Biochemical bases of the interaction of the human basic fibroblast growth factor with glycosaminoglycans: new insights from trypsin digestion studies

Heparins from bovine mucosa and lung, and chemically modified
heparins were assayed for their capacity to: (i) protect human
recombinant basic fibroblast growth factor (bFGF) from tryptic
cleavage; (ii) prevent 1251-bFGF binding to heparan sulphate
proteoglycans present in the extracellular matrix and on the cell
surface of fetal bovine aortic endothelial GM 7373 cell cultures;
(iii) affect 1251-bFGF binding to high-affinity tyrosine kinase
FGF receptors present on the cell membrane of GM 7373 cells;
(iv) inhibit the mitogenic activity exerted by bFGF in the same
cells. The results demonstrate thatthe potency shown by mucosal
heparins in the different assays is a direct function of size, verylow-
molecular-mass heparin (2.0 kDa) being significantly less
effective on a molar basis than unfractionated heparin (13.6 kDa).
Increased flexibility of the backbone structure, as observed in
reduced/oxidized heparins of different size, does not affect the
capacity of the polysaccharide to interact with bFGF. In contrast,
selective 2-O-desulphation, but not 6-O-desulphation, drastically
reduced the capacity of heparin to protect bFGF from proteolytic
cleavage, to affect its interaction with low- and high-affinity sites,
and to inhibit its mitogenic activity. Two preparations of bovine
lung heparin, differing in molecular mass, were as effective as
mucosal heparin in the bFGF-tryptic-digestion assay and the
endothelial-cell proteoglycan-binding assay, but they were highly
inefficient at inhibiting the capacity of bFGF to interact with its
tyrosine kinase receptors. Bovine lung heparins were also less
effective than mucosal heparin as bFGF antagonists in GM
7373-cell-proliferation assays. N-Desulphated/N-acetylated
bovine lung heparin retained only a significant capacity to
protect bFGF from tryptic cleavage. The results demonstrate
that different chemical features of the heparin molecule, including
decrease in molecular mass, selective desulphation, disaccharide
composition and clustering, affect differently the capacity of the
glycosaminoglycan to interact with bFGF and to influence its
biological behaviour in different assays in vitro and in endothelial
cell cultures. Our findings should aid the design of synthetic
oligosaccharides aimed at improving the. bioavailability of bFGF
when administered in vivo as a therapeutic agent.