The 140-kilodalton antiangiogenic fragment of thrombospondin-1 binds to basic fibroblast growth factor.
Thrombospondin-1 (TSP) inhibits the angiogenic activity of basic fibroblast growth factor (bFGF). Here we address the hypothesis of a direct interaction between TSP and bFGF. Gel permeation chromatography and cross-linking experiments demonstrated that bFGF binds to TSP in solution. bFGF also bound to immobilized TSP in a solid-phase assay. Binding was dose-dependent, with a Kd in the nanomolar range, and was inhibited by anti-TSP antibodies. The 140-kDa carboxyl-terminal fragment of TSP, but not the 25-kDa heparin-binding fragment, fully retained the bFGF binding capacity. Accordingly, binding was inhibited by monoclonal antibodies directed against this fragment. Heparin completely blocked bFGF binding to TSP and to the 140-kDa fragment. TSP and its 140-kDa fragment inhibited the binding of bFGF to endothelial cells at concentrations (> or = 100 nM) that inhibited endothelial cell proliferation but not motility. Low-affinity binding was inhibited more than high-affinity binding (up to 76 and 41% inhibition, respectively), and the inhibition was reversed by anti-TSP antibodies. Vitronectin and transforming growth factor beta, potentially associated with TSP, did not affect bFGF binding to endothelial cells. Although TSP did not affect the activation of the high-affinity receptors, it reduced the long-term internalization of bFGF. We conclude that TSP binds to bFGF through a domain within its 140-kDa fragment, a mechanism that might affect bFGF interaction with endothelial cells, activity, and association with the extracellular matrix.
Taraboletti
G
G
Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy.
Belotti
D
D
Borsotti
P
P
Vergani
V
V
Rusnati
M
M
Presta
M
M
Giavazzi
R
R
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002455:Cell Division; D002465:Cell Movement; D004730:Endothelium, Vascular / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D018919:Neovascularization, Physiologic; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D019699:Thrombospondins
D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D019699:Thrombospondins; D016222:Fibroblast Growth Factor 2; D006493:Heparin