title: Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site.
abstract: The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.
authors.affiliation: Department of Biochemistry, the Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Makogonenko
authors.forename: Galina
authors.initials: G
authors.lastname: Tsurupa
authors.forename: Kenneth
authors.initials: K
authors.lastname: Ingham
authors.forename: Leonid
authors.initials: L
authors.lastname: Medved
mesh_terms: D001665:Binding Sites / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D005353:Fibronectins; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; C098000:fibrinogen alphaC; D005337:Fibrin; D005340:Fibrinogen