HEPARIN-DEPENDENT FIBROBLAST GROWTH-FACTOR ACTIVITIES - EFFECTS OF DEFINED HEPARIN OLIGOSACCHARIDES

Heparin and related molecules have been identified as important partic ipants in fibroblast growth factor (FGF) signaling although the mechan isms of action remain unclear. We have used heparin oligosaccharides t o examine steps in the signaling process which could be affected by th e polysaccharide. Immobilized FGF-1 and FGF-2 bound all sizes of oligo saccharides tested, ranging from tetrasaccharide to decasaccharide, at physiological salt concentration. Each group of oligosaccharide,vas e luted from the FGF affinity columns in several peaks, and larger oligo saccharides showed higher apparent affinity for the immobilized growth factors compared to the shorter ones. Heparin hexasaccharides were th e smallest fragments providing complete protection of FGF-1 and FGF-2 against trypsin digestion. Tetrasaccharides, however, were able to pro vide partial protection. The requirement of heparin for ligand-recepto r interaction was evaluated in receptor binding assays using Sf9 insec t cells engineered to overexpress different recombinant FGF receptor ( FGFR) species including FGFR1 beta, FGFR1 alpha or FGFR4 at the cell s urface. In these assays hexasaccharides were the smallest fragments ca pable of stimulating FGF-receptor interaction, Over the range of conce ntrations examined, neither hexasaccharides nor octasaccharides were a ble to stimulate receptor binding to the level attained by intact hepa rin, In fact, these oligosaccharides interfered with the ability of in tact heparin in promoting FGF-receptor binding. The presence of both s timulatory and inhibitory activities in hexasaccharide and octasacchar ide populations could be attributed to structural heterogeneity within the oligosaccharide preparations. However, similar observations were obtained with ''highly-sulfated'' structurally homogeneous preparation s of hexasaccharide and octasaccharide, although these molecules gener ally had greater stimulatory and less inhibitory activity than their s tructurally heterogeneous counterparts. Hexasaccharides were found to be the smallest fragments able to potentiate the FGF-1-induced 3T3 cel l proliferation while their effect on FGF-2 signaling was less clear, These observations suggest that heparin can modulate FGF-signaling at several stages with different end results.