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. 1992 Jul;90(1):238–244. doi: 10.1172/JCI115841

Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia. Homozygous substitution of B beta 68 Ala----Thr.

J Koopman 1, F Haverkate 1, S T Lord 1, J Grimbergen 1, P M Mannucci 1
PMCID: PMC443086  PMID: 1634610

Abstract

In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens.

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Selected References

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

  1. Al-Mondhiry H., Bilezikian S. B., Nossel H. L. Fibrinogen ""New York"--an abnormal fibrinogen associated with thromboembolism: functional evaluation. Blood. 1975 May;45(5):607–619. [PubMed] [Google Scholar]
  2. Berliner L. J., Sugawara Y., Fenton J. W., 2nd Human alpha-thrombin binding to nonpolymerized fibrin-Sepharose: evidence for an anionic binding region. Biochemistry. 1985 Nov 19;24(24):7005–7009. doi: 10.1021/bi00345a038. [DOI] [PubMed] [Google Scholar]
  3. Blombäck B., Hessel B., Hogg D., Therkildsen L. A two-step fibrinogen--fibrin transition in blood coagulation. Nature. 1978 Oct 12;275(5680):501–505. doi: 10.1038/275501a0. [DOI] [PubMed] [Google Scholar]
  4. Blombäck B., Hessel B., Iwanaga S., Reuterby J., Blombäck M. Primary structure of human fibrinogen and fibrin. I. Clevage of fibrinogen with cyanogen bromide. Isolation and characterization of NH 2 -terminal fragments of the ("A") chain. J Biol Chem. 1972 Mar 10;247(5):1496–1512. [PubMed] [Google Scholar]
  5. CLAUSS A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol. 1957 Apr;17(4):237–246. doi: 10.1159/000205234. [DOI] [PubMed] [Google Scholar]
  6. Chung D. W., Que B. G., Rixon M. W., Mace M., Jr, Davie E. W. Characterization of complementary deoxyribonucleic acid and genomic deoxyribonucleic acid for the beta chain of human fibrinogen. Biochemistry. 1983 Jun 21;22(13):3244–3250. doi: 10.1021/bi00282a032. [DOI] [PubMed] [Google Scholar]
  7. Doolittle R. F. Fibrinogen and fibrin. Annu Rev Biochem. 1984;53:195–229. doi: 10.1146/annurev.bi.53.070184.001211. [DOI] [PubMed] [Google Scholar]
  8. Engesser L., Koopman J., de Munk G., Haverkate F., Nováková I., Verheijen J. H., Briët E., Brommer E. J. Fibrinogen Nijmegen: congenital dysfibrinogenemia associated with impaired t-PA mediated plasminogen activation and decreased binding of t-PA. Thromb Haemost. 1988 Aug 30;60(1):113–120. [PubMed] [Google Scholar]
  9. Fenton J. W., 2nd, Fasco M. J., Stackrow A. B. Human thrombins. Production, evaluation, and properties of alpha-thrombin. J Biol Chem. 1977 Jun 10;252(11):3587–3598. [PubMed] [Google Scholar]
  10. Fenton J. W., 2nd, Olson T. A., Zabinski M. P., Wilner G. D. Anion-binding exosite of human alpha-thrombin and fibrin(ogen) recognition. Biochemistry. 1988 Sep 6;27(18):7106–7112. doi: 10.1021/bi00418a066. [DOI] [PubMed] [Google Scholar]
  11. Gustafson S., Proper J. A., Bowie E. J., Sommer S. S. Parameters affecting the yield of DNA from human blood. Anal Biochem. 1987 Sep;165(2):294–299. doi: 10.1016/0003-2697(87)90272-7. [DOI] [PubMed] [Google Scholar]
  12. Haverkate F., Koopman J., Kluft C., D'Angelo A., Cattaneo M., Mannucci P. M. Fibrinogen Milano II: a congenital dysfibrinogenaemia associated with juvenile arterial and venous thrombosis. Thromb Haemost. 1986 Feb 28;55(1):131–135. [PubMed] [Google Scholar]
  13. Haverkate F., Koopman J., Kluft C., D'Angelo A., Cattaneo M., Mannucci P. M. Fibrinogens "Milano II"- and "Naples". Thromb Haemost. 1987 Jun 3;57(3):375–375. [PubMed] [Google Scholar]
  14. Hogg D. H., Blombäck B. The mechanism of the fibrinogen-thrombin reaction. Thromb Res. 1978 Jun;12(6):953–964. doi: 10.1016/0049-3848(78)90051-8. [DOI] [PubMed] [Google Scholar]
  15. Hurlet-Jensen A., Cummins H. Z., Nossel H. L., Liu C. Y. Fibrin polymerization and release of fibrinopeptide B by thrombin. Thromb Res. 1982 Aug 15;27(4):419–427. doi: 10.1016/0049-3848(82)90059-7. [DOI] [PubMed] [Google Scholar]
  16. Kaczmarek E., McDonagh J. Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E. J Biol Chem. 1988 Sep 25;263(27):13896–13900. [PubMed] [Google Scholar]
