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Molecular-size-dependent variations in the proportions of chains with high binding affinities for antithrombin in rat skin heparin proteoglycans.

ABSTRACT: Approximately half of all rat skin heparin proteoglycans have polysaccharide chains that have no sites with high binding affinity for antithrombin. The rest have chains with high-affinity antithrombin-binding-site densities ranging from zero to five sites per chain, with a high degree of variation. Proteoglycans vary in size because of diversity in the number of chains per molecule; the relationship between proteoglycan size and high-affinity antithrombin-binding-site density has not been studied previously. Polydisperse heparin proteoglycans from rat skin, labelled biosynthetically with 35S, were fractionated by gel filtration on Bio-Gel A-150m and arbitrarily divided into five fractions of decreasing average molecular size. Fractionation of these products on antithrombin-agarose showed that the proportion of proteoglycans with high affinity for antithrombin decreased from 39% to 25% as molecular size decreased. However, as the molecular size of high-affinity proteoglycans decreased, the proportion of their chains that had high affinity increased from 29% to 59%. Therefore molecular size is a significant factor in determining the proportion of high-affinity chains in heparin proteoglycans. A model of heparin biosynthesis is proposed in which areas of specific enzyme activity that control the synthesis of the antithrombin-binding-site sequence are sparsely and nonrandomly distributed on mast-cell Golgi membranes. It is postulated that the likelihood of a developing proteoglycan encountering one of these hypothetical areas is molecular-size-dependent.

SUBMITTER: Horner AA

PROVIDER: S-EPMC1133365 | biostudies-other | 1989 Sep

REPOSITORIES: biostudies-other

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Project description:The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

Project description:The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.

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Molecular-size-dependent variations in the proportions of chains with high binding affinities for antithrombin in rat skin heparin proteoglycans.

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