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. 1989 Oct;84(4):1236–1242. doi: 10.1172/JCI114290

Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

P Molho-Sabatier 1, M Aiach 1, I Gaillard 1, J N Fiessinger 1, A M Fischer 1, G Chadeuf 1, E Clauser 1
PMCID: PMC329783  PMID: 2794060

Abstract

The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.

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Selected References

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

  1. Aiach M., François D., Priollet P., Capron L., Roncato M., Alhenc-Gelas M., Fiessinger J. N. An abnormal antithrombin III (AT III) with low heparin affinity: AT III Clichy. Br J Haematol. 1987 Aug;66(4):515–522. doi: 10.1111/j.1365-2141.1987.tb01337.x. [DOI] [PubMed] [Google Scholar]
  2. Aiach M., Nora M., Fiessinger J. N., Roncato M., François D., Gelas M. A. A functional abnormal antithrombin III (AT III) deficiency: AT III Charleville. Thromb Res. 1985 Sep 1;39(5):559–570. doi: 10.1016/0049-3848(85)90236-1. [DOI] [PubMed] [Google Scholar]
  3. Aiach M., Roncato M., Chadeuf G., Dezellus P., Capron L., Fiessinger J. N. Antithrombin III Avranches, a new variant with defective serine-protease inhibition--comparison with antithrombin III Charleville. Thromb Haemost. 1988 Aug 30;60(1):94–96. [PubMed] [Google Scholar]
  4. Bell G. I., Karam J. H., Rutter W. J. Polymorphic DNA region adjacent to the 5' end of the human insulin gene. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5759–5763. doi: 10.1073/pnas.78.9.5759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blinder M. A., Marasa J. C., Reynolds C. H., Deaven L. L., Tollefsen D. M. Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli. Biochemistry. 1988 Jan 26;27(2):752–759. doi: 10.1021/bi00402a039. [DOI] [PubMed] [Google Scholar]
  6. Bock S. C., Marrinan J. A., Radziejewska E. Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site. Biochemistry. 1988 Aug 9;27(16):6171–6178. doi: 10.1021/bi00416a052. [DOI] [PubMed] [Google Scholar]
  7. Bock S. C., Wion K. L., Vehar G. A., Lawn R. M. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acids Res. 1982 Dec 20;10(24):8113–8125. doi: 10.1093/nar/10.24.8113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Borg J. Y., Owen M. C., Soria C., Soria J., Caen J., Carrell R. W. Proposed heparin binding site in antithrombin based on arginine 47. A new variant Rouen-II, 47 Arg to Ser. J Clin Invest. 1988 Apr;81(4):1292–1296. doi: 10.1172/JCI113447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brunel F., Duchange N., Fischer A. M., Cohen G. N., Zakin M. M. Antithrombin III Alger: a new case of Arg 47----Cys mutation. Am J Hematol. 1987 Jun;25(2):223–224. doi: 10.1002/ajh.2830250214. [DOI] [PubMed] [Google Scholar]
  10. Carrell R. W., Owen M. C. Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis. Nature. 1985 Oct 24;317(6039):730–732. doi: 10.1038/317730a0. [DOI] [PubMed] [Google Scholar]
  11. Chandra T., Stackhouse R., Kidd V. J., Woo S. L. Isolation and sequence characterization of a cDNA clone of human antithrombin III. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1845–1848. doi: 10.1073/pnas.80.7.1845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chang J. Y., Tran T. H. Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity. J Biol Chem. 1986 Jan 25;261(3):1174–1176. [PubMed] [Google Scholar]
  13. Chasse J. F., Esnard F., Guitton J. D., Mouray H., Perigois F., Fauconneau G., Gauthier F. An abnormal plasma antithrombin with no apparent affinity for heparin. Thromb Res. 1984 May 15;34(4):297–302. doi: 10.1016/0049-3848(84)90386-4. [DOI] [PubMed] [Google Scholar]
  14. Devraj-Kizuk R., Chui D. H., Prochownik E. V., Carter C. J., Ofosu F. A., Blajchman M. A. Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity. Blood. 1988 Nov;72(5):1518–1523. [PubMed] [Google Scholar]
  15. Duchange N., Chasse J. F., Cohen G. N., Zakin M. M. Molecular characterization of the antithrombin III tours deficiency. Thromb Res. 1987 Jan 1;45(1):115–121. doi: 10.1016/0049-3848(87)90263-5. [DOI] [PubMed] [Google Scholar]
  16. Erdjument H., Lane D. A., Panico M., Di Marzo V., Morris H. R. Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow. J Biol Chem. 1988 Apr 25;263(12):5589–5593. [PubMed] [Google Scholar]
