A Novel Exosite on Coagulation Factor VIIa and Its Molecular Interactions with a New Class of Peptide Inhibitors

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• 作者：Martin Roberge ; Lydia Santell ; Mark S. Dennis ; Charles Eigenbrot ; Mary A. Dwyer ; and Robert A. Lazarus
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：August 14, 2001
• 年：2001
• 卷：40
• 期：32
• 页码：9522 - 9531
• 全文大小：628K
• 年卷期：v.40,no.32(August 14, 2001)
• ISSN：1520-4995

文摘

A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa(FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides,identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M.,Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that isdistinct from both the active site and the exosite 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]. Peptide A-183 prolonged TF-dependent clottingin human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbitsequence differences in the protease domain, localized the binding site to residues in the 60s loop and theC-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showedthat proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium bindingconstants for zymogen FVII, FVIIa and TF·FVIIa were determined using immobilized N-terminalbiotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteaseswas observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FXactivation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of themutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version ofzymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., andUltsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active sitemay lend itself towards the design of new anticoagulants that inhibit FVIIa.