Download PDF
Thromb Haemost 2002; 88(03): 436-443
DOI: 10.1055/s-0037-1613235
Review Article
Schattauer GmbH

Antithrombin ‘DREUX’ (Lys114Glu): A Variant with Complete Loss of Heparin Affinity

Alec Mushunje   *
1   Department of Haematology, University of Cambridge, Cambridge, Institute of Medical Research, Hills Road, Cambridge, UK
,
Aiwu Zhou   *
1   Department of Haematology, University of Cambridge, Cambridge, Institute of Medical Research, Hills Road, Cambridge, UK
,
James A. Huntington
1   Department of Haematology, University of Cambridge, Cambridge, Institute of Medical Research, Hills Road, Cambridge, UK
,
Jacqueline Conard
1   Department of Haematology, University of Cambridge, Cambridge, Institute of Medical Research, Hills Road , ambridge, UK Hemost asis and Thrombosis Unit, Hôtel-Dieu, Paris, France
,
Robin W. Carrell
1   Department of Haematology, University of Cambridge, Cambridge, Institute of Medical Research, Hills Road, Cambridge, UK
› Author Affiliations
Further Information

Publication History

Received 07 January 2002

Accepted after resubmission 28 May 2002

Publication Date:
08 December 2017 (online)

   Permissions and Reprints

Summary

Here we report the finding of a new natural antithrombin mutation that confirms the critical contribution of lysine 114 to the binding of the core heparin pentasaccharide, with the replacement of lysine 114 by glutamate causing a complete loss in affinity. The variant was identified in a father and son, the father having been investigated for an episode of cerebral ischaemia associated with hypercholesterolaemia. The variant forms SDS-stable complexes with activated factor X (fXa) and its thermal stability and rate of factor Xa inhibition in the absence of heparin are identical to those of normal antithrombin. Normal antithrombin binds to the high affinity heparin pentasaccharide with a Kd of 1nM, as detected by a 45% change in intrinsic fluorescence, resulting in a 230-fold increase in rate of factor Xa inhibition. However, no change in fluorescence was detected for the variant when titrated with heparin or the heparin pentasaccharide, nor was there detectable activation towards factor Xa, indicating a complete loss of heparin binding.

Keywords

Antithrombin - heparin - thrombosis - mutant - serpin

* These authors contributed equally to the work


 

