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

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
• 2 Lindsey GG, Brown G, Purves LR. Calcium binding to human fibrinogen -localization of two calcium specific sites. Thromb Res 1978; 13: 345-350
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 18 Furlan M, Rupp C, Beck EA, Svendsen L. Effect of calcium and synthetic peptides on fibrin polymerization. Thromb Haemost 1982; 47: 118-121
• 19 Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672
• 20 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 29 Doolittle RF. A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives. Protein Science 1992; 1: 1563-1577
• 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
• 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
• 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
• 33 Lawrie JS, Kemp G. The presence of a Ca²⁺ bridge within the chain of human fibrinogen. Biochim Biophys Acta 1979; 577: 415-423
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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