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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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Related in: MedlinePlus

Fibrin forms a network within the Col I fibril network(A–C) The secondary network of fibrin within Col I fibrils was observed using SEM. Fibrin fibres were observed in associations with: (i) cell surfaces during gel contraction and (ii) Col I fibrils, suggesting the ‘bridging’ effect of fibrin between cells and Col I fibrils. The white arrows in (B) and (C) point to fibrin fibrils. After 12 h, the fibrin fibres were observed in closer associations with the cell surface (C) compared with at 4 h (B). (D–F) Representative micrographs of C2C12 cells in pure Col I gels. Cells were round and incapable of spreading after 4 h of culture. (G–I) Representative micrographs of C2C12-α2β1 cells in pure Col I gels. No secondary network, similar to fibrin fibrils (white arrows in B and C), was observed after either 4 h (E) or 12 h (F) of contraction.
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Figure 2: Fibrin forms a network within the Col I fibril network(A–C) The secondary network of fibrin within Col I fibrils was observed using SEM. Fibrin fibres were observed in associations with: (i) cell surfaces during gel contraction and (ii) Col I fibrils, suggesting the ‘bridging’ effect of fibrin between cells and Col I fibrils. The white arrows in (B) and (C) point to fibrin fibrils. After 12 h, the fibrin fibres were observed in closer associations with the cell surface (C) compared with at 4 h (B). (D–F) Representative micrographs of C2C12 cells in pure Col I gels. Cells were round and incapable of spreading after 4 h of culture. (G–I) Representative micrographs of C2C12-α2β1 cells in pure Col I gels. No secondary network, similar to fibrin fibrils (white arrows in B and C), was observed after either 4 h (E) or 12 h (F) of contraction.

Mentions: The morphology of C2C12 cells in the reconstituted fibrin/Col I gels was analysed using SEM (Figures 2A–2C). When collagen fibres had been formed, thrombin was added to gels allowing fibrin deposits to form. Fibrin formed a secondary fibrillar matrix interwoven with the collagen fibre network. Predominantly, fibrin formed an independent amorphous mesh between the Col I fibres (Figures 2B and see Figure 6B). However notably, in many instances, fibrin was directly associated with the collagen fibres (Figures 2B and 2C). Even at this low concentration, fibrin formed an amorphous meshwork that most often was not directly associated with the Col I fibres. Even though both collagen and fibrin fibres were observed in association with the cell surface, only fibrin fibres were found in tighter associations with the cell surface during the later time periods, as shown in Figure 2(C) (12 h contraction) compared with Figures 2(B) (4 h contraction). For comparison, C2C12 cells in pure Col I gels were studied. In pure Col I gels, the C2C12 cells were not capable of spreading (Figures 2D–2F). The C2C12-α2β1 cells were also studied in pure Col I gels (Figures 2G–2I). In the Col I gels, no fibril structure similar to the fibrin network (observed in composite gels, as in Figures 2A–2C) was found (Figures 2D–2I). The presence of fibrin in the composite gels was confirmed by immunogold staining of the fibrin/Col I gels. The signal from gold-conjugated Protein A, which was used to detect rabbit anti-fibrinogen IgG, was observed at the junctions of cell protrusions with fibrils of the composite gels (Figures 3A–3D). The C2C12-α2β1 cells in Col I gel samples were treated and analysed in the same way, but no gold particle stain was observed (Figures 3E–3H).


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)

Fibrin forms a network within the Col I fibril network(A–C) The secondary network of fibrin within Col I fibrils was observed using SEM. Fibrin fibres were observed in associations with: (i) cell surfaces during gel contraction and (ii) Col I fibrils, suggesting the ‘bridging’ effect of fibrin between cells and Col I fibrils. The white arrows in (B) and (C) point to fibrin fibrils. After 12 h, the fibrin fibres were observed in closer associations with the cell surface (C) compared with at 4 h (B). (D–F) Representative micrographs of C2C12 cells in pure Col I gels. Cells were round and incapable of spreading after 4 h of culture. (G–I) Representative micrographs of C2C12-α2β1 cells in pure Col I gels. No secondary network, similar to fibrin fibrils (white arrows in B and C), was observed after either 4 h (E) or 12 h (F) of contraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109839&req=5

Figure 2: Fibrin forms a network within the Col I fibril network(A–C) The secondary network of fibrin within Col I fibrils was observed using SEM. Fibrin fibres were observed in associations with: (i) cell surfaces during gel contraction and (ii) Col I fibrils, suggesting the ‘bridging’ effect of fibrin between cells and Col I fibrils. The white arrows in (B) and (C) point to fibrin fibrils. After 12 h, the fibrin fibres were observed in closer associations with the cell surface (C) compared with at 4 h (B). (D–F) Representative micrographs of C2C12 cells in pure Col I gels. Cells were round and incapable of spreading after 4 h of culture. (G–I) Representative micrographs of C2C12-α2β1 cells in pure Col I gels. No secondary network, similar to fibrin fibrils (white arrows in B and C), was observed after either 4 h (E) or 12 h (F) of contraction.
Mentions: The morphology of C2C12 cells in the reconstituted fibrin/Col I gels was analysed using SEM (Figures 2A–2C). When collagen fibres had been formed, thrombin was added to gels allowing fibrin deposits to form. Fibrin formed a secondary fibrillar matrix interwoven with the collagen fibre network. Predominantly, fibrin formed an independent amorphous mesh between the Col I fibres (Figures 2B and see Figure 6B). However notably, in many instances, fibrin was directly associated with the collagen fibres (Figures 2B and 2C). Even at this low concentration, fibrin formed an amorphous meshwork that most often was not directly associated with the Col I fibres. Even though both collagen and fibrin fibres were observed in association with the cell surface, only fibrin fibres were found in tighter associations with the cell surface during the later time periods, as shown in Figure 2(C) (12 h contraction) compared with Figures 2(B) (4 h contraction). For comparison, C2C12 cells in pure Col I gels were studied. In pure Col I gels, the C2C12 cells were not capable of spreading (Figures 2D–2F). The C2C12-α2β1 cells were also studied in pure Col I gels (Figures 2G–2I). In the Col I gels, no fibril structure similar to the fibrin network (observed in composite gels, as in Figures 2A–2C) was found (Figures 2D–2I). The presence of fibrin in the composite gels was confirmed by immunogold staining of the fibrin/Col I gels. The signal from gold-conjugated Protein A, which was used to detect rabbit anti-fibrinogen IgG, was observed at the junctions of cell protrusions with fibrils of the composite gels (Figures 3A–3D). The C2C12-α2β1 cells in Col I gel samples were treated and analysed in the same way, but no gold particle stain was observed (Figures 3E–3H).

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