  • References

  • 1 Carrell RW, Christey PB, Boswell DR. Serpins: antithrombin and other inhibitors of coagulation. Evidence from amino acid sequences. In: Thrombosis and Haemostasis. Verstrate M, Vermylen J. eds. Leuven University Press; 1987: 1-15.
    Search in Google Scholar
  • 2 Huber R, Carrell RW. Implications of the three-dimensional structure of G1-antitrypsin for structure and function of serpins. Biochemistry 1989; 28: 8951-66.
    CrossrefPubMedSearch in Google Scholar
  • 3 Peterson CB, Noyes CM, Pecon JM, Church FC, Blackburn MN. Identification of a lysyl residue in antithrombin which is essential for heparin binding. J Biol Chem 1987; 262: 8061-5.
    PubMedSearch in Google Scholar
  • 4 Gandrille S, Aiach M, Lane DA, Vidaud D, Molho-Sabatier P, Caso R, de Moerloose P, Fiessinger JN, Clauser E. Important role of arginine 129 in heparin-binding site of antithrombin III Identification of a novel mutation arginine 129 to glutamine. J Biol Chem 1990; 265: 18997-9001.
    PubMedSearch in Google Scholar
  • 5 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; 81: 1292-6.
    CrossrefPubMedSearch in Google Scholar
  • 6 Chowdhury V, Lane DA, Mille B, Auberger K, Gandenberger-Bachem S, Pabinger I, Olds RJ, Thein SL. Homozygous antithrombin deficiency: report of two new cases (99 Leu to Phe) associated with arterial and venous thrombosis. Thromb Haemost 1994; 72: 198-202.
    Thieme ConnectPubMedSearch in Google Scholar
  • 7 Okajima K, Abe H, Maeda S, Motomura M, Tsujihata M, Nagataki S, Okabe H, Takatsuki K. Antithrombin III Nagasaki (Ser116-Pro): a heterozygous variant with defective heparin binding associated with thrombosis. Blood 1993; 81: 1300-5.
    PubMedSearch in Google Scholar
  • 8 Jin L, Abrahams JP, Skinner R, Petitou M, Pike RN, Carrell RW. The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci USA 1997; 94: 14683-8.
    CrossrefPubMedSearch in Google Scholar
  • 9 Carrell RW, Stein PE, Fermi G, Wardell MR. Biological implications of a 3Å structure of dimeric antithrombin. Structure 1994; 02: 257-70.
    CrossrefPubMedSearch in Google Scholar
  • 10 Kridel SJ, Knauer DJ. Lysine residue 114 in human antithrombin III is required for heparin pentasaccharide-mediated activation. J Biol Chem 1997; 272: 7656-60.
    CrossrefPubMedSearch in Google Scholar
  • 11 Arocas V, Bock SC, Raja S, Olson ST, Bjork I. Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding. J Biol Chem 2001; 276: 43809-17.
    CrossrefPubMedSearch in Google Scholar
  • 12 Choay J, Petitou M, Lormeau JC, Sinay P, Casu B, Gatti G. Structureactivity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Res Commun 1983; 116: 492-9.
    CrossrefPubMedSearch in Google Scholar
  • 13 Thunberg L, Backstrom G, Lindahl U. Further characterization of the antithrombin-binding sequence in heparin. Carbohydr Res 1982; 100: 393-410.
    CrossrefPubMedSearch in Google Scholar
  • 14 Petitou M, van Boeckel CA. Chemical synthesis of heparin fragments and analogues. Fortschr Chem Org Naturst 1992; 60: 143-210.
    PubMedSearch in Google Scholar
  • 15 Abildgaard U, Lie M, Odegard OR. Antithrombin (heparin cofactor) assay with "new" chromogenic substrates (S-2238 and Chromozym TH). Thromb Res 1977; 11: 549-53.
    CrossrefPubMedSearch in Google Scholar
  • 16 Odegard OR, Lie M, Abildgaard U. Antifactor Xa activity measured with amidolytic methods. Haemostasis 1976; 05: 265-75.
    PubMedSearch in Google Scholar
  • 17 Sas G, Pepper DS, Cash JD. Investigations on antithrombin III in normal plasma and serum. Br J Haematol 1975; 30: 265-72.
    CrossrefPubMedSearch in Google Scholar
  • 18 Fitton HL, Walker ID, Jones L, Brown K, Carrell PRW Coughlin. Five antithrombin variants, four associated with thrombosis. Blood Coag Fibrinol 1997; 08: 145-8.
    CrossrefPubMedSearch in Google Scholar
  • 19 Perry DJ. Antithrombin and its inherited deficiencies. Blood Reviews 1994; 08: 37-55.
    CrossrefPubMedSearch in Google Scholar
  • 20 McKay EJ. A simple two-step procedure for the isolation of antithrombin III from biological fluids. Thromb Res 1981; 21: 375-82.
    CrossrefPubMedSearch in Google Scholar
  • 21 Nordenman B, Nystrom C, Bjork I. The size and shape of human and bovine antithrombin III. Eur J Biochem 1977; 78: 195-203.
    CrossrefPubMedSearch in Google Scholar
  • 22 Olson ST, Shore JD. Binding of high affinity heparin to antithrombin III Characterization of the protein fluorescence enhancement. J Biol Chem 1981; 256: 11065-72.
    PubMedSearch in Google Scholar
  • 23 Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-59.
    PubMedSearch in Google Scholar
  • 24 Desai UR, Petitou M, Bjork I, Olson ST. Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites. Biochemistry 1998; 37: 13033-41.
    CrossrefPubMedSearch in Google Scholar
  • 25 Lawrence DA, Olson ST, Palaniappan S, Ginsburg D. Engineering plasminogen activator inhibitor 1 mutants with increased functional stability. Biochemistry 1994; 33: 3643-8.
    CrossrefPubMedSearch in Google Scholar
  • 26 Beauchamp NJ, Pike RN, Daly M, Butler L, Makris M, Dafforn T, Zhou A, Fitton HL, Preston FE, Peake IR, Carrell RW. Antithrombins wibble and wobble (T85M/K): archetypal conformational diseases with in vivo latenttransition, thrombosis and heparin activation. Blood 1998; 92: 2696-706.
    PubMedSearch in Google Scholar
  • 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedSearch in Google Scholar
  • 28 Zhou A, Huntington JA, Carrell RW. Formation of the antithrombin heterodimer and the onset of thrombosis. Blood 1999; 94: 3388-96.
    CrossrefPubMedSearch in Google Scholar
  • 29 Desai UR, Petitou M, Bjork I, Olson ST. Mechanism of heparin activation of antithrombin Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin. J Biol Chem 1998; 273: 7478-87.
    CrossrefPubMedSearch in Google Scholar
  • 30 Lane DA, Bayston T, Olds RJ, Fitches AC, Cooper DN, Millar DS, Jochmans K, Perry DJ, Okajima K, Thein SL, Emmerich J. Antithrombin mutation database: 2nd (1997) update For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1997; 77: 197-211.
    Thieme ConnectPubMedSearch in Google Scholar