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. 2002 Apr 15;363(Pt 2):387–393. doi: 10.1042/0264-6021:3630387

Fusion of two distinct peptide exosite inhibitors of Factor VIIa.

Martin Roberge 1, Mark Peek 1, Daniel Kirchhofer 1, Mark S Dennis 1, Robert A Lazarus 1
PMCID: PMC1222490  PMID: 11931669

Abstract

Highly potent bifunctional inhibitors of Factor VIIa (FVIIa) were generated by linking two distinct peptides, recently shown to bind to two discrete exosites on the FVIIa protease domain [Dennis, Eigenbrot, Skelton, Ultsch, Santell, Dwyer, O'Connell and Lazarus (2000) Nature (London) 404, 465-470; Dennis, Roberge, Quan and Lazarus (2001) Biochemistry 40, 9513-9521; Roberge, Santell, Dennis, Eigenbrot, Dwyer and Lazarus (2001) Biochemistry 40, 9522-9531]. Fusion peptides consisting of an N-terminal A-series peptide followed by flexible linkers, an E-series peptide, and the Z-domain of protein A were expressed in Escherichia coli and purified using IgG-Sepharose affinity chromatography. The fusion peptides were potent anticoagulants and had steep concentration dependence curves in tissue factor-dependent prothrombin time (PT) assays in comparison to the individual peptides or their noncovalent combination. This phenomenon was dependent on the length of the linker joining the A- and E-peptides. The fusion of the peptides increased the extent of inhibition of Factor X (FX) activation to 100% at saturating peptide concentrations, but did not improve the binding affinity for Factor VIIa (FVIIa) at the A- and E- binding sites or the IC(50) for the inhibition of FX activation. Differences between the peptides in the PT fold prolongation in normal and FVII-deficient plasma, in conjunction with the inhibition of (125)I-FVII activation, suggest that the enhanced effects of the fusion peptides involve the inhibition of FVII autoactivation.

