Edward A. Johnson

Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration

Heparin purified by affinity chromatography on anti-thrombin III-Sepharose has been studied by various methods. The specific activities of the materials obtained were in the range 170–230 units/mg as determined by a whole plasma clotting method and 360–780 units/mg as determined by a F.Xa inhibition method. Gel filtration of the material showed that there was a definite molecular size dependency of the specific activities and the activity profiles were markedly different when assay -ed by different methods. These features were also observed (at generally lower activities) with gel filtration fractions of commercial heparin. The possible conclusions regarding the mechanism of heparin anticoagulant action are discussed.

Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma

Abstract Heparin purified by affinity chromatography on antithrombin III-Sepharose was fractionated according to molecular size by gel filtration. The various fractions obtained were studied with respect to their ability to potentiate the inhibition of thrombin and of Factor Xa in plasma and in a purified system containing antithrombin III. Clotting assays and chromogenic peptide substrate assays were used in parallel. The inhibition of thrombin showed a dependency on the molecular size of the heparin that was similar to that shown earlier for the APTT assay. The inhibition of Factor Xa showed a different dependency, indicating that the mechanisms for heparin-potentiated inhibition of thrombin and Factor Xa by antithrombin III are not entirely the same. The inhibition of thrombin and Factor Xa in plasma differed from that in the pure system and this difference was shown to be dependent on heparin neutralization effects.

The molecular-weight range of mucosal-heparin preparations.

A recently reported method describes the determination of the molecular-weight range of heparins by using an electrofocusing procedure to isolate individual molecular species. Commercially available heparins were fractionated on a column of polyacrylamide-agarose gel to give fractions whose molecular weights were estimated by viscometry. Fractions with mutually exclusive molecllar-weight ranges gave an appreciable number of common bands when subjected to the electrofocusing procedure; therefore, each of these bands cannot be formed from a single molecular species of heparin. Other mucopolysaccharides also gave band sequences indistinguishable from those of heparin; they differed in position and intensity with different ampholyte batches, and probably arose from individual molecular species of the ampholyte rather than the mucopolysaccharide. The molecular-weight range of the heparin was observed to be broader than that usually reported.

Anticoagulant Activities of High and Low Molecular Weight Heparin Fractions

Summary. The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti‐Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti‐Xa assays, but in APTT assays the LMW fraction had only half the activity of the HMW fraction. Anti‐Xa assays using synthetic substrate S‐222 gave about 20% lower levels than anti‐Xa clotting assays for both heparins. Complete protamine neutralization of the post‐injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti‐Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three in vitro. This non‐neutralizable activity probably accounts for the difference between the anti‐Xa clotting and synthetic substrate assays. Studies by crossed immunoclectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.

The effect of variation of substitution on the solution conformation of heparin: a spectroscopic and molecular modelling study

The effect of variations in substitution on the conformation of iduronate-containing sequences in heparin and heparan sulphate has been studied using a series of chemically-modified heparins in which substitution with O- and N-sulphate and N-acetyl substituents has been systematically altered. Monosaccharides corresponding to residues in these modified heparins have also been investigated. The conformations of the glycosidic linkages in O- and N-desulphated re-N-acetylated heparin, O-desulphated re-N-sulphated heparin, and 6-O-desulphated re-N-sulphated heparin have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have previously been reported [B. Mulloy, M.J. Forster, C. Jones, and D.B. Davies, Biochem. J., 293 (1993) 849-858]. The overall conformation of all the polysaccharides is shown to be similar, regardless of substitution pattern. The conformational equilibrium of the pyranose ring of iduronic acid residues in the polysaccharides has been monitored by the use of 13C NMR chemical shift temperature coefficients, and shown to be similar for all the modified heparins with the exception of N-desulphated re-N-acetylated heparin. Circular dichroism spectra of all the polysaccharides are reported, and their variations attributed to differences in the proportions of pyranose ring forms in the iduronate conformational equilibrium.

Anticoagulant activities of lung and mucous heparins

Abstract It is generally assumed that heparins prepared from different tissues have equivalent actions. However, significant differences were found between lung and mucosal heparins when they were examined in an anti-Factor Xa assay, both in vivo and in vitro . These differences were not seen when the heparins were assayed by an APTT assay. With both types of heparin, anti-Xa activity increased with low molecular weight fractions, while the APTT activity decreased. However, with lung heparin the specific activities by anti-Xa assay were much lower at all molecular weights examined. It is believed that these observations have important implications for the assay and clinical use of heparin.

