Heparin Structure Analysis Essay

1. Introduction

Low molecular weight heparins (LMWHs) are heterogeneous mixtures of sulfated glycosaminoglycans with notable pharmacological activity. They were developed as alternative therapies to heparin in the prophylaxis and treatment of venous and arterial thrombotic disorders, to overcome its uncommon but potentially serious side effects, such as bleeding and thrombocytopenia, and relative unpredictability [1]. LMWHs are derived from unfractionated heparin (UFH) through controlled chemical or enzymatic depolymerization processes to yield fragments which are approximately one third the size of the original chains with Mws ranging from 1000 to 10,000 Da [2,3]. Owing to their lower molecular size they typically possess more predictable pharmacological action, better bioavailability and longer half-life [4,5]. Depending on manufacturing process, marketed LMWHs mainly differ in degree of depolymerisation, and in the chemical structure of their terminal units, as well as their therapeutic and pharmacological properties.

Apart from characteristic terminal residues [6], their linear internal sequences are primarily composed by 1,4-linked repeating trisulfated disaccharides units containing 2-O-sulfated iduronic acid (I2S) and N-sulfated glucosamine 6-O-sulfated (ANS,6S). These trisulfated regions alternate with undersulfated sequences containing non-sulfated uronic acids—iduronic (I) and in lower proportion glucuronic (G)—which are usually preceded by N-acetylated glucosamine units (ANAc). The specific pentasaccharide sequence -ANAc,6S-G-ANS,3S,6S-I2S-ANS,6S-(AGA*IA), present only in some of the chains, constitutes the antithrombin binding site (AT-bs), essential for a high anticoagulant and antithrombotic activity of LMWHs, such as that of UFH. LMWH structures can also include traces of the linkage region (LR), the non-sulfated sequence G-Gal-Gal-Xyl which links the original heparin chain to the core protein of its natural proteoglycan precursor through a serine (Ser) residue [7].

Due to the structural diversity and heterogeneity arising from the different methods of preparation, the LMWHs available for clinical use are regarded as chemically and pharmacologically distinct entities, each one with its own unique efficacy and safety profile, and their therapeutic interchange is considered inappropriate. Correlating the biological properties with particular structural motifs has been the most important challenge in the design of new LMWHs as well as in the development of generic versions of these drugs [6].

For the LMWH dalteparin, approved indications include the prevention of venous thromboembolism (VTE) in patients undergoing hip replacement or abdominal surgery and in acutely ill medical patients with severely restricted mobility, in long term secondary prevention of VTE, in patients with VTE and cancer, and in acute coronary syndromes as unstable angina and non-Q wave myocardial infarction [8].

Dalteparin is produced from porcine intestinal mucosa through a relatively simple and low cost method based on a controlled deaminative cleavage with nitrous acid followed by reduction, resulting in the formation of an anhydromannitol (aM.ol) ring at the reducing end. The complete structural characterization of dalteparin, such as of all LMWHs, should supply information on the size of the chains, the monosaccharide composition in terms of iduronic/glucuronic acid, N-sulfated/N-acetylated glucosamine content and sulfation pattern and the sequence of these residues along the chains. No single technique is able to fulfil all these requirements and only a combination of orthogonal analytical approaches can provide a thorough characterization. Among the analytical approaches used for structure investigation of LMWHs, NMR spectroscopy and mass spectrometry (MS) represent the most effective techniques [3]. In particular, a growing interest has been registered in the last two decades in the application of MS, by increasingly sophisticated instruments with higher mass resolution and accuracy. A relevant contribution to the analysis of very complex mixtures such as LMWHs was provided by the direct connection of mass spectrometry to liquid chromatography systems (LC-MS) [9,10,11]. Among the most recent academic studies, fingerprint analysis by reversed-phase ion-pairing ultra-performance liquid chromatography coupled to high resolution mass spectrometry (RPIP-UHPLC–MS), has been proved to be a very important analytical method to ensure, in a very fast way, drug quality and collection of extremely interesting information about oligosaccharide structure, chain length and chemical modification [12,13]. Nevertheless, due to the extremely high complexity of samples exhibiting large polydispersity over a large molecular weight distribution together with several isomers, further approaches are necessary to obtain a detailed understanding of the samples and make ensure the comparability between different samples and lot-to-lot variability. Heparin/heparan sulfate fragment mapping by enzymatic digestion either with either heparinase I, II and III or using a mixture of them is a very common strategy reported in several studies and different analytical methods to run the depolymerized solutions and record interesting information in function to the technique used are often described [14,15,16,17,18]. Moreover, it was recently described that the quantitative NMR-HSQC analysis, applied to determine the mono- and disaccharide composition of heparin and LMWHs, can be used for detailed structural comparison studies between generic or biosimilar drugs with the reference product [19].