  17. Kaminski M., McDonagh J. Inhibited thrombins. Interactions with fibrinogen and fibrin. Biochem J. 1987 Mar 15;242(3):881–887. doi: 10.1042/bj2420881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaminski M., McDonagh J. Studies on the mechanism of thrombin. Interaction with fibrin. J Biol Chem. 1983 Sep 10;258(17):10530–10535. [PubMed] [Google Scholar]
  19. Kehl M., Lottspeich F., Henschen A. Analysis of human fibrinopeptides by high-performance liquid chromatography. Hoppe Seylers Z Physiol Chem. 1981 Dec;362(12):1661–1664. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Liu C. Y., Koehn J. A., Morgan F. J. Characterization of fibrinogen New York 1. A dysfunctional fibrinogen with a deletion of B beta(9-72) corresponding exactly to exon 2 of the gene. J Biol Chem. 1985 Apr 10;260(7):4390–4396. [PubMed] [Google Scholar]
  22. Liu C. Y., Nossel H. L., Kaplan K. L. The binding of thrombin by fibrin. J Biol Chem. 1979 Oct 25;254(20):10421–10425. [PubMed] [Google Scholar]
  23. Lord S. T., Byrd P. A., Hede K. L., Wei C., Colby T. J. Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates. J Biol Chem. 1990 Jan 15;265(2):838–843. [PubMed] [Google Scholar]
  24. Mancini G., Carbonara A. O., Heremans J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 1965 Sep;2(3):235–254. doi: 10.1016/0019-2791(65)90004-2. [DOI] [PubMed] [Google Scholar]
  25. Marsh H. C., Jr, Meinwald Y. C., Thannhauser T. W., Scheraga H. A. Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10. Biochemistry. 1983 Aug 30;22(18):4170–4174. doi: 10.1021/bi00287a002. [DOI] [PubMed] [Google Scholar]
  26. Naski M. C., Shafer J. A. Alpha-thrombin-catalyzed hydrolysis of fibrin I. Alternative binding modes and the accessibility of the active site in fibrin I-bound alpha-thrombin. J Biol Chem. 1990 Jan 25;265(3):1401–1407. [PubMed] [Google Scholar]
  27. Ni F., Konishi Y., Frazier R. B., Scheraga H. A., Lord S. T. High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen. Biochemistry. 1989 Apr 4;28(7):3082–3094. doi: 10.1021/bi00433a052. [DOI] [PubMed] [Google Scholar]
  28. Ni F., Meinwald Y. C., Vásquez M., Scheraga H. A. High-resolution NMR studies of fibrinogen-like peptides in solution: structure of a thrombin-bound peptide corresponding to residues 7-16 of the A alpha chain of human fibrinogen. Biochemistry. 1989 Apr 4;28(7):3094–3105. doi: 10.1021/bi00433a053. [DOI] [PubMed] [Google Scholar]
  29. Nieuwenhuizen W., Emeis J. J., Vermond A., Kurver P., van der Heide D. Studies on the catabolism and distribution of fibrinogen in rats. Application of the Iodogen labelling technique. Biochem Biophys Res Commun. 1980 Nov 17;97(1):49–55. doi: 10.1016/s0006-291x(80)80132-x. [DOI] [PubMed] [Google Scholar]
  30. Rixon M. W., Chung D. W., Davie E. W. Nucleotide sequence of the gene for the gamma chain of human fibrinogen. Biochemistry. 1985 Apr 9;24(8):2077–2086. doi: 10.1021/bi00329a041. [DOI] [PubMed] [Google Scholar]
  31. Ruf W., Bender A., Lane D. A., Preissner K. T., Selmayr E., Müller-Berghaus G. Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization. Biochim Biophys Acta. 1988 May 12;965(2-3):169–175. doi: 10.1016/0304-4165(88)90053-0. [DOI] [PubMed] [Google Scholar]
  32. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  33. Southan C., Lane D. A., Bode W., Henschen A. Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution. Eur J Biochem. 1985 Mar 15;147(3):593–600. doi: 10.1111/j.0014-2956.1985.00593.x. [DOI] [PubMed] [Google Scholar]
  34. Vali Z., Scheraga H. A. Localization of the binding site on fibrin for the secondary binding site of thrombin. Biochemistry. 1988 Mar 22;27(6):1956–1963. doi: 10.1021/bi00406a023. [DOI] [PubMed] [Google Scholar]
  35. Van Ruijven-Vermeer I. A., Nieuwenhuizen W. Purification of rat fibrinogen and its constituent chains. Biochem J. 1978 Mar 1;169(3):653–658. doi: 10.1042/bj1690653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilner G. D., Danitz M. P., Mudd M. S., Hsieh K. H., Fenton J. W., 2nd Selective immobilization of alpha-thrombin by surface-bound fibrin. J Lab Clin Med. 1981 Mar;97(3):403–411. [PubMed] [Google Scholar]

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