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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 native Col I(A) Biotinylated fibrinogen bound to plates coated with 10 μg/ml native Col I, but not denatured Col I. Plates were incubated for 2 h at 37°C and bound fibrinogen was detected as described in the Experimental section. (B) Addition of non-biotinylated fibrinogen competed with the binding of biotinylated fibrinogen (bio.Fib) (~90 nM) to immobilized Col I (left-hand panel). To investigate the reversibility of the binding reaction, bound biotin-labelled fibrinogen was allowed to bind for 2 h and then the plates were washed and incubated for another 2 h with 300 nM unlabelled fibrinogen (right-hand panel). Remaining bound biotin-labelled fibrinogen was detected as described in the Experimental section. Values are expressed as a percentage of the binding of ~90 nM biotinylated fibrinogen to immobilized Col I. (C) Using SPR, maximal binding of fibrinogen to immobilized Col I was detected at a fibrinogen concentration of approximately 10 nM. The on-rate was high, but the off-rates during wash-out were slow and incomplete within the investigated time period. (D) Biotinylated Col I bound to immobilized fibrinogen in a concentration-dependent manner. Values are the means from at least three independent experiments that were performed in triplicate, and the error bars are S.E.M.
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Figure 4: Fibrinogen binds to native Col I(A) Biotinylated fibrinogen bound to plates coated with 10 μg/ml native Col I, but not denatured Col I. Plates were incubated for 2 h at 37°C and bound fibrinogen was detected as described in the Experimental section. (B) Addition of non-biotinylated fibrinogen competed with the binding of biotinylated fibrinogen (bio.Fib) (~90 nM) to immobilized Col I (left-hand panel). To investigate the reversibility of the binding reaction, bound biotin-labelled fibrinogen was allowed to bind for 2 h and then the plates were washed and incubated for another 2 h with 300 nM unlabelled fibrinogen (right-hand panel). Remaining bound biotin-labelled fibrinogen was detected as described in the Experimental section. Values are expressed as a percentage of the binding of ~90 nM biotinylated fibrinogen to immobilized Col I. (C) Using SPR, maximal binding of fibrinogen to immobilized Col I was detected at a fibrinogen concentration of approximately 10 nM. The on-rate was high, but the off-rates during wash-out were slow and incomplete within the investigated time period. (D) Biotinylated Col I bound to immobilized fibrinogen in a concentration-dependent manner. Values are the means from at least three independent experiments that were performed in triplicate, and the error bars are S.E.M.

Mentions: The possibility of a direct interaction between fibrinogen/fibrin and Col I was raised based on the EM analysis of fibrin/Col I composite gels, in which fibrin fibres were observed in a close association with the Col I fibrils. The potential binding of fibrinogen to Col I was investigated using a solid-phase assay. The biotinylated fibrinogen bound to immobilized native, but not to denatured, Col I in a saturable fashion using solid-phase assays performed for 2 h at 37°C (Figure 4A). Half-maximal binding was achieved at a fibrinogen concentration of ~70 nM. Addition of unlabelled fibrinogen competed with the biotinylated fibrinogen in binding to the immobilized Col I (Figure 4B). Bound biotinylated fibrinogen was displaced by unlabelled fibrinogen added at a 15-fold molar excess, suggesting an equilibrium type of binding (Figure 4B). Furthermore, investigations of the binding reaction using SPR showed that fibrinogen bound to immobilized Col I with a high on-rate (Figure 4C). Half-maximal binding in this system was achieved at a fibrinogen concentration of approximately 10 nM. The off-rates during wash-out were, however, slow and incomplete within the investigated time frame (Figure 4C). Biotinylated Col I on the other hand bound to immobilized fibrinogen in the same solid-phase setting, i.e. at a temperature allowing formation of collagen microfibrils (Figure 4D). Although no direct binding of fibrinogen to denatured collagen was detected in the solid-phase assays (Figure 4A), addition of increasing concentrations of fibronectin mediated binding of biotinylated fibrinogen to immobilized denatured Col I, which is in agreement with the reported ability of fibronectin to bind to fibrinogen [2] and collagen [38].