  17. Finazzi G., Caccia R., Barbui T. Different prevalence of thromboembolism in the subtypes of congenital antithrombin III deficiency: review of 404 cases. Thromb Haemost. 1987 Dec 18;58(4):1094–1094. [PubMed] [Google Scholar]
  18. Fischer A. M., Beguin S., Sternberg C., Dautzenberg M. D. Comparative effect of heparin and heparan sulphate on two abnormal antithrombin III type 3 variants. Br J Haematol. 1987 Jun;66(2):213–217. doi: 10.1111/j.1365-2141.1987.tb01301.x. [DOI] [PubMed] [Google Scholar]
  19. Fischer A. M., Cornu P., Sternberg C., Mériane F., Dautzenberg M. D., Chafa O., Beguin S., Desnos M. Antithrombin III Alger: a new homozygous AT III variant. Thromb Haemost. 1986 Apr 30;55(2):218–221. [PubMed] [Google Scholar]
  20. Hultin M. B., McKay J., Abildgaard U. Antithrombin Oslo: type Ib classification of the first reported antithrombin-deficient family, with a review of hereditary antithrombin variants. Thromb Haemost. 1988 Jun 16;59(3):468–473. [PubMed] [Google Scholar]
  21. Koide T., Foster D., Odani S. The heparin-binding site(s) of histidine-rich glycoprotein as suggested by sequence homology with antithrombin III. FEBS Lett. 1986 Jan 6;194(2):242–244. doi: 10.1016/0014-5793(86)80092-8. [DOI] [PubMed] [Google Scholar]
  22. Koide T., Odani S., Takahashi K., Ono T., Sakuragawa N. Antithrombin III Toyama: replacement of arginine-47 by cysteine in hereditary abnormal antithrombin III that lacks heparin-binding ability. Proc Natl Acad Sci U S A. 1984 Jan;81(2):289–293. doi: 10.1073/pnas.81.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lane D. A., Erdjument H., Flynn A., Di Marzo V., Panico M., Morris H. R., Greaves M., Dolan G., Preston F. E. Antithrombin Sheffield: amino acid substitution at the reactive site (Arg393 to His) causing thrombosis. Br J Haematol. 1989 Jan;71(1):91–96. doi: 10.1111/j.1365-2141.1989.tb06280.x. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  26. Owen M. C., Beresford C. H., Carrell R. W. Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity. FEBS Lett. 1988 Apr 25;231(2):317–320. doi: 10.1016/0014-5793(88)80841-x. [DOI] [PubMed] [Google Scholar]
  27. Owen M. C., Borg J. Y., Soria C., Soria J., Caen J., Carrell R. W. Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His. Blood. 1987 May;69(5):1275–1279. [PubMed] [Google Scholar]
  28. Prochownik E. V., Bock S. C., Orkin S. H. Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily. J Biol Chem. 1985 Aug 15;260(17):9608–9612. [PubMed] [Google Scholar]
  29. Prochownik E. V., Markham A. F., Orkin S. H. Isolation of a cDNA clone for human antithrombin III. J Biol Chem. 1983 Jul 10;258(13):8389–8394. [PubMed] [Google Scholar]
  30. Rosenberg R. D. Actions and interactions of antithrombin and heparin. N Engl J Med. 1975 Jan 16;292(3):146–151. doi: 10.1056/NEJM197501162920307. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Sakuragawa N., Takahashi K., Kondo S., Koide T. Antithrombin III Toyama: a hereditary abnormal antithrombin III of a patient with recurrent thrombophlebitis. Thromb Res. 1983 Jul 15;31(2):305–317. doi: 10.1016/0049-3848(83)90333-x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Shanbaky N. M., Ahn Y., Jy W., Harrington W., Fernandez L., Haynes D. H. Abnormal aggregation accompanies abnormal platelet Ca2+ handling in arterial thrombosis. Thromb Haemost. 1987 Feb 3;57(1):1–10. [PubMed] [Google Scholar]
  35. Smith J. W., Knauer D. J. A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence. J Biol Chem. 1987 Sep 5;262(25):11964–11972. [PubMed] [Google Scholar]
  36. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  37. Stephens A. W., Thalley B. S., Hirs C. H. Antithrombin-III Denver, a reactive site variant. J Biol Chem. 1987 Jan 25;262(3):1044–1048. [PubMed] [Google Scholar]
  38. Wolf M., Boyer C., Lavergne J. M., Larrieu M. J. A new familial variant of antithrombin III: 'antithrombin III Paris'. Br J Haematol. 1982 Jun;51(2):285–295. doi: 10.1111/j.1365-2141.1982.tb02782.x. [DOI] [PubMed] [Google Scholar]
  39. Youssoufian H., Kazazian H. H., Jr, Phillips D. G., Aronis S., Tsiftis G., Brown V. A., Antonarakis S. E. Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. 1986 Nov 27-Dec 3Nature. 324(6095):380–382. doi: 10.1038/324380a0. [DOI] [PubMed] [Google Scholar]

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