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Thromb Haemost 1977; 37(01): 144-153
DOI: 10.1055/s-0038-1649212
Original Article
Schattauer GmbH

Soluble Fibrin Complexes: Separation as a Function of pH and Characterization

Y Benabid
1   Laboratoire d’Hématologie, Institut de Recherche Fondamentale, Centre d’Études Nucléaires de Grenoble, 85 X, Grenoble, France
,
E Concord
1   Laboratoire d’Hématologie, Institut de Recherche Fondamentale, Centre d’Études Nucléaires de Grenoble, 85 X, Grenoble, France
,
M Suscillon
1   Laboratoire d’Hématologie, Institut de Recherche Fondamentale, Centre d’Études Nucléaires de Grenoble, 85 X, Grenoble, France
› Author Affiliations
Further Information

Publication History

Received 10 July 1976

Accepted 25 October 1976

Publication Date:
03 July 2018 (online)

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Summary

1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH.

2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aα, Bβ and γ chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aα, α, Bβ and γ chains were found. In the peak containing the high polymers, only the presence of α, Bβ and γ chains was demonstrated.

3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.

 

  • References

  • 1 Arnesen H, Ly B, Ödegard O.R. Improved quantitation of serum FDP after coagulation at low pH. Thrombosis Research 1973; 3: 643
    CrossrefPubMedSearch in Google Scholar
  • 2 Bachmann L, Schmitt-Fumian W.W, Hammel R, Lederer D. Size and shape of fibrinogen. Electron microscopy of the hydrated molecule. Makro molecular Chemistry 1975; 173: 2603
    PubMedSearch in Google Scholar
  • 3 Belitzer V.A, Khodorova E.L, Smekhova E.A. Induction of rate and pathway-length changes in fibrin polymerisation. Thrombosis Research 1975; 7: 25
    CrossrefPubMedSearch in Google Scholar
  • 4 Benabid Y, Laurent M, Concord E, et Suscillon M. Etude des dérivés du fibrinogène apparaissant dans le plasma de veau après action de la thrombine et de l’ endotoxine. Pathologie et Biologie 1973; 21 suppl. 24
    PubMedSearch in Google Scholar
  • 5 Blombäck B, Laurent T.C. N-terminal and lightscattering studies on fibrinogen and its transformation to fibrin. Arkiv for Kemi 1958; 12: 137
    PubMedSearch in Google Scholar
  • 6 Budzinski A.Z, Latallo Z.S, Lipinski B, Kowalski E. The effect of pH on the interaction of fibrinogen degradation products (FDP) with the fibrinogen-fibrin conversion. Bulletin de l’Académie Polonaise des Sciences 1963; 11: 477
    PubMedSearch in Google Scholar
  • 7 Curtis C.G, Brown K.L, Credo R.B, Domanik R.A, Gray A, Stenberg P, Lorand L. Calcium-dependant unmasking of active center cysteine during activation of fibrin stabilizing factor. Biochemistry 1974; 13: 3774
    CrossrefPubMedSearch in Google Scholar
  • 8 Doolittle R.F. Structural aspects of the fibrinogen to fibrin conversion. Advances in Proteins Chemistry 1973; 23: 1
    PubMedSearch in Google Scholar
  • 9 Hall C.E, Slayter H.S. The fibrinogen molecule: its size, shape and mode of polymerisation. Journal of Biophysics, Biochemistry and Cytology 1959; 5: 11
    PubMedSearch in Google Scholar
  • 10 Haschemeyer A.E.V. A collapsed structure of fibrinogen? New observations of transient electric birefringence. Thrombosis Research 1975; 7: 59
    CrossrefPubMedSearch in Google Scholar
  • 11 Hudry-Clergeon G, Marguerie G, Pouit L, Suscillon M. Models proposed for the fibrinogen molecule and for the polymerisation process. Thrombosis Research 1975; 6: 533
    CrossrefPubMedSearch in Google Scholar