A Comparison of the Binding Specificities of Lectins from Ulex Europaeus and Lotus Tetragonolobus

The L-fucose-binding lectins present in the seeds of Ulex europaeus and Lotus tetragonolobus have been isolated by affinity chromatography. The binding specificities of the lectins were compared by hemagglutination inhibition analysis. The lectins behaved similarly with respect to a variety of synthetic saccharides. D-Fucose derivatives were inactive as inhibitors. In general, the alpha -L-fucopyranosyl derivatives were better inhibitors than the beta-L-fucopyranosyl derivatives. Aromatic aglycones increased the inhibitory activity of the saccharides with p-nitrophenyl-alpha-L-fucopyranoside being one of the best inhibitors. The major difference between the two lectins was the observation that the Ulex lectin required higher concentrations of saccharides to bring about complete inhibition of hemagglutination than did the Lotus lectin.

A simple procedure for the isolation of L-fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus.

L-Fucose-binding lectins from Ulex europeaus and Lotus tetragonolobus were isolated by affinity chromatography on columns of L-fucose-Sepharose 6B. L-Fucose was coupled to Sepharose 6B after divinyl sulfone-activation of the gel to give an affinity adsorbent capable of binding more than 1.2 mg of Ulex lextin/ml of gel, which could then be eluted with 0.1M or 0.05M L-fucose. Analysis of the isolated lectins by hemagglutination assay, by gel filtration, and polyacrylamide disc-electrophoresis revealed the presence of isolectins, or aggregated species, or both. The apparent mol. wt. of the major lectin fraction from Lotus was 35000 when determined on Sephadex G-200 or Ultrogel AcA 34. In contrast, the apparent mol. wt. of the major lectin fraction from Ulex was 68 000 when chromatographed on Sephadex G-200 and 45 000 when chromatographed on Ultrogel AcA 34. The yields of lectins were 4.5 mg/100 g of Ulex seeds and 394 mg/100 g of Lotus seeds.

Assignment of the 1H-N.M.R. spectra of heparin and heparan sulphate

Resonances from the main repeating unit of heparan, ----4)-beta-D-GlcA-(1----4)-alpha-D-GlcNAc-(1----, have been assigned by using a sample of the capsular polysaccharide of E. coli K5. Comparison of the spectra of heparan sulphate samples before and after O- and/or N-desulphation, with re-N-acetylation or re-N-sulphation, allowed assignment of some of the H-1 doublets in terms of sequence effects. Chemical shifts for H-1 of unsulphated uronic acid residues are influenced by 6-sulphation of the nearest neighbour GlcN on the reducing side; those of GlcN residues vary according to whether they have IdoA or GlcA as the nearest neighbour on the reducing side. The H-1 doublets due to residues in the binding sequence for antithrombin have been assigned by comparison of the spectra of heparins having high and low affinities for immobilised antithrombin.

Binding-site specificity of lectins from Bauhinia purpurea alba, Sophora japonica, and Wistaria floribunda

Abstract The binding-site specificities of lectins isolated from the seeds of Baihinia purpurea alba, Sophora japonica , and Wistaria floribunda were studied by hemagglutination-inhibition assays utilizing a variety of saccharides as inhibitors. For Bauhinia lectin, 2-acetamido-2-deoxy- d -galactose was found to be the best monosaccharide inhibitor and the free monosaccharide inhibitor was as active as its glycosides. d -Galactose was a weak inhibitor and so were some of its glycosides. Some of the oligosaccharides having a d -galactose nonreducing terminus were good inhibitors, but substitution on the d -galactose or 2-acetamido-2-deoxy- d -galactose residues with other saccharides abolished the inhibitory activity. No specificity for anomeric configuration or linkage position could be demonstrated. The presence of aromatic aglycon groups did not enhance inhibitory activity of the saccharides tested and, in some cases, the inhibitory activity was decreased. In contrast to the results for the Bauhinia lectin, compounds having aromatic aglycon groups were markedly better inhibitors for Sophora and Wistaria lectins than the corresponding compounds without aromatic aglycons. d -Galactose was a weak inhibitor for Sophora and Wistaria lectins, whereas 2-acetamido- d -galactose was a poor inhibitor of Sophora lectin but a good inhibitor of Wistaria lectin. Sophora and Wistaria lectins were somewhat similar in their activity as some of the saccharides having a d -galactose in penultimate position to an l -fucose residue were weak inhibitors. However, Sophora lectin has a binding preference for β anomers, whereas Wistaria lectin did not demonstrate a clear preference for α or β anomers. For some pairs of compounds, the α was a better inhibitor than, the β anomer; in other cases, the reverse was true.