Mast cell glycosaminoglycans.

Mast cells contain granules packed with a mixture of proteins that are released on degranulation. The proteoglycan serglycin carries an array of glycosaminoglycan (GAG) side chains, sometimes heparin, sometimes chondroitin or dermatan sulphate. Tight packing of granule proteins is dependent on the presence of serglycin carrying these GAGs. The GAGs of mast cells were most intensively studied in the 1970s and 1980s, and though something is known about the fine structure of chondroitin sulphate and dermatan sulphate in mast cells, little is understood about the composition of the heparin/heparan sulphate chains. Recent emphasis on the analysis of mast cell heparin from different species and tissues, arising from the use of this GAG in medicine, lead to the question of whether variations within heparin structures between mast cell populations are as significant as variations in the mix of chondroitins and heparins.

Collaborative study for the establishment of replacement batches of Ph. Eur. Heparin Low-Molecular-Mass for Calibration CRS.

An international collaborative study was run within the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the Commission of the European Union to establish replacement batches for European Pharmacopoeia (Ph. Eur.) Heparin Low-Molecular-Mass (LMM) for calibration Chemical Reference Substance batch 3 (CRS3) used for the characterisation of LMM heparins by high performance size-exclusion chromatography. Two candidate batches (A, cCRS4 and B, cCRS5) were filled using the same material as the existing official calibrants, adopted with either an assigned number-average molecular mass (Mna) or a broad standard table (BST). Fifteen laboratories evaluated the suitability of these candidate batches for use as calibrants with the pharmacopoeial dual refractive index/ultraviolet (RI/UV) detector calibration method, as well as with a modified mobile phase and the BST calibration method. Seven preparations of LMM heparin were tested. The results confirmed that the proposed batches are suitable for use with the same characteristic Mna as CRS3 and with the BST established for the World Health Organization (WHO) 2nd International Standard (IS). The BST calibration method gave comparable results to the RI/UV method, while showing better reproducibility, being easier to perform and requiring no calibrant with UV absorbance. The modified mobile phase had no impact on the calculated values while improving separation between the calibrant and salt peaks. The two candidate batches were adopted as Ph. Eur. Heparin LMM for calibration CRS batches 4 and 5, respectively, with the assigned Mna value of 3800 and a BST. In anticipation of the depletion of the calibrant required for use with the RI/UV method, and taking into account the unlikely procurement of a new lot of suitable starting material, it was recommended to include the BST method in Ph. Eur. monograph 0828, Heparins, low-molecular-mass. In order to improve peak separation, it was also recommended to include the use of ammonium acetate solution as mobile phase in the monograph, both for the Ph. Eur. RI/UV and the proposed BST calibration methods. Further to this study, Ph. Eur. monograph 0828 was revised to replace the RI/UV method by the BST method. This contributed to the harmonisation of methods across regions, thereby facilitating a concerted global action for the development and establishment of the next batches of calibrants for the quality control of LMM heparins.

Assays and reference materials for current and future applications of heparins.

Heparin is widely used in the prevention and treatment of thrombosis. However, this complex polysaccharide is biologically active in many systems other than coagulation, due to its structural similarity to the cell surface and matrix glycan heparan sulphate. These properties give rise to a number of potential therapeutic applications, such as those involving the anti-inflammatory activity of heparin. The anticoagulant activity of heparin is used to determine the potency of heparin preparations for use as antithrombotics. Several types of assay are used, and reference materials are available for their calibration. There is no equivalent measure of heparin's activity in other applications. For new types of heparin preparation, physicochemical methods of ensuring consistency and stability will be important, and new in vitro assays will have to be developed, all of which will require reference materials.

Determination of Oversulphated Chondroitin Sulphate and Dermatan Sulphate in unfractionated heparin by (1)H-NMR - Collaborative study for quantification and analytical determination of LoD.

Oversulphated Chondroitin Sulphate (OSCS) and Dermatan Sulphate (DS) in unfractionated heparins can be identified by nuclear magnetic resonance spectrometry (NMR). The limit of detection (LoD) of OSCS is 0.1% relative to the heparin content. This LoD is obtained at a signal-to-noise ratio (S/N) of 2000:1 of the heparin methyl signal. Quantification is best obtained by comparing peak heights of the OSCS and heparin methyl signals. Reproducibility of less than 10% relative standard deviation (RSD) has been obtained. The accuracy of quantification was good.

