Specific binding of human fibrinogen to cultured human fibroblasts: Evidence for the involvement of the E domain

Elisabetta DEJANA, Margarita VERGARA‐DAUDEN, Giovanna BALCONI, Attilio PIETRA, Ghislaine CHEREL, Maria Benedetta DONATI, Marie‐José ‐J LARRIEU, Gérard MARGUERIE

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Abstract

Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37°C with 125I‐fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I‐fibrinogen and MRC5 was observed. The binding was time‐dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non‐labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3% at a 50/1 molar ratio while non‐immune Fab produced a 1.5% inhibition at similar concentration. Autoradiography of the display of fibroblast‐bound 125I on a 7.5% polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B β and γ chains of the fibrinogen molecule. An apparent affinity constant, Ka= 6.7 ± 0.2 × 106 M−1, was estimated from binding isotherms. After a 30‐min incubation time the interaction between 125I‐fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non‐labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I‐fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin‐degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I‐fibrinogen/fragment molar ratio, fragment E inhibited binding by 30% while fragment D produced a 3% inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.

Original languageEnglish
Pages (from-to)657-662
Number of pages6
JournalEuropean Journal of Biochemistry
Volume139
Issue number3
DOIs
Publication statusPublished - 1984

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Fibroblasts
Fibrinogen
Monolayers
Embryonic Structures
Immunoglobulin Fab Fragments
Molecules
Myoglobin
Ovalbumin
Molecular mass
Autoradiography
Fibronectins
Cell culture
Iodine
Isotherms
Buffers
Cell Culture Techniques
Cell Count
Epithelial Cells
Phosphates
Display devices

ASJC Scopus subject areas

  • Biochemistry

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Specific binding of human fibrinogen to cultured human fibroblasts : Evidence for the involvement of the E domain. / DEJANA, Elisabetta; VERGARA‐DAUDEN, Margarita; BALCONI, Giovanna; PIETRA, Attilio; CHEREL, Ghislaine; DONATI, Maria Benedetta; LARRIEU, Marie‐José ‐J; MARGUERIE, Gérard.

In: European Journal of Biochemistry, Vol. 139, No. 3, 1984, p. 657-662.

Research output: Contribution to journalArticle

DEJANA, Elisabetta ; VERGARA‐DAUDEN, Margarita ; BALCONI, Giovanna ; PIETRA, Attilio ; CHEREL, Ghislaine ; DONATI, Maria Benedetta ; LARRIEU, Marie‐José ‐J ; MARGUERIE, Gérard. / Specific binding of human fibrinogen to cultured human fibroblasts : Evidence for the involvement of the E domain. In: European Journal of Biochemistry. 1984 ; Vol. 139, No. 3. pp. 657-662.
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abstract = "Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37°C with 125I‐fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I‐fibrinogen and MRC5 was observed. The binding was time‐dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non‐labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3{\%} at a 50/1 molar ratio while non‐immune Fab produced a 1.5{\%} inhibition at similar concentration. Autoradiography of the display of fibroblast‐bound 125I on a 7.5{\%} polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B β and γ chains of the fibrinogen molecule. An apparent affinity constant, Ka= 6.7 ± 0.2 × 106 M−1, was estimated from binding isotherms. After a 30‐min incubation time the interaction between 125I‐fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non‐labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I‐fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin‐degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I‐fibrinogen/fragment molar ratio, fragment E inhibited binding by 30{\%} while fragment D produced a 3{\%} inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.",
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T2 - Evidence for the involvement of the E domain

AU - DEJANA, Elisabetta

AU - VERGARA‐DAUDEN, Margarita

AU - BALCONI, Giovanna

AU - PIETRA, Attilio

AU - CHEREL, Ghislaine

AU - DONATI, Maria Benedetta

AU - LARRIEU, Marie‐José ‐J

AU - MARGUERIE, Gérard

PY - 1984

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N2 - Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37°C with 125I‐fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I‐fibrinogen and MRC5 was observed. The binding was time‐dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non‐labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3% at a 50/1 molar ratio while non‐immune Fab produced a 1.5% inhibition at similar concentration. Autoradiography of the display of fibroblast‐bound 125I on a 7.5% polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B β and γ chains of the fibrinogen molecule. An apparent affinity constant, Ka= 6.7 ± 0.2 × 106 M−1, was estimated from binding isotherms. After a 30‐min incubation time the interaction between 125I‐fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non‐labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I‐fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin‐degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I‐fibrinogen/fragment molar ratio, fragment E inhibited binding by 30% while fragment D produced a 3% inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.

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