Download PDF
Thromb Haemost 1993; 70(03): 438-442
DOI: 10.1055/s-0038-1649601
Original Article
Coagulation
Schattauer GmbH Stuttgart

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

B Grøn
The Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway
,
C Filion-Myklebust
The Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway
,
S Bjørnsen
The Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway
,
P Haidaris
1   The Medical Center, Hematology Unit, University of Rochester, Rochester, Ny USA
,
F Brosstad
The Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway
› Author Affiliations
Further Information

Publication History

Received 23 October 1992

Accepted after revision 10 March 1993

Publication Date:
05 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Fibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

 

  • References

  • 1 Lorand L, Konishi K, Jacobsen A. Transpeptidation mechanism in blood clotting. Nature 1962; 194: 1148-1149
    CrossrefPubMedSearch in Google Scholar
  • 2 Fuller SM, Doolittle RF. The formation of crosslinked fibrins. Evidence for the involvement of lysine-amino groups. Biochem Biophys Res Commun 1966; 25: 694-700
    CrossrefPubMedSearch in Google Scholar
  • 3 McKee PA, Mattock P, Hill RL. Subunit structure of human fibrinogen, soluble fibrin, and cross-linked insoluble fibrin. Proc Natl Acad Sci 1970; 66: 738-744
    CrossrefPubMedSearch in Google Scholar
  • 4 McKee PA, Schwartz MC, Pizzo SV, Hill RL. Cross-linking of fibrin by fibrin-stabilizing factor. Ann NY Acad Sci 1972; 202: 127-148
    CrossrefPubMedSearch in Google Scholar
  • 5 Sobel JH, Thibodeau CA, Canfield R. Early alpha chain crosslinking in human fibrin preparations. Thromb Haemostas 1988; 60: 153-159
    PubMedSearch in Google Scholar
  • 6 Grøn B, Filion-Myklebust C, Bennick A, Nieuwenhuizen W, Matsueda G, Brosstad F. Early cross-linked fibrin in human plasma contains β-polymers with intact fibrinopeptide A. Blood Coagul Fibrinolys. (accepted, September) 1992
    PubMedSearch in Google Scholar
  • 7 Lorand L, Chenoweth D, Domanik RA. Chain pairs in the crosslinking of fibrin. Biochem Biophys Res Commun 1969; 37: 219-224
    CrossrefPubMedSearch in Google Scholar
  • 8 Chen R, Doolittle RF. Identification of the polypeptide chains involved in the crosslinking of fibrin. Proc Natl Acad Sci 1969; 63: 420-427
    CrossrefPubMedSearch in Google Scholar
  • 9 Shainoff JR, Urbanic DA, DiBello PM. Immunoelectrophoretic characterization of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. J Biol Chem 1991; 266: 6429-6437
    PubMedSearch in Google Scholar
  • 10 Murthy SNP, Wilson J, Guy SL, Lorand L. Intramolecular crosslinking of monomeric fibrinogen by tissue transglutaminase. Proc Natl Acad Sci USA 1991; 88: 10601-10604
    CrossrefPubMedSearch in Google Scholar
  • 11 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to fibrin-like peptide bind to human fibrin but not to fibrinogen. Science 1983; 222: 1129-1132
    CrossrefPubMedSearch in Google Scholar
  • 12 Liau CS, Haber E, Matsueda GR. Evaluation of monoclonal antibodies by their binding to human blood clots. Thromb Haemostas 1987; 57: 49-54
    PubMedSearch in Google Scholar
  • 13 Koppert PW, Huijsmans CMG, Nieuwenhuizen W. A monoclonal antibody, specific for human fibrinogen, fibronopeptide A-containing fragments and not reacting with free fibrinopeptide A. Blood 1985; 66: 503-507
    PubMedSearch in Google Scholar
  • 14 Hoegee-de NobelE, Voskuilen E, Briët E, Brommer EJP, Nieuwenhuizen W. A monoclonal antibody-based quantitative enzyme immunoassay for the determination of plasma fibrinogen concentrations. Thromb Haemostas 1988; 60: 415-418
    PubMedSearch in Google Scholar
  • 15 Haidaris PJ, Francis CW, Sporn LA, Arvan DS, Collichio FA, Marder J. Megacaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of ϒ chain variant polypeptides. Blood 1989; 2: 743-750
    PubMedSearch in Google Scholar
  • 16 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedSearch in Google Scholar
  • 17 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogenes. Acta Haematol (Basel) 1957; 17: 237-246
    CrossrefPubMedSearch in Google Scholar
