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Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels.

Reyhani V, Seddigh P, Guss B, Gustafsson R, Rask L, Rubin K - Biochem. J. (2014)

Bottom Line: This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin.A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels.Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

View Article: PubMed Central - PubMed

Affiliation: *Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, BMC Box 582, SE-751 23 Uppsala, Sweden.

ABSTRACT
The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVβ3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

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Fibrinogen binds to matrix-binding site of Col I fibres and this binding is essential for fibrin/Col I gel contraction(A) The collagen-binding protein CNE at 100 μg/ml completely inhibited the binding of biotinylated fibrinogen (~90 nM) to immobilized native Col I using a solid-phase assay. (B) No binding of biotinylated CNE to immobilized fibrinogen was detected (solid-phase assay). (C) Biotinylated fibrinogen only bound to immobilized collagen peptide II-44 from Toolkit II. Peptides II-1 and II-44 are ligands for CNE and peptides II-37 and (GPP)10 were used as controls. (D) C2C12 cells were allowed to contract either pure Col I gels or fibrin/Col I gels. Addition of 25 μg/ml CNE (~375 nM) abolished the fibrin-enhanced contraction of composite gels. Left-hand panel, values are relative to the original gel area after 4 h of contraction (*P<0.05). Right-hand panel, values are the relative weight loss of gels after 24 h of contraction by C2C12 cells. In all panels the values are the means from at least three independent experiments performed in at least triplicate, and the error bars are S.E.M. (E) The amino acid sequence of peptides II-1, II-44, II-37 and (GPP)10 from human collagen Toolkit II, as well as the region of human (Homo sapiens) pro-collagen α1(II) and bovine (Bos taurus) pro-collagen α1(I) that are covered by peptide II-44. Peptide II-44 has 93% homology with bovine pro-collagen α1(I).
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Figure 5: Fibrinogen binds to matrix-binding site of Col I fibres and this binding is essential for fibrin/Col I gel contraction(A) The collagen-binding protein CNE at 100 μg/ml completely inhibited the binding of biotinylated fibrinogen (~90 nM) to immobilized native Col I using a solid-phase assay. (B) No binding of biotinylated CNE to immobilized fibrinogen was detected (solid-phase assay). (C) Biotinylated fibrinogen only bound to immobilized collagen peptide II-44 from Toolkit II. Peptides II-1 and II-44 are ligands for CNE and peptides II-37 and (GPP)10 were used as controls. (D) C2C12 cells were allowed to contract either pure Col I gels or fibrin/Col I gels. Addition of 25 μg/ml CNE (~375 nM) abolished the fibrin-enhanced contraction of composite gels. Left-hand panel, values are relative to the original gel area after 4 h of contraction (*P<0.05). Right-hand panel, values are the relative weight loss of gels after 24 h of contraction by C2C12 cells. In all panels the values are the means from at least three independent experiments performed in at least triplicate, and the error bars are S.E.M. (E) The amino acid sequence of peptides II-1, II-44, II-37 and (GPP)10 from human collagen Toolkit II, as well as the region of human (Homo sapiens) pro-collagen α1(II) and bovine (Bos taurus) pro-collagen α1(I) that are covered by peptide II-44. Peptide II-44 has 93% homology with bovine pro-collagen α1(I).

Mentions: To characterize further the binding of fibrinogen to Col I, the streptococcal protein CNE that specifically binds to fibronectin and to two sites in collagen type II (one at the N-terminus of the triple-helical part of collagen and one at a site known to bind matrix metalloproteinase-1 and discoidin domain receptor-2), was used [36,38–40]. CNE effectively inhibited the binding of fibrinogen to immobilized Col I in a dose-dependent manner (Figure 5A). Soluble biotinylated CNE did not bind to immobilized fibrinogen (Figure 5B). To investigate which of the two CNE-binding sites on collagen interacts with fibrinogen, synthetic native collagen peptides derived from the Toolkit collagen II library were used [39]. Biotinylated fibrinogen bound to immobilized peptide II-44, but not to immobilized peptides II-1 or II-37, nor to the control peptide (GPP)10 (Figure 5C). In summary, these findings show that fibrinogen specifically recognizes a site in the collagen triple helix and that it also specifically binds to immobilized native Col I.


Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels.

