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Thromb Haemost 1990; 63(03): 439-444
DOI: 10.1055/s-0038-1645062
Original Article
Schattauer GmbH Stuttgart

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

C Kuyas
The Laboratory for Thrombosis Research, Department of Medicine, University of Bern, Inselspital, Bern, Switzerland
,
A Haeberli
The Laboratory for Thrombosis Research, Department of Medicine, University of Bern, Inselspital, Bern, Switzerland
,
P Walder
The Laboratory for Thrombosis Research, Department of Medicine, University of Bern, Inselspital, Bern, Switzerland
,
P W Straub
The Laboratory for Thrombosis Research, Department of Medicine, University of Bern, Inselspital, Bern, Switzerland
› Author Affiliations
Further Information

Publication History

Received 15 September 1989

Accepted after revision 16 February 1990

Publication Date:
30 June 2018 (online)

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Summary

With an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

 

  • References

  • 1 Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark Kemi 1956; 10: 415-43
    PubMedGoogle Scholar
  • 2 Takeda Y. Studies of the metabolism and distribution of fibrinogen in healthy men with autologous 125I-labelled fibrinogen. J Clin Invest 1966; 45: 103-11
    CrossrefPubMedGoogle Scholar
  • 3 Jakobsen E, Kierulf P. A modified β-alanine precipitation to prepare fibrinogen free of antithrombin III and plasminogen. Thromb Res 1973; 3: 145-59
    CrossrefPubMedGoogle Scholar
  • 4 Matthias FR, Hocke G, Lasch HG. Isolation of fibrinogen from plasma by affinity chromatography on insolubilized fibrinogen (FG-ag) and immobilized fibrin monomer (FM-ag). Thromb Res 1975; 7: 861-70
    CrossrefPubMedGoogle Scholar
  • 5 Laudano AP, Doolittle RF. Studies on synthetic peptides that bind to fibrinogen and prevent fibrm polymerization. Structural requirements, number of binding sites and species differences. Biochemistry 1980; 19: 1013-9
    CrossrefPubMedGoogle Scholar
  • 6 Moroder L, Hallett A, Wünsch E, Keller O, Wersin G. Di-tert-butyldicarbonate, a useful tert-butyloxycarbonylating reagent. Hoppe-Seyler’s Z Physiol Chem 1976; 357: 1651-3
    PubMedGoogle Scholar
  • 7 König W, Geiger R. A new method for synthesis of peptides: Activation of the carboxylgroup with dicyclohexylcarbodiimide using 1-hydroxybenzotriazoles as additives. Chem Ber 1970; 103: 788-98
    CrossrefPubMedGoogle Scholar
  • 8 Weber K, Osborne M. The reliability of molecular weight determinations by dodecylsulphate polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 4406-12
    PubMedGoogle Scholar
  • 9 Haverkate F, Timan G. Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragments D. Thromb Res 1977; 10: 803-12
    CrossrefPubMedGoogle Scholar
  • 10 Nieuwenhuizen W, Voskuilen M, Vermond A, Havcrkate E, Hermans J. A fibrinogen bagmen I D (D intermediate) with calcium binding but without anticlotting properties. Biochim Biophys Acta 1982; 707: 190-2
    CrossrefPubMedGoogle Scholar
  • 11 Galanakis DK, Mosesson MW, Stathakis NE. Human fibrinogen heterogeneities: Distribution and charge characteristics of chains of Aα origin. J Lab Clin Med 1978; 92: 376-86
    PubMedGoogle Scholar
  • 12 Holm B, Godal HC. Quantitation of the three normally occurring plasma fibrinogens in health and during so-called “acute phase” by SDS electrophoresis of fibrin obtained from EDTA-plasma. Thromb Res 1984; 35: 279-90
    CrossrefPubMedGoogle Scholar
  • 13 Southan C, Thompson E, Panico M, Etienne T, Morris HR, Lane DA. Characterization of peptides cleaved by plasmin from C-terminal polymerization domain of human fibrinogen. J Biol Chem 1985; 260: 13095-101
    PubMedGoogle Scholar
  • 14 Varadi A, Scheraga HA. Localization of segments essential for polymerization and for calcium binding in the γ-chain of human fibrinogen. Biochemistry 1986; 25: 519-28
    CrossrefPubMedGoogle Scholar
  • 15 Laudano AP, Cottrell BA, Doolittle RF. Synthetic peptides modeled on fibrin polymerization sites. Ann N Y Acad Sei 1983; 408: 315-29
    CrossrefPubMedGoogle Scholar
  • 16 Holm B, Nilsen DW T, Kierulf P, Godal HC. Purification and characterization of 3 fibrinogens with different molecular weights obtained from normal human plasma. Thromb Res 1985; 37: 165-76
    CrossrefPubMedGoogle Scholar