title: Interaction of the fibronectin COOH-terminal Fib-2 regions with fibrin: further characterization and localization of the Fib-2-binding sites.
abstract: Incorporation of fibronectin into fibrin clots is important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. Previous studies revealed that this incorporation occurs through noncovalent interaction between two NH2-terminal Fib-1 regions of fibronectin (one on each chain) and the alphaC-regions of fibrin, and is further reinforced by factor XIIIa-mediated covalent cross-linking of fibronectin to the fibrin matrix. To clarify the role of another pair of fibrin-binding regions, Fib-2, located at the disulfide-linked COOH-terminal ends of fibronectin, we prepared by limited proteolysis a dimeric 140 kDa (Fib-2)2 fragment containing both Fib-2 regions and tested its interaction with recombinant fragments corresponding to the alphaC regions of fibrin(ogen). In both ELISA and surface plasmon resonance (SPR) experiments 140 kDa (Fib-2)2 bound to the immobilized Aalpha221-610 alphaC-fragment. However, the affinity of binding was substantially lower than that for Fib-1. Ligand blotting and ELISA established that the Fib-2 binding site is located in the connector part of the alphaC region including residues Aalpha221-391. Analysis of the SPR-detected binding of fibronectin to the immobilized Aalpha221-610 alphaC-fragment revealed two types of fibronectin-binding sites, one with high affinity and another one with much lower affinity. Competition experiments revealed about 30% inhibition of the Fib-2 mediated binding by increasing concentrations of Fib-1 fragment suggesting partial overlap of the two sets of binding sites. Based on these results and our previous studies we propose a mechanism of interaction of fibronectin with fibrin in which both Fib-1 and Fib-2 play a role.
authors.affiliation: Center for Vascular and Inflammatory Diseases and the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Makogonenko
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Ingham
authors.forename: Leonid
authors.initials: L
authors.lastname: Medved
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D006487:Hemostasis / Q000502:physiology; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology
chemical_list: D005353:Fibronectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005337:Fibrin