Thromb Haemost 1988; 60(03): 471-475
DOI: 10.1055/s-0038-1646993
Original Article
Schattauer GmbH Stuttgart

Antithrombin Milano, Single Amino Acid Substitution at the Reactive Site, Arg393 to Cys

H Erdjument
The Department of Haematology, Charing Cross and Westminster Medical School, London
,
D A Lane
The Department of Haematology, Charing Cross and Westminster Medical School, London
,
H Ireland
The Department of Haematology, Charing Cross and Westminster Medical School, London
,
V Di Marzo
*   The Department of Biochemistry, Imperial College, London, UK
,
M Panico
*   The Department of Biochemistry, Imperial College, London, UK
,
H R Morris
*   The Department of Biochemistry, Imperial College, London, UK
,
A Tripodi
**   The A. Bianchi Bonomi Haemophilia and Thrombosis Centre and Institute of Internal Medicine, University of Milano, Italy
,
P M Mannucci
**   The A. Bianchi Bonomi Haemophilia and Thrombosis Centre and Institute of Internal Medicine, University of Milano, Italy
› Author Affiliations
Further Information

Publication History

Received 02 June 1988

Accepted after revision 05 August 1988

Publication Date:
23 July 2018 (online)

Summary

Antithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; anti thrombin Milano peak 1 of Mr ∼60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr ∼120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the ∼120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr ∼60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the ∼120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxy-methylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, North-wick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.

 
  • References

  • 1 Damus PS, Hicks M, Rosenberg RD. Anticoagulant action of heparin. Nature 1973; 246: 355-357
  • 2 Petersen TE, Dudek-Wojciechowska G, Sottrup-Jensen L, Mag-nusson S. Primary structure of antithrombin III (Heparin cofactor). Partial homology between αl-antitrypsin and antithrombin III. In: The Physiological Inhibitors of Blood Coagulation and Fibrinolysis Collen D, Wiman B, Verstraete M. (eds) Elsevier/North-Holland Biomed. Press; Amsterdam: 1979. pp 43-54
  • 3 Bock SC, Wion KL, Vehar GA, Lawn RM. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acid Res 1982; 10: 8113-8125
  • 4 Chandra T, Stackhouse R, Kidd VJ, Woo SL C. Isolation and sequence characterisation of a DNA clone of human antithrombin III. Proc Natl Acad Sci USA 1983; 80: 1845-1848
  • 5 Prochownik EV, Markham AF, Orkin SH. Isolation of a cDNA clone for human antithrombin III. J Biol Chem 1983; 258: 8389-8394
  • 6 Carrell RW, Boswell DR. Serpins: The superfamily of plasma serine proteinase inhibitors. In: Proteinase Inhibitors Barrett AJ, Salvesen G. (eds) Elsevier Publishing Co; Amsterdam: 1986. pp 403-420
  • 7 Shieh B-H, Travis J. The reactive site of human α22-antiplasmin. J Biol Chem 1987; 262: 6055-6059
  • 8 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
  • 9 Marcum JA, Me Kenney JB, Rosenberg RD. Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparin-like molecules bound to the endothelium. J Clin Invest 1984; 74: 341-350
  • 10 Holmer E, Söderström G, Andersson LO. Studies on the mechanism of the rate enhancing effect of heparin on the thrombin-antithrombin III reactions. Eur J Biochem 1979; 93: 1-5
  • 11 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 lack heparin-binding ability. Proc Natl Acad Sci USA 1984; 81: 289-293
  • 12 Brunel F, Duchange N, Fischer AM, Cohen GN, Zakin MM. Antithrombin III Alger: A new case of Arg 47-Cys mutation. Am J Hemat 1987; 25: 223-224
  • 13 Duchange N, Chasse JF, Cohen GN, Zakin M. Antithrombin III Tours gene: Identification of a point mutation leading to an arginine-cysteine replacement in a silent deficiency. Nucleic Acid Res 1986; 14: 2408
  • 14 Chang JY, Tran TH. Antithrombin III Basel: Identification of a ProLeu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity. J Biol Chem 1986; 261: 1174-1176
  • 15 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; 69: 1275-1279
  • 16 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. 47Arg to Ser. J Clin Invest 1988 in press
  • 17 Blackburn MN, Sibley CC. The heparin binding site of antithrombin III. Evidence for a critical tryptophan residue. J Biol Chem 1980; 255: 824-826
  • 18 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: 8061-8065
  • 19 Gettins P, Wooten EW. On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR Spectroscopy. Biochemistry 1987; 26: 4403-4408
  • 20 Bjork I, Jackson CM, Jornvall H, Lavine KK, Nordling K, Salsgiver W. The active site of antithrombin. Release of the same proteolyti-cally cleaved form of the inhibitor from complexes with factor IXa, factor Xa and thrombin. J Biol Chem 1982; 257: 2406-2411
  • 21 Stephens AW, Thalley BS, Hirs CH W. Antithrombin III Denver, a reactive site variant. J Biol Chem 1987; 262: 1044-1048
  • 22 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; 263: 5589-5593
  • 23 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 1988 in press
  • 24 Wolf M, Boyer A, Tripodi A, Meyer D, Larrieu MJ, Mannucci PM. Antithrombin Milano: A new variant with monomeric and dimeric inactive antithrombin III. Blood 1985; 65: 496-500
  • 25 Wolf M, Boyer-Neumann C, Meyer D, Tripodi A, Mannucci PM, Larrieu MJ. Purification and further characterisation of antithrombin III Milano: Lack of reactivity with thrombin. Thromb Haemostas 1987; 58: 888-892
  • 26 Erdjument H, Lane DA, Ireland H, Panico M, Di Marzo V, Blench I, Morris HR. Formation of a covalent disulfide-linked antithrombinalbumin complex by an antithrombin variant, antithrombin “North-wick Park”. J Biol Chem 1987; 262: 13381-13384
  • 27 McKay EJ. A simple two-step procedure for the isolation of antithrombin III from biological fluids. Thromb Res 1981; 21: 375-382
  • 28 Lane DA, Lowe GD O, Flynn A, Thompson E, Ireland H, Erdjument H. Antithrombin III Glasgow: A variant with increased heparin affinity and reduced ability to inactivate thrombin associated with familial thrombosis. Br J Haematol 1987; 66: 523-527
  • 29 Lane DA, Flynn A, Ireland H, Erdjument H, Samson D, Howarth D, Thompson E. Antithrombin III Northwick Park: Demonstration of an inactive high MW complex with increased affinity for heparin. Br J Haematol 1987; 65: 451-456
  • 30 Morris HR, Panico M. Fast atom bombardment: A new mass spectrometric method for peptide analysis. Biochem Biophys Res Commun 1981; 101: 623-631
  • 31 Longas MO, Ferguson WS, Finlay TH. Studies on the interaction of heparin with thrombin, antithrombin, and other plasma proteins. Arch Biochem Biophys 1980; 200: 595-602