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Title: Synthesis, characterisation and applications of chemically modified heparins
Author: Moffat, C. F.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1987
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Heparin is a highly sulphated glycosaminoglycan. Approximately 70% of the polymer structure is represented by the disaccharide repeat unit (IdA-2S→GlcNS-6S). Variations with respect to the degree of sulphation, acetylation of the amino group and configuration of the uronic acid introduces extensive microheterogeneity within the polymer primary sequence. Heparin is not just a blood anticoagulant but has a wide ranging capability to interact with inorganic ions, proteins and drugs. Such interactions have been increasingly studied with specific reference to structure-function relationships. Heparin was subjected to a variety of chemical modifications including de-N-sulphation, de-N/O-sulphation, N-acetylation, N-propionylation, N-sulphation and carboxyl reduction. Partially modified polymers were synthesised using either less stringent solvolytic conditions or hydrochloric acid. The modified polymers were characterised using high-resolution ^1 3 C-NMR spectroscopy, Ir spectroscopy and acid-base titrations. Prior analysis of native heparin provided a set of reference spectra and titration profiles. Specific ion replacement and polarimetry were employed to study the interactions between heparin and cations. Polarimetry was further utilised to investigate the effect of polymer modification on the interaction with calcium and copper (II) ions. A study was conducted on the ability of the modified polymers to potentiate antithrombin inhibition of the cleavage of synthetic substrates by Factor Xa and thrombin. In addition, the influence of the modified heparin on capillary vessel growth in the chick CAM was investigated. Heparin and the chemically modified heparins were subjected to the bacterial enzymes heparinase II and heparinase III. Analysis of the degradation products was conducted using molecular exclusion chromatography and HPLC. The substrate specificity of the two lyases was assessed together with the potential of employing the enzymes, in conjunction with HPLC, to characterise heparins from various sources. The major conclusion are: 1) The interaction of calcium and copper (II) ions with modified polymers are fundamentally different with the amino group playing an important role in the copper (II) interaction. 2) Polysaccharide-catalysed inhibition of thrombin by antithrombin is more pronounced than for Factor AXa. The polysaccharides appear to play a subsidiary role through the formation of a simple electrostatic interaction with thrombin and antithrombin, thus bringing the protease and inhibitor together. 3) Heparinase II is active against a wide range of heparin-like polymers producing a variety of disaccharides and tetrasaccharides. Two specific glycosidic linkages are, however, resistant to the action of the enzyme. 4) Heparinase III catalyses the degradation of heparin-like polymers that are N-sulphated or N-acetylated but is inactive in those parts of the molecule in which a 2-0-sulphated uronic acid is present. 5) Enzyme-catalysed degradation followed by HPLC analysis is a viable method of characterising the disaccaride composition of heparins and heparin-like polymers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Heparins' biosynthesis