Reyhani V, Seddigh P, Guss B, Gustafsson R, Rask L, Rubin K - Biochem. J. (2014)

Fibrinogen binds to matrix-binding site of Col I fibres and this binding is essential for fibrin/Col I gel contraction(A) The collagen-binding protein CNE at 100 μg/ml completely inhibited the binding of biotinylated fibrinogen (~90 nM) to immobilized native Col I using a solid-phase assay. (B) No binding of biotinylated CNE to immobilized fibrinogen was detected (solid-phase assay). (C) Biotinylated fibrinogen only bound to immobilized collagen peptide II-44 from Toolkit II. Peptides II-1 and II-44 are ligands for CNE and peptides II-37 and (GPP)10 were used as controls. (D) C2C12 cells were allowed to contract either pure Col I gels or fibrin/Col I gels. Addition of 25 μg/ml CNE (~375 nM) abolished the fibrin-enhanced contraction of composite gels. Left-hand panel, values are relative to the original gel area after 4 h of contraction (*P<0.05). Right-hand panel, values are the relative weight loss of gels after 24 h of contraction by C2C12 cells. In all panels the values are the means from at least three independent experiments performed in at least triplicate, and the error bars are S.E.M. (E) The amino acid sequence of peptides II-1, II-44, II-37 and (GPP)10 from human collagen Toolkit II, as well as the region of human (Homo sapiens) pro-collagen α1(II) and bovine (Bos taurus) pro-collagen α1(I) that are covered by peptide II-44. Peptide II-44 has 93% homology with bovine pro-collagen α1(I).
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: Fibrinogen binds to matrix-binding site of Col I fibres and this binding is essential for fibrin/Col I gel contraction(A) The collagen-binding protein CNE at 100 μg/ml completely inhibited the binding of biotinylated fibrinogen (~90 nM) to immobilized native Col I using a solid-phase assay. (B) No binding of biotinylated CNE to immobilized fibrinogen was detected (solid-phase assay). (C) Biotinylated fibrinogen only bound to immobilized collagen peptide II-44 from Toolkit II. Peptides II-1 and II-44 are ligands for CNE and peptides II-37 and (GPP)10 were used as controls. (D) C2C12 cells were allowed to contract either pure Col I gels or fibrin/Col I gels. Addition of 25 μg/ml CNE (~375 nM) abolished the fibrin-enhanced contraction of composite gels. Left-hand panel, values are relative to the original gel area after 4 h of contraction (*P<0.05). Right-hand panel, values are the relative weight loss of gels after 24 h of contraction by C2C12 cells. In all panels the values are the means from at least three independent experiments performed in at least triplicate, and the error bars are S.E.M. (E) The amino acid sequence of peptides II-1, II-44, II-37 and (GPP)10 from human collagen Toolkit II, as well as the region of human (Homo sapiens) pro-collagen α1(II) and bovine (Bos taurus) pro-collagen α1(I) that are covered by peptide II-44. Peptide II-44 has 93% homology with bovine pro-collagen α1(I).
Mentions: To characterize further the binding of fibrinogen to Col I, the streptococcal protein CNE that specifically binds to fibronectin and to two sites in collagen type II (one at the N-terminus of the triple-helical part of collagen and one at a site known to bind matrix metalloproteinase-1 and discoidin domain receptor-2), was used [36,38–40]. CNE effectively inhibited the binding of fibrinogen to immobilized Col I in a dose-dependent manner (Figure 5A). Soluble biotinylated CNE did not bind to immobilized fibrinogen (Figure 5B). To investigate which of the two CNE-binding sites on collagen interacts with fibrinogen, synthetic native collagen peptides derived from the Toolkit collagen II library were used [39]. Biotinylated fibrinogen bound to immobilized peptide II-44, but not to immobilized peptides II-1 or II-37, nor to the control peptide (GPP)10 (Figure 5C). In summary, these findings show that fibrinogen specifically recognizes a site in the collagen triple helix and that it also specifically binds to immobilized native Col I.

Bottom Line: This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin.A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels.Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

View Article: PubMed Central - PubMed

Affiliation: *Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, BMC Box 582, SE-751 23 Uppsala, Sweden.

ABSTRACT
The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVβ3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

Show MeSH
Related in: MedlinePlus