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Peptide exosite inhibitors of factor VIIa as anticoagulants

Abstract

Potent anticoagulants have been derived by targeting the tissue factor–factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric ‘switch’ mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.

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Figure 1: Inhibition of TF–FVIIa-catalysed activation of FX and chromogenic activity by E-76.
Figure 2: Lineweaver–Burk plots for E-76 inhibition of FX activation and amidolytic activity.
Figure 3: Fold prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) of E-76 in human plasma.
Figure 4: Solution structure of E-76.
Figure 5: Location of the E-76 binding site.
Figure 6: Stereo view of the E-76–FVIIa complex.

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Acknowledgements

We thank R. Kelley for TF and helpful discussions; R. Artis, R. McDowell, C. Refino and D. Kirchhofer for helpful discussions; R. Smyth and S. Bullens for clotting assays; H. Lowman, B. Cunningham and G. Nakamura for help with library constructions; C. Quan, J. Tom and M. Struble for peptide synthesis; M. Beresini, A. Hebert and L. Caris for ELISA assays; W. Prince for inhibition assays; P. Jhurani, P. Ng and M. Vasser for DNA synthesis; M. Hamner, A. Zhong and A. Goddard for DNA sequencing; D. Stafford and J. Toomey for plasmids encoding FVII and FVII/FIX chimaeras; B. J. Clarke for a preprint on rabbit FVIIa expression and characterization; and A. de Vos, J. Burnier and D. Lowe for their support. This work is based in part upon research conducted at the Stanford Synchrotron Radiation Laboratory (SSRL), which is funded by the Department of Energy, Office of Basic Energy Sciences.

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Dennis, M., Eigenbrot, C., Skelton, N. et al. Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature 404, 465–470 (2000). https://doi.org/10.1038/35006574

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