  • 18 Grøn B, Bennick A, Nieuwenhuizen W, Bjørnsen S, Brosstad F. Immunovisualization of fibrinogen Aβ-chain heterogeneity in normal plasma and plasma from patients with DIC or on streptokinasc therapy. Thromb Res 1988; 52: 413-424
    CrossrefPubMedSearch in Google Scholar
  • 19 Henschen A, Edman P. Large scale preparations of S-carboxymethylated chains of human fibrin and fibrinogen and the occurrence of ϒ-chain variants. Biochim Biophys Acta 1972; 263: 351-367
    CrossrefPubMedSearch in Google Scholar
  • 20 Weinstein MJ, Deykin D. Low solubility fibrinogen examined by two-dimensional sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Thromb Res 1978; 13: 361-377
    CrossrefPubMedSearch in Google Scholar
  • 21 Kuyas C, Haeberli A, Straub PW. Sialic acid dependent polypeptide chain heterogeneity of human fibrinogen demonstrated by two-dimensional electrophoresis. Thromb Haemostas 1982; 47: 19-21
    PubMedSearch in Google Scholar
  • 22 Teige B, Brosstad F, Olaisen B, Helland R. Two-dimensional electrophoresis of fibrinogen and fibrin subunit chains. In: Electrophoresis ’82: Advanced Methods, Biochemical and Clinical Applications. Stathakos D. (ed) Walter de Gruyter & Co; Berlin, New York: 1983: 413-421
  • 23 Carrell NA, Holahan GC, McDonagh J. High resolution electrophoretic Analysis of human fibrinogen and its crosslinked intermediates. Thromb Haemostas 1983; 49: 47-50
    PubMedSearch in Google Scholar
  • 24 Sherman LA, Mosesson MW, Sherry S. Isolation and characterization of the clottable low molecular weight fibrinogen derived by limited plasmin hydrolysis and human fraction 1–4. Biochemistry 1969; 8: 1515-1523
    CrossrefPubMedSearch in Google Scholar
  • 25 Mills D, Karpatkin S. Heterogeneity of human fibrinogen: possible relation to proteolysis by thrombin and plasmin as studied by SDS-polyacrylamide gel electrophoresis. Biochem Biophys Res Commun 1970; 40: 206-211
    CrossrefPubMedSearch in Google Scholar
  • 26 Mosesson MW, Finlayson JS, Umfleet RA, Galanakis D. Human fibrinogen heterogeneities. J Biol Chem 1972; 274: 5210-5219
    PubMedSearch in Google Scholar
  • 27 Sakata Y, Aoki N. Cross-linking of β2-plasmin inhibitor to fibrin by fibrin-stabilizing factor. J Clin Invest 1980; 65: 290-297
    CrossrefPubMedSearch in Google Scholar
  • 28 Mosher DF. Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor. J Biol Chem 1975; 250: 6614-6621
    PubMedSearch in Google Scholar
  • 29 Hada M, Kaminski M, Bockenstedt P, McDonagh J. Covalent crosslinking of von Willebrand factor to fibrin. Blood 1986; 63: 95-101
    PubMedSearch in Google Scholar
  • 30 Brosstad F, Teige B, Olaisen B, Gravem K, Godal HC, Sormorken H. Two-dimensional electrophoretic characterization (ISO-DALT) of fibrinogen and fibrin subunit chains from four different genetic dysfibrinogen variants. In: Fibrinogen-Structure, Functional Aspects, Metabolism. Haverkate F. et al. (eds) Walter de Gruyter & Co; Berlin, New York: 1983. 2: pp 145-153
    Search in Google Scholar
  • 31 Bombäck B, Grøndahl NJ, Hessel B, Iwanaga S. Wallen. Primary structure of fibrinogen and fibrin II. Structural studies on NH2-terminal part of ϒ chain. J Biol Chem 1973; 384: 5806-5820
    PubMedSearch in Google Scholar
  • 32 Henschen A, Lottspeich F, Hessel B. Preliminary note on the completion of the intermediate part of the β-chain sequence. Amino acid sequence of human fibrin. Hoppe-Seyler’s Z Physiol Chem 1979; 360: 1951-1956
    PubMedSearch in Google Scholar
  • 33 Doolittle RF, Watt KWK, Cottrell BA, Strong DD, Riley M. The amino acid sequence of the β-chain of human fibrinogen. Nature 1979; 280: 464-468
    CrossrefPubMedSearch in Google Scholar
  • 34 Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gels. J Biol Chem 1969; 244: 4406-4412
    PubMedSearch in Google Scholar
  • 35 Mosesson MW, Siebenlist KR, Amrani DL, DiOrio JP. Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin. Proc Natl Acad Sci USA 1989; 86: 1113-1117
    CrossrefPubMedSearch in Google Scholar
  • 36 Siebenlist KR, Mosesson MW. Factors affecting ϒ-chain multimer formation in cross-linked fibrin. Biochemistry 1991; 31: 936-941
    PubMedSearch in Google Scholar