Download PDF
Thromb Haemost 1996; 76(03): 377-383
DOI: 10.1055/s-0038-1650587
Original Article
Schattauer GmbH Stuttgart

Normal Binding of Calcium to Five Fibrinogen Variants with Mutations in the Carboxy Terminal Part of the γ-Chain

Miha Furlan
The Central Hematology Laboratory, Inselspital University Hospital of Bern, Bern, Switzerland
,
Bettina Stucki
The Central Hematology Laboratory, Inselspital University Hospital of Bern, Bern, Switzerland
,
Colette Steinmann
The Central Hematology Laboratory, Inselspital University Hospital of Bern, Bern, Switzerland
,
Myriam Jungo
The Central Hematology Laboratory, Inselspital University Hospital of Bern, Bern, Switzerland
,
Bernhard Lämmle
The Central Hematology Laboratory, Inselspital University Hospital of Bern, Bern, Switzerland
› Author Affiliations
Further Information

Publication History

Received 27 November 1995

Accepted after resubmission 22 May 1996

Publication Date:
10 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

Calcium ions are known to accelerate polymerization of fibrin monomers. Each of the two carboxy terminal domains of normal fibrinogen contains one high-affinity calcium binding site that seems to be situated close to the polymerization site in the γ-chain. Most hitherto described functionally defective fibrinogen variants showed impaired clot formation. Since the tightly bound calcium ions may influence the conformation of the polymerization site, the question arises whether the abnormal clotting of a dysfibrinogen might be due to defective calcium binding. We investigated binding of calcium to fibrinogen and the effect of calcium on the clotting properties of five heterozygous fibrinogen variants showing normal thrombin-induced fibrinopeptide release but abnormal polymerization of fibrin monomers. Each of these dysfibrinogens has one single amino acid substitution in the carboxy-terminal part of the γ-chain: fibrinogen Claro (γ 275 Arg ⟶ His), Milano V (γ 275 Arg Cys), Milano I (γ 330 Asp ⟶ Val), Bern I (γ 337 Asn Lys), and Milano VII (γ 358 Ser Cys). The shortest thrombin clotting time and the earliest onset of turbidity increase were observed in fibrinogen γ 358 Ser ⟶ Cys; both parameters were little affected by calcium concentration. In the variant γ 337 Asn ⟶ Lys, the thrombin time was abnormally prolonged at 0.01 mM Ca2+, but it was normalized at 1 mM calcium. In contrast, the abnormal fibrin polymerization of fibrinogen γ 330 Asp ⟶ Val was barely improved at increasing calcium concentrations. Both variants with the substitution of γ 275 Arg, the residue indispensable for normal D:D interactions, showed the slowest rate of fibrin polymerization and the lowest turbidity of fibrin clots at any Ca2+ concentration used. High affinity calcium binding was found to be normal in all five fibrinogen variants studied, suggesting that their abnormal clotting was not due to defective binding of calcium. The γ-chain in the fragment D1 derived from the variant γ 337 Asn ⟶ Lys was further degraded by plasmin in the presence and in the absence of calcium, whereas fragments D1 from the other four γ-chain variants as well as from normal fibrinogen were protected against plasmic degradation in the presence of 1 mM Ca2+.

 

