THE MECHANISM OF AN INHIBITORY ANTIBODY ON TF-INITIATED BLOOD-COAGULATION REVEALED BY THE CRYSTAL-STRUCTURES OF HUMAN TISSUE FACTOR, FAB5G9AND TF-CENTER-DOT-5G9 COMPLEX

The tissue factor (TF)-initiated blood coagulation protease cascade ca n be greatly inhibited in vivo by a potent anti-human-TF monoclonal an tibody, 5G9. This antibody binds the carboxyl module of the extracellu lar domain of TF with a nanomolar binding constant and inhibits the fo rmation of the TF.VIIa.X ternary initiation complex. We have determine d the crystal structures of the extra-cellular modules of human TF, Fa b 5G9, and their complex (TF.5G9) to 2.4 Angstrom, 2.5 Angstrom, and 3 .0 Angstrom, respectively, and measured the apparent inhibition consta nts of 5G9 on a panel of TF mutants. In our unliganded TF structure, a 7 degrees change in the relative orientation between the D1 and D2 mo dules was observed when compared with other published TF structures. C omparison of the free and bound Fab 5G9 indicates that small segmental and side-chain variation of the antibody complementarity determining regions occurred on complexation with TF. The antibody-antigen recogni tion involves 18 TF antigen residues and 19 Fab residues from six CDRs with one of the largest buried surface areas seen to date. A combinat ion of structural and mutagenesis data indicate that Tyr156, Lys169, A rg200, and Lys201 play the major role in the antibody recognition. The TF.5G9 structure provides insights into the mechanism by which the an tibody 5G9 inhibits formation of the TF.VIIa.X ternary complex. (C) 19 98 Academic Press Limited.