The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

Order out of complexity--protein structures that interact with heparin.

Many proteins of widely differing functionality and structure are capable of binding heparin. Structural characterisations of the many types of such complexes are being reported in ever-increasing number and at improved resolution. Several crystal structures of complexes formed through the interaction of heparin-derived oligosaccharides with one or more protein partners have been described.

Establishment of replacement batches for heparin low-molecular-mass for calibration CRS, and the International Standard Low Molecular Weight Heparin for Calibration.

An international collaborative study involving fourteen laboratories has taken place, organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) with National Institute for Biological Standards & Control (NIBSC) (in its capacity as a World Health Organisation (WHO) Laboratory for Biological Standardisation) to provide supporting data for the establishment of replacement batches of Heparin Low-Molecular-Mass (LMM) for Calibration Chemical Reference Substance (CRS), and of the International Reference Reagent (IRR) Low Molecular Weight Heparin for Molecular Weight Calibration. A batch of low-molecular-mass heparin was donated to the organisers and candidate preparations of freeze-dried heparin were produced at NIBSC and EDQM. The establishment study was organised in two phases: a prequalification (phase 1, performed in 3 laboratories in 2005) followed by an international collaborative study (phase 2). In phase 2, started in March 2006, molecular mass parameters were determined for seven different LMM heparin samples using the current CRS batch and two batches of candidate replacement material with a defined number average relative molecular mass (Mn) of 3,700, determined in phase 1. The values calculated using the candidates as standard were systematically different from values calculated using the current batch with its assigned number-average molecular mass (Mna) of 3,700. Using raw data supplied by participants, molecular mass parameters were recalculated using the candidates as standard with values for Mna of 3,800 and 3,900. Values for these parameters agreed more closely with those calculated using the current batch supporting the fact that the candidates, though similar to batch 1 in view of the production processes used, differ slightly in terms of molecular mass distribution. Therefore establishment of the candidates was recommended with an assigned Mna value of 3,800 that is both consistent with phase 1 results and guarantees continuity with the current CRS batch. In phase 2, participants also determined molecular weight parameters for the seven different LMM heparin samples using both the 1st IRR (90/686) and its Broad Standard Table and the candidate World Health Organization (WHO) 2nd International Standard (05/112) (2nd IS) using a Broad Standard Table established in phase 1. Mean molecular weights calculated using 2nd IS were slightly higher than with 1st IRR, and participants in the study indicated that this systematic difference precluded establishment of 2nd IS with the table supplied. A replacement Broad Standard Table has been devised on the basis of the central recalculations of raw data supplied by participants; this table gives improved agreement between values derived using the 1st IRR and the candidate 2nd IS. On the basis of this study a recommendation was made for the establishment of 2nd IS and its proposed Broad Standard Table as a replacement for the 1st International Reference Reagent Low Molecular Weight Heparin for Molecular Weight Calibration. Unlike the 1st IRR however, the candidate material 2nd IS is not suitable for use with the method of Nielsen. The candidate materials were established as heparin low-molecular-mass for calibration batches 2 and 3 by the Ph. Eur. Commission in March 2007 and as 2nd IS low-molecular-weight heparin for molecular weight calibration (05/112) by the Expert Committee on Biological Standardization in November 2007.

Molecular modelling study of HIV p17gag (MA) protein shell utilising data from electron microscopy and X-ray crystallography.

The matrix protein p17gag (MA) is a product of proteolytic cleavage of the gag gene encoded polyprotein (pr55gag) and is formed when HIV particles undergo the process of maturation. The MA protein is associated with the inner surface of the viral membrane and determines the overall shape of the virion. Previous studies have shown the existence of trimers of MA in solution and in the crystalline state. Here, we used molecular modelling methods to identify feasible interactions between pairs of MA trimers and have related this to structural data from electron microscopy. A systematic search docking procedure was able to identify many energetically favourable conformations for a pair of trimers, including some which have been previously reported. These conformations were used to generate several networks of MA trimers, which were then evaluated against structural observations of the MA network. The model suggested here provides a good match with experimental data such as the spacing between gag protein rings, the number and disposition of glycoprotein (gp41-gp120) knobs and the number of copies of MA in a virus particle. It also rationalizes the observed distribution of sizes of virus particles and is consistent with the presence of icosahedral organisation in mature HIV. Energy minimisation performed with explicit water and counter ions, was used to identify residues participating in inter-trimer interactions. The nature of these interactions is discussed in relation to the conservation of these residues in reported variants of the HIV and SIV MA protein sequences.