  • References

  • 1 Marguerie G, Chagniel G, Suscillon M. The binding of calcium to bovine fibrinogen. Biochim Biophys Acta 1977; 490: 94-103
    CrossrefPubMedGoogle Scholar
  • 2 Lindsey GG, Brown G, Purves LR. Calcium binding to human fibrinogen -localization of two calcium specific sites. Thromb Res 1978; 13: 345-350
    CrossrefPubMedGoogle Scholar
  • 3 Nieuwenhuizen W, van Ruijven-Vermeer AM, Nooijen WJ, Vermond A, Haverkate F. Recalculation of calcium-binding properties of human and rat fibrin(ogen) and their degradation products. Thromb Res 1981; 22: 653-657
    CrossrefPubMedGoogle Scholar
  • 4 Marguerie G, Ardaillou N. Potential role of the Aa chain in the binding of calcium to human fibrinogen. Biochim Biophys Acta 1982; 701: 410-412
    CrossrefPubMedGoogle Scholar
  • 5 Nieuwenhuizen W, Vermond A, Hermans J. Evidence for the localization of a calcium-binding site in the amino-terminal disulphide knot of fibrin(ogen). Thromb Res 1983; 31: 81-86
    CrossrefPubMedGoogle Scholar
  • 6 Nieuwenhuizen W, Vermond A, Nooijen WJ, Haverkate F. Calcium-binding properties of human fibrin(ogen) and degradation products. FEBS Lett 1979; 98: 257-259
    CrossrefPubMedGoogle Scholar
  • 7 Dang CV, Shin CK, Bell WR, Nagaswami C, Weisel JW. Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly. J Biol Chem 1989; 264: 15104-15108
    PubMedGoogle Scholar
  • 8 Langer BG, Weisel JW, Dinauer PA, Nagaswami C, Bell WR. Deglyco-sylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers. J Biol Chem 1988; 263: 15056-15063
    PubMedGoogle Scholar
  • 9 Okude M, Yamanaka A, Morimoto Y, Akihama S. Sialic acid in fibrinogen. Effects of sialic acid on fibrinogen-fibrin conversion by thrombin and properties of asialofibrin clot. Biol Pharm Bull 1993; 16: 448-452
    PubMedGoogle Scholar
  • 10 Dang CV, Ebert RF, Bell WR. Localization of a fibrinogen calcium binding site between γ-subunit positions 311 and 336 by terbium fluorescence. J Biol Chem 1985; 260: 9713-9719
    PubMedGoogle Scholar
  • 11 Shimizu A, Nagel GM, Doolittle RF. Photoaffinity labeling of the primary fibrin polymerization site: Isolation and characterization of a labeled cyanogen bromide fragment corresponding to γ-chain residues 337-379. Proc Natl Acad Sci USA 1992; 89: 2888-2892
    CrossrefPubMedGoogle Scholar
  • 12 Reber P, Furlan M, Rupp C, Kehl A, Henschen A, Mannucci PM, Beck EA. Characterization of fibrinogen Milano I: amino acid exchange γ 330 Asp ⟶ Val impairs fibrin polymerization. Blood 1986; 67: 1751-1756
    PubMedGoogle Scholar
  • 13 Reber P, Furlan M, Henschen A, Kaudewitz H, Barbui T, Hilgard P, Nenci GG, Berrettini M, Beck EA. Three abnormal fibrinogen variants with the same amino acid substitution (γ 275 Arg ⟶ His): fibrinogens Bergamo II, Essen and Perugia. Thromb Haemost 1986; 56: 401-406
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Steinmann C, Reber P, Jungo M, Lammle B, Heinemann G, Wermuth B, Furlan M. Fibrinogen Bern I: substitution γ 337 Asn ⟶ Lys is responsible for defective fibrin monomer polymerization. Blood 1993; 82: 2104-2108
    PubMedGoogle Scholar
  • 15 Steinmann C, Bogli C, Jungo M, Lammle B, Heinemann G, Wermuth B, Redaelli R, Baudo F, Furlan M. Fibrinogen Milano V: a congenital dys-fibrinogenemia with a γ 275 Arg Cys substitution. Blood Coag Fibrinol 1994; 5: 463-471
    PubMedGoogle Scholar
  • 16 Steinmann C, Bogli C, Jungo M, Lammle B, Heinemann G, Wermuth B, Redaelli R, Baudo F, Furlan M. A new substitution, γ 358 Ser ⟶ Cys, in fibrinogen Milano VII causes defective fibrin polymerization. Blood 1994; 84: 1874-1880
    PubMedGoogle Scholar
  • 17 Steinmann C, Jungo M, Beck EA, Lammle B, Furlan M. Fibrinogen Claro - another dysfunctional fibrinogen variant with y 275 arginine ⟶ histidine substitution. Thromb Res 1996; 81: 145-150
    CrossrefPubMedGoogle Scholar
  • 18 Furlan M, Rupp C, Beck EA, Svendsen L. Effect of calcium and synthetic peptides on fibrin polymerization. Thromb Haemost 1982; 47: 118-121
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672
    CrossrefPubMedGoogle Scholar
  • 20 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 21 Haverkate F, Timan G. Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragments D. Thromb Res 1977; 10: 803-812
    CrossrefPubMedGoogle Scholar
  • 22 Nieuwenhuizen W, Voskuilen M, Vermond A, Haverkate F, Hermans J. A fibrinogen fragment D (D intermediate) with calcium binding but without anticlotting properties. Biochim Biophys Acta 1982; 707: 190-192
    CrossrefPubMedGoogle Scholar
  • 23 Yamazumi K, Doolittle RF. Photoaffinity labeling of the primary fibrin polymerization site: localization of the label to γ-chain Tyr-363. Proc Natl Acad Sci USA 1992; 89: 2893-2896
    CrossrefPubMedGoogle Scholar
