title: Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain.
abstract: Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.
authors.affiliation: Department of Oncology, Mario Negri Institute for Pharmacological Research, Bergamo, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Margosio
authors.forename: Marco
authors.initials: M
authors.lastname: Rusnati
authors.forename: Katiuscia
authors.initials: K
authors.lastname: Bonezzi
authors.forename: Blue-Leaf A
authors.initials: BL
authors.lastname: Cordes
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Annis
authors.forename: Chiara
authors.initials: C
authors.lastname: Urbinati
authors.forename: Raffaella
authors.initials: R
authors.lastname: Giavazzi
authors.forename: Marco
authors.initials: M
authors.lastname: Presta
authors.forename: Domenico
authors.initials: D
authors.lastname: Ribatti
authors.forename: Deane F
authors.initials: DF
authors.lastname: Mosher
authors.forename: Giulia
authors.initials: G
authors.lastname: Taraboletti
mesh_terms: D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D019700:Thrombospondin 1 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020533:Angiogenesis Inhibitors; D019700:Thrombospondin 1; D016222:Fibroblast Growth Factor 2; D002118:Calcium