Gel permeation chromatography of heparins.

Gel permeation chromatography of heparins using water as solvent has recently been used to obtain fractions for biological investigation. We find that heparin fractions obtained in this way are not, necessarily, differentiated simply on a molecular size basis as has been alleged. Correlations between biological activity and molecular size obtained using such fractions must therefore be treated with caution.

Characterization of unfractionated heparin: comparison of materials from the last 50 years.

Physicochemical and anticoagulant characteristics of 27 samples from recent batches of commercially produced unfractionated heparin have been determined as part of the process of establishment of the 5th International Standard Unfractionated Heparin. They have been compared with current heparin standards (European Pharmacopoeia, United States Pharmacopoeia, Chinese), with the 4th International Standard Unfractionated Heparin. and with the three predecessor International Standards. The results indicate that the 4th International Standard Unfractionated Heparin, established in 1982, has significantly lower molecular weight and specific activity than recently produced heparin; this is also true of all preceding International Standard Heparins and of the United States Pharmacopoeial standard. The composition of commercial unfractionated heparin may therefore have changed over time; reasons for this are discussed.

A collaborative study to establish the 5th International Standard for Unfractionated Heparin.

Twenty-four laboratories participated in a collaborative study to calibrate a replacement for the 4th International Standard for Unfractionated Heparin (82/502). Both candidate materials A and B, gave excellent intra- and inter-laboratory variations (majority of mean %gcv <10%) when assayed against the 4th International Standard. No major differences of potency estimates were found between methods, although the USP method generally gave lower potencies than the other methods and candidate B gave a greater variation between methods than A. Overall, this study showed that the differences between the candidates are marginal. Based on its narrower molecular weight profile, higher specific activity and slightly lower inter-method variation, candidate A, 97/578, was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 5th International Standard for Unfractionated Heparin with an assigned potency of 2031 IU/ampoule.

A collaborative study of proposed European Pharmacopoeia reference preparations of low molecular mass heparin.

A European collaborative study, in which 16 laboratories participated, was carried out to assess the performance of the European Pharmacopoeia (EP) monograph methods for anticoagulant activities (anti-Xa and anti-IIa assays) of low molecular mass (LMM) heparin and to assess the suitability of six candidate materials as the EP working standard for LMM heparin. There was good interlaboratory agreement for both types of assays as indicated by most gcy's being less than 10%, indicating acceptable performance of the EP assay methods. All the candidate preparations gave dose-response curves parallel to the 1st International Standard for Low Molecular Weight heparin and to each other. All preparations, possibly with the exception of E and F, gave similar performance as measured by interlaboratory agreement and would be suitable as working standards. Based on these data, preparations A, B, C and D have been established by the EP as official EP Biological Reference Preparations and they will be issued as successive batches.

Molecular weight measurements of low molecular weight heparins by gel permeation chromatography.

The molecular weight profiles of low molecular weight heparin samples have been measured by high-performance gel permeation chromatography using as calibrant the heparinase-degraded material (90/686) now established as the 1st International Reference Preparation (IRP) Low Molecular Weight Heparin for Molecular Weight Calibration Use of the calibrant as a broad molecular weight standard is described and a calibration table provided based on data collected over several years in one laboratory. In order to confirm the assignment of degree of polymerisation to resolved oligosaccharide peaks in the calibrant, molecular weights of oligosaccharides fractionated from the 1st IRP were independently determined by fast atom bombardment mass spectrometry (FAB MS). The molecular weight distributions of commercial low molecular weight heparins have been characterized. Measurements of molecular weight parameters of heparin molecular weight standards from several sources provide comparisons between the molecular weight scales of this and other studies.

Low anticoagulant heparin retains anti-HIV type 1 activity in vitro.

Heparin is a potent inhibitor of HIV-1 replication, in addition to being a well-established inhibitor of blood coagulation. The major anticoagulant activity of heparin results from binding to the plasma protein antithrombin (AT). The high-affinity binding site for AT is a specific pentasaccharide sequence that is of low abundance and completely absent from the majority of heparin chains. We have examined the anti-HIV-1 activity of both conventional and low molecular weight heparins fractionated according to affinity for AT. The high- and low-affinity fractions, despite differing markedly in anticoagulant activity, are identical in their ability to bind to the envelope glycoprotein of HIV-1, and in their inhibitory effect on HIV-1 replication in vitro (EC50 1 and 8 micrograms/ml for conventional and low molecular weight fractions, respectively). Our study shows that the anti-HIV activity of heparin is independent of its antithrombin-mediated inhibition of coagulation proteases. Therefore, heparin preparations retaining full anti-HIV-1 activity in vitro but with greatly reduced anticoagulant activity may be readily produced for further clinical investigation in the prophylaxis and therapy of HIV infection.