  • 24 Koopman J, Haverkate F, Briet E, Lord ST. A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the γ-chain gene, causing defective calcium binding and impaired fibrin polymerization. J Biol Chem 1991; 266: 13456-13461
    PubMedGoogle Scholar
  • 25 Yoshida N, Hirata H, Morigami Y, Imaoka S, Matsuda M, Yamazumi K, Asakura S. Characterization of an abnormal fibrinogen Osaka V with the replacement of γ-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis. J Biol Chem 1992; 267: 2753-2759
    PubMedGoogle Scholar
  • 26 Yoshida N, Terukina S, Okuma M, Moroi M, Aoki N, Matsuda M. Characterization of an apparently lower molecular weight γ-chain variant in fibrinogen Kyoto I. The replacement of γ-asparagine 308 by lysine which causes accelerated cleavage of fragment Dj by plasmin and the generation of a new plasmin cleavage site. J Biol Chem 1988; 263: 13848-13856
    PubMedGoogle Scholar
  • 27 Soria J, Soria C, Samama M, Tabori S, Kehl M, Henschen A, Nieuwen-huizen W, Riman A, Tatarski I. Fibrinogen Haifa: fibrinogen variant with absence of protective effect of calcium on plasmin degradation of gamma chains. Thromb Haemost 1987; 57: 310-313
    Article in Thieme ConnectPubMedGoogle Scholar
  • 28 Bantia S, Bell WR, Dang CV. Polymerization defect of fibrinogen Baltimore III due to a γ Asn308 ⟶lie mutation. Blood 1990; 75: 1659-1663
    PubMedGoogle Scholar
  • 29 Doolittle RF. A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives. Protein Science 1992; 1: 1563-1577
    CrossrefPubMedGoogle Scholar
  • 30 Olexa SA, Budzynski AZ. Evidence for four different polymerization sites involved in human fibrin formation. Proc Natl Acad Sci USA 1980; 77: 1374-1378
    CrossrefPubMedGoogle Scholar
  • 31 Mosesson MW, Siebenlist KR, DiOrio JP, Matsuda M, Hainfeld JF, Wall JS. The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (γ 275 Arg ⟶ Cys). J Clin Invest 1995; 96: 1053-1058
    CrossrefPubMedGoogle Scholar
  • 32 Matsuda M, Baba M, Morimoto K, Nakamikawa C. “Fibrinogen Tokyo II”: an abnormal fibrinogen with an impaired polymerization site on the aligned DD domain of fibrin molecules. J Clin Invest 1983; 72: 1034-1041
    CrossrefPubMedGoogle Scholar
  • 33 Lawrie JS, Kemp G. The presence of a Ca2+ bridge within the chain of human fibrinogen. Biochim Biophys Acta 1979; 577: 415-423
    CrossrefPubMedGoogle Scholar
  • 34 Yoshida N, Imaoka S, Hirata H, Matsuda M, Asakura S. Heterozygous abnormal fibrinogen Osaka III with the replacement of arginine-275 by histidine has an apparently higher molecular weight γ-chain variant. Thromb Haemost 1992; 68: 534-538
    Article in Thieme ConnectPubMedGoogle Scholar
  • 35 Terukina S, Matsuda M, Hirata H, Takeda Y, Miyata T, Takao T, Shimonishi Y. Substitution of γ Arg-275 by Cys in an abnormal fibrinogen, “fibrinogen Osaka II”. Evidence for a unique solitary cystine structure at the mutation site. J Biol Chem 1988; 263: 13579-13587
    PubMedGoogle Scholar
  • 36 Yoshida N, Ota K, Moroi M, Matsuda M. An apparently higher molecular weight γ-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of y arginine-275 by cysteine. Blood 1988; 71: 480-487
    PubMedGoogle Scholar
  • 37 Yamazumi K, Shimura K, Terukina S, Takahashi N, Matsuda M. A γ methionine-310 to threonine substitution and consequent N-glycosylation at y asparagine-308 identified in a congenital dysfibrinogenemia associated with posttraumatic bleeding, fibrinogen Asahi. J Clin Invest 1989; 83: 1590-1597
    CrossrefPubMedGoogle Scholar
  • 38 Grailhe P, Boyer-Neumann C, Haverkate F, Grimbergen J, Larrieu MJ, Angles-Cano E. The mutation in fibrinogen Bicetre II (γ Asn308 ⟶ Lys) does not affect the binding of t-PA and plasminogen to fibrin. Blood Coag Fibrinol 1993; 4: 679-687
    CrossrefPubMedGoogle Scholar
  • 39 Miyata T, Furukawa K, Iwanaga S, Takamatsu J, Saito H. Fibrinogen Nagoya, a replacement of glutamine-329 by arginine in the γ-chain that impairs the polymerization of fibrin monomer. J Biochem 1989; 105: 10-14
    CrossrefPubMedGoogle Scholar
  • 40 Terukina S, Yamazumi K, Okamoto K, Yamashita H, Ito Y, Matsuda M. Fibrinogen Kyoto III: a congenital dysfibrinogen with a γ aspartic acid-330 to tyrosine substitution manifesting impaired fibrin monomer polymerization. Blood 1989; 74: 2681-2687
    PubMedGoogle Scholar
  • 41 Rupp C, Kuyas C, Haberli A, Furlan M, Perret BA, Beck EA. Fibrinogen Bern I: a hereditary fibrinogen variant with defective conformational stabilization by calcium ions. In: Fibrinogen: Recent Biochemical and Medical Aspects Henschen A, Graeff H, Lottspeich F. eds W de Gruyter; Berlin: 1982: 167-181
  • 42 Yamazumi K, Terukina S, Onohara S, Matsuda M. Normal plasmic cleavage of the γ-chain variant of “fibrinogen Saga” with an Arg-275 to His substitution. Thromb Haemost 1988; 60: 476-480
    Article in Thieme ConnectPubMedGoogle Scholar