Gudrun Bäckström

Further characterization of the antithrombin-binding sequence in heparin

An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin and previously found to have the following predominant structure (see formula in text) has been studied further. High-voltage, paper electrophoresis of the 3H-labelled disaccharides obtained by deamination with HNO2 (pH 1.5) followed by reduction with Na[3H]BH4 showed approximately 25% of mono-O-sulfated components, in addition to L-iduronic acid(2-O-SO3)-2,5-anhydro-D-[3H]mannitol (6-O-SO3). The monosulfated disaccharides were identified by high pressure, ion-exchange chromatography as L-iduronic acid(2-O-SO3)-2,5-anhydro-D-[3H]mannitol, L-Iduronic acid-2,5-anhydro-D-[3H]mannitol(6-O-SO3). and D-glucuronic acid-2,5-anhydro-D-[3H]-mannitol L, iduronic acid 2,5-anhydro-D-[3H]mannitol(6-O-SO3), and D-glucuronic acid-2,5-anhydro-D-[3H]-mannitol. These components originated from the reducing, terminal disaccharide residue (units 7 and 8), as indicated by selective labelling with Na[3H]-BH4. The structural variability within this region suggests that it is not part of the antithrombin-binding sequence. Neither enzymic removal of the non-sulfated L-iduronic acid unit 1 nor N-deacetylation (by hydrazinolysis) at unit 2 had any significant effect on the affinity of the octasaccharide for antithrombin. However, removal of the disaccharide corresponding to units 1 and 2, by selective deamination of the N-deacetylated octasaccharide, yielded a low-affinity hexasaccharide. In addition, a high-affinity deamination product was formed, presumably an octasaccharide containing a 6-sulfated 2-deoxy-2-C-formyl-D-pentofuranosyl unit due to ring contraction in unit 2. These results suggest that the 6-sulfate group in unit 2 may be involved in antithrombin binding. It is concluded that the antithrombin binding site in heparin is represented by the pentasaccharide sequence extending from unit 2 to unit 6 of the octasaccharide studied.

The molecular size of the antithrombin-binding sequence in heparin

The blood anticoagulant activity of heparin depends on its ability to bind, and thereby activate, antithrombin, a plasma protein that inhibits the proteinases of the so-called coagulation cascade [ 11. Only a fraction (-1/3rd) of the molecules in heparin preparations binds with high affinity to antithrombin and this fraction accounts for most of the anticoagulant activity of the unfractionated material [2-41. Attempts to define the structural basis for the interaction between heparin and antithrombin led to partially conflicting results. While it was claimed that the heparin structure required for binding to antithrombin is contained within a tetrasaccharide sequence [5], we proposed a more extended binding region [6]. Our conclusion was based on the isolation of oligosaccharides with high affinity for antithrombin, following partial depolymerization of heparin with bacterial heparinase [7] or with nitrous acid [6]. The oligosaccharides were tentatively identified as dodecaor tetradecasaccharides. However, no attempt was made to define the extent of the actual binding sequence by selecting for the smallest possible oligosaccharide yet capable of binding with high affinity to antithrombin. Here, such a component has been isolated and identified as an octasaccharide. The location in the octasaccharide molecule of a tetrasaccharide structure (IdUA+GlcNac+GlcUA+GlcNSOi), implicated in the antithrombin-binding sequence [5,6], has been determined.

Biosynthesis of Heparin: Tritium Incorporation into Chemically Modified Heparin Catalyzed by C-5-Uronosylepimerase

Publisher Summary The epimerization of D-glucuronic to L-iduronic acid residues in the course of heparin biosynthesis is accompanied by the exchange of C-5 hydrogen atom. This feature of the reaction serves as the basis for an assay of epimerase activity, in which the release of tritium into the water of the incubation medium is measured with a D-[5-3H]glucosyluronic acid-labeled precursor polysaccharide as a substrate. This chapter discusses an experiment to study the enzymatic incorporation of radioactivity from T2O into chemically modified heparin, yielding a product suitable as a substrate in the epimerase assay. In the assay, heparin from hog mucosa was purified by repeated precipitation with cetylpyridinium chloride from 1.4 M NaCl essentially. The chapter illustrates the result by a graph providing the time course of tritium incorporation into modified heparin.