Antimicrobial effect of human milk on Bordetella pertussis.

It has been demonstrated that human milk, unlike bovine milk, can reduce the viability of Bordetella pertussis. This antibacterial activity was not due to the presence of antibiotics or antibodies in the human milk. Reducing the level of available iron or increasing the concentration of lysozyme in bovine milk did not induce anti-B. pertussis activity. Analysis of total fatty acids revealed that human milk contained significantly more linoleic acid than bovine milk. However, the addition of linoleic acid to bovine milk did not inhibit the growth of B. pertussis.

High-field NMR as a technique for the determination of polysaccharide structures.

NMR spectroscopy has played a developing role in the study of polysaccharide structures for over 30 years. Many new bacterial polysaccharide repeat unit structures have recently been published as a result of the application of modern NMR techniques. NMR can also be used to elucidate the structures of both regular and heterogeneous polysaccharides from fungal and plant sources, as well as complex glycosaminoglycans of animal origin. In addition to covalent structure, conformation and dynamics of polysaccharides are susceptible to NMR analysis, both in solution and in the solid state. Improvements in NMR technology with potential applications to polysaccharide studies hold promise for the future.

Structure/function studies of anticoagulant sulphated polysaccharides using NMR.

Sulphated polysaccharides have many biological functions, which depend on binding of highly specific carbohydrate structures to proteins. NMR spectroscopy is a technique capable of detailed structural elucidation of these polysaccharides, and can be used in applications ranging from routine analysis to research into covalent and conformational aspects of polysaccharide structure. This technique can be used to characterise sequence variations in heparin samples. The NMR-determined solution conformation of heparin has been used to predict binding sites on the surface of heparin-binding proteins. Sulphation patterns for dermatan sulphates of marine invertebrates have been determined. Their anticoagulant effects depend on an exact pattern of sulphate substitution. A small alteration in dermatan sulphate structure, from 4-O-sulphated to 6-O-sulphated galactosamine, leads to almost complete loss of anticoagulant activity in spite of an overall high level of sulphation. A fucosylated chondroitin sulphate isolated from sea cucumber has anticoagulant and antithrombotic activity depending on its sulphated fucose branches. The anticoagulant activity of algal fucans has been compared with that of regular, linear sulphated fucans from marine echinoderms; again high activity appears to correlate with the presence of sulphated fucose branches.

Introduction to nuclear magnetic resonance.

This brief guide is not intended as a full explanation of the theory and practice of nuclear magnetic resonance (NMR), on which there are a large number of excellent texts (1-3), but as an introduction to the terms used in the subsequent chapters. The section as a whole does not provide a comprehensive outline of the NMR of organic compounds, which would be out of place in this volume, but is a selection of particular applications likely to be of use to molecular biologists and biochemists. Over the last few years, the number of publications dealing with NMR determinations of protein and peptide conformation in solution has increased dramatically, and this is reflected in the amount of space given here to the subject in Chapters 2 and Chapters 3 . The use of NMR in the study of internal mobility in proteins and in interactions between molecules is covered in Chapter 7 . Chapters 5 and Chapters 6 deal with structural studies on complex carbohydrates, which have thrived on recent advances in NMR. Nucleic acids and their interactions are covered in Chapter 4 .

The application of nuclear magnetic resonance to structural studies of polysaccharides.

Polysaccharides are ubiquitous components of living tissues. They are storage compounds in both animals and plants, and form important structural elements in, for example, plant cell walls, insect exoskeletons, and animal connective tissues. In bacteria, they are important both as structural elements in the cell wall (the teichoic and teichuronic acids) and as surface antigens, such as the O-antigenic oligo- or polysaccharide chain of the lipopolysaccharides (LPS) of gram-negative species, and the capsular polysaccharides (CPS) found on many pathogenic bacteria. These extracellular bacterial polysaccharides have a protective function, preventing desiccation of the organism, and are important determinants of virulence, since they shield the bacterium from the body's defenses.

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.