that partial redu ction and S-carboxamidomethylation of disulfide bonds in VIIa led to l osses of amidolytic activity and the binding ability to sTF. To determ ine the sites of modification that associate with the loss of function s, partially S-carboxamidomethylated Ma was separated on a column of i mmobilized sTF, Each of the sTF-bound and sTF-unbound fractions and na tive 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 bin ding ability with sTF, and the modification of a disulfide bond betwee n Cys-340 and Cys-368 of VIIa led to loss of the amidolytic activity. In the three-dimensional structures of trypsinogen and trypsin, the di sulfide bonds corresponding to Cys-340-Cys-368 and Cys-310-Cys-329 of Ma are, respectively, in and adjacent to the activation domain, which has flexible conformation in trypsinogen but not in trypsin. Furthermo re, 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 th at 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 struct ure in a conformation similar to that of the active state of VIIa.