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Selected References

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  1. Butenas S., Mann K. G. Kinetics of human factor VII activation. Biochemistry. 1996 Feb 13;35(6):1904–1910. doi: 10.1021/bi951768c. [DOI] [PubMed] [Google Scholar]
  2. Davie E. W. Biochemical and molecular aspects of the coagulation cascade. Thromb Haemost. 1995 Jul;74(1):1–6. [PubMed] [Google Scholar]
  3. Dennis M. S., Eigenbrot C., Skelton N. J., Ultsch M. H., Santell L., Dwyer M. A., O'Connell M. P., Lazarus R. A. Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature. 2000 Mar 30;404(6777):465–470. doi: 10.1038/35006574. [DOI] [PubMed] [Google Scholar]
  4. Dennis M. S., Roberge M., Quan C., Lazarus R. A. Selection and characterization of a new class of peptide exosite inhibitors of coagulation factor VIIa. Biochemistry. 2001 Aug 14;40(32):9513–9521. doi: 10.1021/bi010591l. [DOI] [PubMed] [Google Scholar]
  5. Eigenbrot C., Kirchhofer D., Dennis M. S., Santell L., Lazarus R. A., Stamos J., Ultsch M. H. The factor VII zymogen structure reveals reregistration of beta strands during activation. Structure. 2001 Jul 3;9(7):627–636. doi: 10.1016/s0969-2126(01)00624-4. [DOI] [PubMed] [Google Scholar]
  6. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  7. Girard T. J., MacPhail L. A., Likert K. M., Novotny W. F., Miletich J. P., Broze G. J., Jr Inhibition of factor VIIa-tissue factor coagulation activity by a hybrid protein. Science. 1990 Jun 15;248(4961):1421–1424. doi: 10.1126/science.1972598. [DOI] [PubMed] [Google Scholar]
  8. Hirsh J. Heparin. N Engl J Med. 1991 May 30;324(22):1565–1574. doi: 10.1056/NEJM199105303242206. [DOI] [PubMed] [Google Scholar]
  9. Johnson K., Hung D. Novel anticoagulants based on inhibition of the factor VIIa/tissue factor pathway. Coron Artery Dis. 1998;9(2-3):83–87. [PubMed] [Google Scholar]
  10. Kirchhofer D., Eigenbrot C., Lipari M. T., Moran P., Peek M., Kelley R. F. The tissue factor region that interacts with factor Xa in the activation of factor VII. Biochemistry. 2001 Jan 23;40(3):675–682. doi: 10.1021/bi002013v. [DOI] [PubMed] [Google Scholar]
  11. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  12. Lee G. F., Lazarus R. A., Kelley R. F. Potent bifunctional anticoagulants: Kunitz domain-tissue factor fusion proteins. Biochemistry. 1997 May 13;36(19):5607–5611. doi: 10.1021/bi970388j. [DOI] [PubMed] [Google Scholar]
  13. Mann K. G. Biochemistry and physiology of blood coagulation. Thromb Haemost. 1999 Aug;82(2):165–174. [PubMed] [Google Scholar]
  14. Morrissey J. H., Macik B. G., Neuenschwander P. F., Comp P. C. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood. 1993 Feb 1;81(3):734–744. [PubMed] [Google Scholar]
  15. Nakagaki T., Foster D. C., Berkner K. L., Kisiel W. Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII. Biochemistry. 1991 Nov 12;30(45):10819–10824. doi: 10.1021/bi00109a001. [DOI] [PubMed] [Google Scholar]
  16. Nemerson Y. The reaction between bovine brain tissue factor and factors VII and X. Biochemistry. 1966 Feb;5(2):601–608. doi: 10.1021/bi00866a029. [DOI] [PubMed] [Google Scholar]
  17. Neuenschwander P. F., Fiore M. M., Morrissey J. H. Factor VII autoactivation proceeds via interaction of distinct protease-cofactor and zymogen-cofactor complexes. Implications of a two-dimensional enzyme kinetic mechanism. J Biol Chem. 1993 Oct 15;268(29):21489–21492. [PubMed] [Google Scholar]
  18. Osterud B., Rapaport S. I. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5260–5264. doi: 10.1073/pnas.74.12.5260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Presta L., Sims P., Meng Y. G., Moran P., Bullens S., Bunting S., Schoenfeld J., Lowe D., Lai J., Rancatore P. Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic. Thromb Haemost. 2001 Mar;85(3):379–389. [PubMed] [Google Scholar]
  20. Radcliffe R., Nemerson Y. Activation and control of factor VII by activated factor X and thrombin. Isolation and characterization of a single chain form of factor VII. J Biol Chem. 1975 Jan 25;250(2):388–395. [PubMed] [Google Scholar]
  21. Rao L. V., Rapaport S. I. Activation of factor VII bound to tissue factor: a key early step in the tissue factor pathway of blood coagulation. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6687–6691. doi: 10.1073/pnas.85.18.6687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rapaport S. I., Rao L. V. The tissue factor pathway: how it has become a "prima ballerina". Thromb Haemost. 1995 Jul;74(1):7–17. [PubMed] [Google Scholar]
  23. Refino C. J., Himber J., Burcklen L., Moran P., Peek M., Suggett S., Devaux B., Kirchhofer D. A human antibody that binds to the gamma-carboxyglutamic acid domain of factor IX is a potent antithrombotic in vivo. Thromb Haemost. 1999 Sep;82(3):1188–1195. [PubMed] [Google Scholar]
  24. Roberge M., Santell L., Dennis M. S., Eigenbrot C., Dwyer M. A., Lazarus R. A. A novel exosite on coagulation factor VIIa and its molecular interactions with a new class of peptide inhibitors. Biochemistry. 2001 Aug 14;40(32):9522–9531. doi: 10.1021/bi010592d. [DOI] [PubMed] [Google Scholar]
  25. Rydel T. J., Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C., Fenton J. W., 2nd The structure of a complex of recombinant hirudin and human alpha-thrombin. Science. 1990 Jul 20;249(4966):277–280. doi: 10.1126/science.2374926. [DOI] [PubMed] [Google Scholar]
  26. Seymour J. L., Lindquist R. N., Dennis M. S., Moffat B., Yansura D., Reilly D., Wessinger M. E., Lazarus R. A. Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. Biochemistry. 1994 Apr 5;33(13):3949–3958. doi: 10.1021/bi00179a022. [DOI] [PubMed] [Google Scholar]
  27. Weitz J. I. Low-molecular-weight heparins. N Engl J Med. 1997 Sep 4;337(10):688–698. doi: 10.1056/NEJM199709043371007. [DOI] [PubMed] [Google Scholar]
  28. Wiiger M. T., Prydz H. Cellular effects of initiation of the extrinsic pathway of blood coagulation. Trends Cardiovasc Med. 2000 Nov;10(8):360–365. doi: 10.1016/s1050-1738(01)00074-3. [DOI] [PubMed] [Google Scholar]
  29. van de Locht A., Lamba D., Bauer M., Huber R., Friedrich T., Kröger B., Höffken W., Bode W. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin. EMBO J. 1995 Nov 1;14(21):5149–5157. doi: 10.1002/j.1460-2075.1995.tb00199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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