Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 May;85(9):3165–3169. doi: 10.1073/pnas.85.9.3165

Localization of a factor VIII-inhibiting antibody epitope to a region between residues 338 and 362 of factor VIII heavy chain.

J Ware 1, J R Toomey 1, D W Stafford 1
PMCID: PMC280164  PMID: 2452445

Abstract

We have used a recombinant DNA epitope library to localize the binding region of a factor VIII (FVIII) monoclonal antibody that neutralizes coagulant activity. The antibody, C5, has previously been described and has been shown to have a FVIII neutralizing potency of 1488 Bethesda units per mg of purified immunoglobulin. A recombinant DNA epitope library was constructed from short, random FVIII cDNA fragments and immunologically screened with C5 to identify bacteriophage expressing the antigenic determinant. The isolation and characterization of immunoreactive bacteriophage restricted the C5 epitope to the overlapping or shared DNA sequence of nine different clones and corresponded to amino acid residues 338-362 of the mature FVIII peptide. The defined epitope is between the proposed activated protein C cleavage site (Arg-336) and thrombin cleavage site (Arg-372) on the amino-terminal 90-kDa FVIII heavy-chain subunit. The identification of the epitope of an inhibiting anti-FVIII antibody between two critical cleavage sites suggests that this amino acid sequence plays a role in regulating FVIII coagulant activity.

Full text

PDF
3165

Images in this article

3166Image
on p.3166

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burke R. L., Pachl C., Quiroga M., Rosenberg S., Haigwood N., Nordfang O., Ezban M. The functional domains of coagulation factor VIII:C. J Biol Chem. 1986 Sep 25;261(27):12574–12578. [PubMed] [Google Scholar]
  3. Eaton D. L., Vehar G. A. Factor VIII structure and proteolytic processing. Prog Hemost Thromb. 1986;8:47–70. [PubMed] [Google Scholar]
  4. Eaton D., Rodriguez H., Vehar G. A. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 1986 Jan 28;25(2):505–512. doi: 10.1021/bi00350a035. [DOI] [PubMed] [Google Scholar]
  5. Eaton D., Rodriguez H., Vehar G. A. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 1986 Jan 28;25(2):505–512. doi: 10.1021/bi00350a035. [DOI] [PubMed] [Google Scholar]
  6. Fulcher C. A., Gardiner J. E., Griffin J. H., Zimmerman T. S. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood. 1984 Feb;63(2):486–489. [PubMed] [Google Scholar]
  7. Fulcher C. A., Roberts J. R., Holland L. Z., Zimmerman T. S. Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes. J Clin Invest. 1985 Jul;76(1):117–124. doi: 10.1172/JCI111933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fulcher C. A., Zimmerman T. S. Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1648–1652. doi: 10.1073/pnas.79.5.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gitschier J., Wood W. I., Goralka T. M., Wion K. L., Chen E. Y., Eaton D. H., Vehar G. A., Capon D. J., Lawn R. M. Characterization of the human factor VIII gene. Nature. 1984 Nov 22;312(5992):326–330. doi: 10.1038/312326a0. [DOI] [PubMed] [Google Scholar]
  10. Goodall A. H., Meyer D. Registry of monoclonal antibodies to factor VIII and von Willebrand factor. International Committee on Thrombosis and Haemostasis. Thromb Haemost. 1985 Dec 17;54(4):878–891. [PubMed] [Google Scholar]
  11. Goto T., Wang J. C. Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell. 1984 Apr;36(4):1073–1080. doi: 10.1016/0092-8674(84)90057-6. [DOI] [PubMed] [Google Scholar]
  12. Guinto E. R., Esmon C. T. Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C. J Biol Chem. 1984 Nov 25;259(22):13986–13992. [PubMed] [Google Scholar]
  13. Jenny R. J., Pittman D. D., Toole J. J., Kriz R. W., Aldape R. A., Hewick R. M., Kaufman R. J., Mann K. G. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4846–4850. doi: 10.1073/pnas.84.14.4846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koschinsky M. L., Funk W. D., van Oost B. A., MacGillivray R. T. Complete cDNA sequence of human preceruloplasmin. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5086–5090. doi: 10.1073/pnas.83.14.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lamb J. R., Ivanyi J., Rees A. D., Rothbard J. B., Howland K., Young R. A., Young D. B. Mapping of T cell epitopes using recombinant antigens and synthetic peptides. EMBO J. 1987 May;6(5):1245–1249. doi: 10.1002/j.1460-2075.1987.tb02360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lawn R. M. The molecular genetics of hemophilia: blood clotting factors VIII and IX. Cell. 1985 Sep;42(2):405–406. doi: 10.1016/0092-8674(85)90094-7. [DOI] [PubMed] [Google Scholar]
  17. Mann K. G. Membrane-bound enzyme complexes in blood coagulation. Prog Hemost Thromb. 1984;7:1–23. [PubMed] [Google Scholar]
  18. McGraw R., Frazier D., de Serres M., Reisner H., Stafford D. Antigenic determinant in human coagulation factor IX: immunological screening and DNA sequence analysis of recombinant phage map a monoclonal antibody to residues 111 through 132 of the zymogen. Blood. 1986 May;67(5):1344–1348. [PubMed] [Google Scholar]
  19. Mehra V., Sweetser D., Young R. A. Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7013–7017. doi: 10.1073/pnas.83.18.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Melgar E., Goldthwait D. A. Deoxyribonucleic acid nucleases. II. The effects of metals on the mechanism of action of deoxyribonuclease I. J Biol Chem. 1968 Sep 10;243(17):4409–4416. [PubMed] [Google Scholar]
  21. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  22. Nunberg J. H., Rodgers G., Gilbert J. H., Snead R. M. Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3675–3679. doi: 10.1073/pnas.81.12.3675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reinach F. C., Fischman D. A. Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2. J Mol Biol. 1985 Feb 5;181(3):411–422. doi: 10.1016/0022-2836(85)90229-3. [DOI] [PubMed] [Google Scholar]
  24. Rotblat F., O'Brien D. P., O'Brien F. J., Goodall A. H., Tuddenham E. G. Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies. Biochemistry. 1985 Jul 30;24(16):4294–4300. doi: 10.1021/bi00337a007. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toole J. J., Knopf J. L., Wozney J. M., Sultzman L. A., Buecker J. L., Pittman D. D., Kaufman R. J., Brown E., Shoemaker C., Orr E. C. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature. 1984 Nov 22;312(5992):342–347. doi: 10.1038/312342a0. [DOI] [PubMed] [Google Scholar]
  27. Truett M. A., Blacher R., Burke R. L., Caput D., Chu C., Dina D., Hartog K., Kuo C. H., Masiarz F. R., Merryweather J. P. Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA. DNA. 1985 Oct;4(5):333–349. doi: 10.1089/dna.1985.4.333. [DOI] [PubMed] [Google Scholar]
  28. Vehar G. A., Keyt B., Eaton D., Rodriguez H., O'Brien D. P., Rotblat F., Oppermann H., Keck R., Wood W. I., Harkins R. N. Structure of human factor VIII. Nature. 1984 Nov 22;312(5992):337–342. doi: 10.1038/312337a0. [DOI] [PubMed] [Google Scholar]
  29. Wood W. I., Capon D. J., Simonsen C. C., Eaton D. L., Gitschier J., Keyt B., Seeburg P. H., Smith D. H., Hollingshead P., Wion K. L. Expression of active human factor VIII from recombinant DNA clones. Nature. 1984 Nov 22;312(5992):330–337. doi: 10.1038/312330a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES