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Logo of jcinvestThe Journal of Clinical Investigation
J Clin Invest. 1989 October; 84(4): 1236–1242.
PMCID: PMC329783

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


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

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  • Shanbaky NM, Ahn Y, Jy W, Harrington W, Fernandez L, Haynes DH. Abnormal aggregation accompanies abnormal platelet Ca2+ handling in arterial thrombosis. Thromb Haemost. 1987 Feb 3;57(1):1–10. [PubMed]
  • Rosenberg RD. Actions and interactions of antithrombin and heparin. N Engl J Med. 1975 Jan 16;292(3):146–151. [PubMed]
  • Bock SC, Wion KL, Vehar GA, Lawn RM. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acids Res. 1982 Dec 20;10(24):8113–8125. [PMC free article] [PubMed]
  • Prochownik EV, Markham AF, Orkin SH. Isolation of a cDNA clone for human antithrombin III. J Biol Chem. 1983 Jul 10;258(13):8389–8394. [PubMed]
  • Prochownik EV, Bock SC, Orkin SH. 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]
  • Chandra T, Stackhouse R, Kidd VJ, Woo SL. 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. [PubMed]
  • Wolf M, Boyer C, Lavergne JM, Larrieu MJ. A new familial variant of antithrombin III: 'antithrombin III Paris'. Br J Haematol. 1982 Jun;51(2):285–295. [PubMed]
  • Fischer AM, Beguin S, Sternberg C, Dautzenberg MD. Comparative effect of heparin and heparan sulphate on two abnormal antithrombin III type 3 variants. Br J Haematol. 1987 Jun;66(2):213–217. [PubMed]
  • Aiach M, François D, Priollet P, Capron L, Roncato M, Alhenc-Gelas M, Fiessinger JN. An abnormal antithrombin III (AT III) with low heparin affinity: AT III Clichy. Br J Haematol. 1987 Aug;66(4):515–522. [PubMed]
  • Fischer AM, Cornu P, Sternberg C, Mériane F, Dautzenberg MD, 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]
  • Aiach M, Nora M, Fiessinger JN, Roncato M, François D, Gelas MA. A functional abnormal antithrombin III (AT III) deficiency: AT III Charleville. Thromb Res. 1985 Sep 1;39(5):559–570. [PubMed]
  • Aiach M, Roncato M, Chadeuf G, Dezellus P, Capron L, Fiessinger JN. 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]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Bell GI, Karam JH, Rutter WJ. 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. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • 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. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Brunel F, Duchange N, Fischer AM, Cohen GN, Zakin MM. Antithrombin III Alger: a new case of Arg 47----Cys mutation. Am J Hematol. 1987 Jun;25(2):223–224. [PubMed]
  • 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. [PubMed]
  • Youssoufian H, Kazazian HH, Jr, Phillips DG, Aronis S, Tsiftis G, Brown VA, Antonarakis SE. Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature. 324(6095):380–382. [PubMed]
  • 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. [PubMed]
  • Blinder MA, Marasa JC, Reynolds CH, Deaven LL, Tollefsen DM. Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli. Biochemistry. 1988 Jan 26;27(2):752–759. [PubMed]
  • Borg JY, Owen MC, Soria C, Soria J, Caen J, Carrell RW. 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. [PMC free article] [PubMed]
  • Smith JW, Knauer DJ. A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence. J Biol Chem. 1987 Sep 5;262(25):11964–11972. [PubMed]
  • 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]
  • Hultin MB, 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]
  • Carrell RW, Owen MC. Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis. Nature. 1985 Oct 24;317(6039):730–732. [PubMed]
  • Lane DA, Erdjument H, Flynn A, Di Marzo V, Panico M, Morris HR, Greaves M, Dolan G, Preston FE. Antithrombin Sheffield: amino acid substitution at the reactive site (Arg393 to His) causing thrombosis. Br J Haematol. 1989 Jan;71(1):91–96. [PubMed]
  • Erdjument H, Lane DA, Panico M, Di Marzo V, Morris HR. 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]
  • Owen MC, Beresford CH, Carrell RW. 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. [PubMed]
  • Stephens AW, Thalley BS, Hirs CH. Antithrombin-III Denver, a reactive site variant. J Biol Chem. 1987 Jan 25;262(3):1044–1048. [PubMed]
  • Bock SC, Marrinan JA, 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. [PubMed]
  • Devraj-Kizuk R, Chui DH, Prochownik EV, Carter CJ, Ofosu FA, Blajchman MA. 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]
  • Chang JY, Tran TH. 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]
  • Chasse JF, Esnard F, Guitton JD, 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. [PubMed]
  • Duchange N, Chasse JF, Cohen GN, Zakin MM. Molecular characterization of the antithrombin III tours deficiency. Thromb Res. 1987 Jan 1;45(1):115–121. [PubMed]
  • 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. [PubMed]
  • Owen MC, Borg JY, Soria C, Soria J, Caen J, Carrell RW. Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His. Blood. 1987 May;69(5):1275–1279. [PubMed]

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