A novel exosite on coagulation Factor VIIa and its molecular interactions with a new class of peptide inhibitors

A new inhibitory peptide binding exosite on the protease domain of coagulat ion Factor VIIa (FVIIa) has been identified. A novel series of peptide inhi bitors of FVIIa, termed the "A-series" peptides, identified from peptide ph age libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifica lly bind at a site that is distinct from both the active site and the exosi te of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-470]. Pept ide A- 183 prolonged TF-dependent clotting in human. but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit seque nce differences in the protease domain, localized the binding site to resid ues in the 60s loop and the C-terminus. The location of the exosite was ref ined by a series of FVIIa alanine mutants, which showed that proximal resid ues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium b inding constants for zymogen FVII, FVIIa and TF-FVIIa were determined using immobilized N-terminal biotinylated A- 183 by surface plasmon resonance. N o peptide binding to nine other human serine proteases was observed. Key re sidues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A- 183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymoge n FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9. 627-6361. The sha pe and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.