Specificities of heparin-binding sites from the amino-terminus and type 1 repeats of thrombospondin-1

Interactions of heparin with intact human thrombospondin-l (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically m odified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avid ity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units, The dependence on oligosaccharid e size for binding to the recombinant amino-terminal heparin-binding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, sugge sting that the interaction between intact TSP1 and heparin is primarily med iated by the aminoterminal domain. Based on activities of chemically modifi ed heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation o f glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl re sidues of the uronic acids. In contrast, all of these modifications were re quired for binding of heparin to the type 1 repeat peptides. Affinity purif ication of heparin octasaccharides on immobilized TSP1 type 1 repeat peptid es revealed a preference for oligosaccharides containing the disaccharide s equence IdoA(2-OSO3)alpha 1-4-GlcNS(6-OSO3). Binding of these oligosacchari des to the peptide required the Trp residues. These data demonstrate that t he heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and ar e consistent with the ability of these TSP1-derived molecules to inhibit FG F2-stimulated angiogenesis. (C) 2000 Academic Press.