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 native Col I(A) Biotinylated fibrinogen bound to plates coated with 10 μg/ml native Col I, but not denatured Col I. Plates were incubated for 2 h at 37°C and bound fibrinogen was detected as described in the Experimental section. (B) Addition of non-biotinylated fibrinogen competed with the binding of biotinylated fibrinogen (bio.Fib) (~90 nM) to immobilized Col I (left-hand panel). To investigate the reversibility of the binding reaction, bound biotin-labelled fibrinogen was allowed to bind for 2 h and then the plates were washed and incubated for another 2 h with 300 nM unlabelled fibrinogen (right-hand panel). Remaining bound biotin-labelled fibrinogen was detected as described in the Experimental section. Values are expressed as a percentage of the binding of ~90 nM biotinylated fibrinogen to immobilized Col I. (C) Using SPR, maximal binding of fibrinogen to immobilized Col I was detected at a fibrinogen concentration of approximately 10 nM. The on-rate was high, but the off-rates during wash-out were slow and incomplete within the investigated time period. (D) Biotinylated Col I bound to immobilized fibrinogen in a concentration-dependent manner. Values are the means from at least three independent experiments that were performed in triplicate, and the error bars are S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Fibrinogen binds to native Col I(A) Biotinylated fibrinogen bound to plates coated with 10 μg/ml native Col I, but not denatured Col I. Plates were incubated for 2 h at 37°C and bound fibrinogen was detected as described in the Experimental section. (B) Addition of non-biotinylated fibrinogen competed with the binding of biotinylated fibrinogen (bio.Fib) (~90 nM) to immobilized Col I (left-hand panel). To investigate the reversibility of the binding reaction, bound biotin-labelled fibrinogen was allowed to bind for 2 h and then the plates were washed and incubated for another 2 h with 300 nM unlabelled fibrinogen (right-hand panel). Remaining bound biotin-labelled fibrinogen was detected as described in the Experimental section. Values are expressed as a percentage of the binding of ~90 nM biotinylated fibrinogen to immobilized Col I. (C) Using SPR, maximal binding of fibrinogen to immobilized Col I was detected at a fibrinogen concentration of approximately 10 nM. The on-rate was high, but the off-rates during wash-out were slow and incomplete within the investigated time period. (D) Biotinylated Col I bound to immobilized fibrinogen in a concentration-dependent manner. Values are the means from at least three independent experiments that were performed in triplicate, and the error bars are S.E.M.
Mentions: The possibility of a direct interaction between fibrinogen/fibrin and Col I was raised based on the EM analysis of fibrin/Col I composite gels, in which fibrin fibres were observed in a close association with the Col I fibrils. The potential binding of fibrinogen to Col I was investigated using a solid-phase assay. The biotinylated fibrinogen bound to immobilized native, but not to denatured, Col I in a saturable fashion using solid-phase assays performed for 2 h at 37°C (Figure 4A). Half-maximal binding was achieved at a fibrinogen concentration of ~70 nM. Addition of unlabelled fibrinogen competed with the biotinylated fibrinogen in binding to the immobilized Col I (Figure 4B). Bound biotinylated fibrinogen was displaced by unlabelled fibrinogen added at a 15-fold molar excess, suggesting an equilibrium type of binding (Figure 4B). Furthermore, investigations of the binding reaction using SPR showed that fibrinogen bound to immobilized Col I with a high on-rate (Figure 4C). Half-maximal binding in this system was achieved at a fibrinogen concentration of approximately 10 nM. The off-rates during wash-out were, however, slow and incomplete within the investigated time frame (Figure 4C). Biotinylated Col I on the other hand bound to immobilized fibrinogen in the same solid-phase setting, i.e. at a temperature allowing formation of collagen microfibrils (Figure 4D). Although no direct binding of fibrinogen to denatured collagen was detected in the solid-phase assays (Figure 4A), addition of increasing concentrations of fibronectin mediated binding of biotinylated fibrinogen to immobilized denatured Col I, which is in agreement with the reported ability of fibronectin to bind to fibrinogen [2] and collagen [38].

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