  • 12 Keckwick R.A, McKay M.E, Nance M.H, Record B.R. The preparation of protein fractions from human plasma with ether. Biochemical Journal 1955; 60: 671
    PubMedSearch in Google Scholar
  • 13 Krakow W, Endres G.F, Siegel B.M, Scheraga H.A. An electron microscopic investigation of the polymerisation of bovine fibrin monomer. Journal of molecular Biology 1972; 71: 95
    CrossrefPubMedSearch in Google Scholar
  • 14 Köppel G. Electron microscopic investigation of the shape of fibrinogen nodules: a model for certain proteins. Nature 1966; 212: 1608
    PubMedSearch in Google Scholar
  • 15 Latallo Z.S, Fletcher A.P, Alkjaersig N, Sherry S. Influence of pH, ionic strength, neutral ions, and thrombin on fibrin polymerisation. American Journal of Physiology 1962; 202: 675
    PubMedSearch in Google Scholar
  • 16 Loewy A.G, Dunathan K, Kriel R, Wolfinger H.L. Fibrinase – I – Purification of substrate and enzyme. Journal Biological Chemistry 1961; 236: 2625
    PubMedSearch in Google Scholar
  • 17 Ly B, Arnesen H. Influence of pH on the incorporation of fibrinogen degradation products into fibrin clots as studied by tracer technique. Haemostasis 1974; 2: 198
    PubMedSearch in Google Scholar
  • 18 Ly B, Kierulf P, Jacobsen E. Stabilisation of soluble fibrin/fibrinogen complexes by fibrin stabilizing factor (FSF). Thrombosis Research 1974; 4: 509
    CrossrefPubMedSearch in Google Scholar
  • 19 Marguerie G, Pouit L, Suscillon M. Macromolecular associations in dilute solutions of activated fibrinogen. Thrombosis Research 1973; 3: 675
    CrossrefPubMedSearch in Google Scholar
  • 20 Marder V.J, Shulman N.R. High molecular weight derivatives of human fibrinogen produced by plasmin – II – Mechanism of their anticoagulant effect. Journal of Biological Chemistry 1969; 244: 2120
    PubMedSearch in Google Scholar
  • 21 Mihalhi E. Transformation of fibrinogen into fibrin. II – Changes in pH during clotting of fibrinogen. Journal of Biology and Chemistry 1954; 209: 733
    PubMedSearch in Google Scholar
  • 22 Pouit L, Marcille G, Suscillon M, et Hollard D. Etude en microscopie électronique de différentes étapes de la fibrinoformation. Thrombosis et Diathesis Haemorrhagica 1972; 27: 559
    PubMedSearch in Google Scholar
  • 23 Sasaki T, Page I.H, Shainoff J.R. Stable complex of fibrinogen and fibrin. Science 1966; 152: 1069
    CrossrefPubMedSearch in Google Scholar
  • 24 Shainoff J.R, Page I.H. Significance of cryoprofibrin in the fibrinogen-fibrin conversion. Journal of Experimental Medicine 1962; 116: 687
    CrossrefPubMedSearch in Google Scholar
  • 25 Sheraga H.A, Laskowski M. The fibrinogen-fibrin conversion. Advances in Protein Chemistry 1957; 12: 1
    PubMedSearch in Google Scholar
  • 26 Shulman S, Ferry J.D, Tinoco J. The conversion of fibrinogen to fibrin XII – Influence of pH, ionic strength and hexamethylene glycol concentration on the polymerization of fibrinogen. Archives of Biochemistry and Biophysics 1953; 42: 24
    PubMedSearch in Google Scholar
  • 27 Slayter E.M. Electron microscopy of globular proteins. In Physical Principles and techniques of protein chemistry. Part A. Ed. Leach, S.J. Academic Press; 1969
    Search in Google Scholar
  • 28 Tranqui-Pouit L. Etude en microscopie électronique de la molécule de fibrinogène et de ses fonctions. Thèse Université Scientifique et Médicale de Grenoble; France: 1975
    Search in Google Scholar
  • 29 Weber K, Osborne H. The reliability of molecular weight determination by dodecyl sul-fate-polyacrylamide gel electrophoresis. Journal Biological Chemistry 1969; 244: 4406
    PubMedSearch in Google Scholar