Further Purification of Heparin Reduces Its Bleeding Effects in the Mesenteric Vessels of Rats

Journal of Biological Chemistry

Oreste³

et al. 1997

Self Cite

179

Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-Odesulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was 1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST- Tat. The results demonstrate that Tat interacts in a sizedependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Interaction of HIV-1 Tat Protein with Heparin

Journal of Biological Chemistry

Oreste³

et al. 1997

Self Cite

179

“…Mucosal heparin (13.6 kDa) was obtained from a commercial batch preparation of unfractionated sodium heparin from bovine mucosa (1131/900 from Laboratori Derivati Organici S.p.A., Milan, Italy) which was purified as previously described [28]. Bovine lung heparin (9.7 kDa) was obtained from Kabi-Pharmacia-Hepar (Franklin, OH, U.S.A.).…”

Section: Experimental Materialsmentioning

confidence: 99%

Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor

Zoppetti

et al. 1994

Biochemical Journal

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 125I-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 125I-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 that the potency shown by mucosal heparins in the different assays is a direct function of size, very-low-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.

“…A., Milan, Italy) which was purified from contaminants according to described methodologies [30]. Low-molecular-mass (4-kDa) heparin and very low-molecular-mass (2-kDa) heparin were obtained by controlled degradation with nitrous acid of conventional heparin, as described elsewhere [31,321.…”

Section: Gagsmentioning

confidence: 99%

Biochemical bases of the interaction of human basic fibroblast growth factor with glycosaminoglycans

European Journal of Biochemistry

Zoppetti

et al. 1993

Self Cite

In the present study we have attempted a characterization of the biochemical bases of the interaction of human basic fibroblast growth factor (bFGF) with glycosaminoglycans (GAGs) in solution. This interaction has been evidenced as the capacity of different GAGs and various sulfated compounds to protect bFGF and different bFGF mutants from tryptic cleavage.Heparin protects bFGF from trypsin digestion in a dose-dependent fashion. Substitution by sitedirected mutagenesis of two or more basic residues with neutral glutamine residues in the aminoterminal region bFGF(27 -32) or in the carboxyl-terminal region bFGF(118 -129) does not significantly affect the protective effect exerted by heparin. In contrast, heparin protection is abolished when the full region bFGF(27 -32) is deleted.The capacity of different GAGs to protect bFGF from proteolytic cleavage decreases in the following order: heparin > heparan sulfate > dermatan sulfate = chondroitin sulfates A and C > hyaluronic acid = K5 polysaccharide, indicating that both the degree of sulfation and the backbone structure of GAG modulate its interaction with bFGF. This is confirmed by the different capacity of various sulfated compounds (including dextran sulfates, suramin, trypan blue, and sulfate ion) to protect bFGF from tryptic digestion. Moreover, tryptic digestion studies performed with various heparin molecules and dextran sulfates of different size, ranging from 2.0 kDa to 500 kDa, indicate that the number of bFGF molecules which interact with a single molecule of polysaccharide is related to the molecular mass of the GAG and that six hexose residues are sufficient to protect 1 -2 molecules bFGF.In conclusion, our findings indicate that the capacity of GAGs to protect bFGF from tryptic cleavage depends upon their size, sulfation, distribution of the anionic sites along the chain, and structural requirements of the bFGF molecule. These studies will help to design synthetic oligosaccharides endowed with different bFGF agonist and/or antagonist activities.