The present work describes the in-depth structural characterization of dalteparin by 2D-NMR and LC-MS techniques, applied both to intact chains and to their enzymatic products. The sample depolymerization by two strategies involving the exhaustive digestion with a cocktail of heparinases I, II and III and heparinase III bottom up fragment mapping, allows one to obtain samples of reduced complexity and at the same time point out eventual structural motifs. As a further level of detailed analysis, investigation of appropriate size-exclusion chromatography fractions, such as octa- and decasaccharides, and of their sub-fractions endowed of high affinity (HA) or no affinity (NA) for antithrombin was performed. The overall analytical approach here described represents an effective strategy for comparative purposes of structural features of different lots of dalteparin samples either concerning the appraisal of batch-to-batch variability or the evaluation of biosimilar products.

2. Results and Discussion

2.1. NMR Characterization

The mono/disaccharide percentage content of eleven different batches of Fragmin® was determined by applying a recently validated method [19]. The HSQC spectrum reported in Figure 1 shows all major and minor signals used for integration. Results are presented in Table 1.

The main structural features of dalteparin revealed by HSQC NMR analysis were the presence of an anhydromannitol ring as the only detectable residue at the reducing end, the relatively high content of sulfated monosaccharides and a substantial percentage of G-(A*) marker of the binding site for antithrombin, in agreement with previous results [20], the absence of N-acetylated glucosamine linked to non-sulfated iduronic acid [ANAc-(I)] and a modest presence of linkage region. Moreover, traces of anomalous structures were also detected, such as epoxide and 2-deoxy-2-C-hydroxymethylpentafuranosidic residues. The former is the result of alkaline treatments possibly suffered by the parent heparin and are identified by typical H2/C2 and H3/C3 signals at 3.74/54.2 and 3.82/53.3 ppm, respectively. Such epoxides can convert to l-galacturonic acid (GalA) during the processes for preparation of both unfractionated heparin and LMWH and this structure was identified too. The latter structure is a side effect of nitrous acid treatment of heparin used to obtain dalteparin. Actually, some internal glucosamine residues can undergo a deamination to give, by ring contraction (Rc), a 2-deoxy-2-C-hydroxymethylpentafuranosidic residue, which hexocyclic CH2 groups are identified by the typical chemical shift at 5.45/104.5 ppm [21].

2.2. Chain Mapping

The eleven Fragmin® samples were subjected to LC-MS analysis of the whole intact mixture. The UHPLC method, using pentylamine as ion reagent in the ion pair reversed phase (IPRP) liquid chromatography, provided a good chromatographic separation of numerous components in a relatively short time range (less than 30 min). Mass spectral information allowed us to calculate oligosaccharide composition, in terms of chain length, number of sulfate and/or acetyl groups, and chemical modifications induced by the production process. The structure hypothesis was expressed using a code consisting of a letter indicating the non-reducing residue, such as U (uronic acid) or A (glucosamine) followed by three numbers indicating monosaccharide residues, sulfate groups and N-acetyl groups, respectively, followed by the aM.ol symbol for the fragment residues terminating with 2,5-anhydro-D-mannitol. All samples displayed highly similar UHPLC/MS profiles in terms of number and shape of peaks; in Figure 2 a representative base peak chromatogram (BPC) is reported. Mass spectra details of the main signals detected in all dalteparin samples are reported in Table S1.

Twentythree main species ranging from pentamer to tetradecamer were detected. It is important to underline that the intensity of peaks observable in Figure 2 is not related to the content of oligomeric species. Actually, the ion desorption efficiency in mass spectrometry ionization source is strongly affected by structure and molecular weight of components. In general, the higher the molecular weight of a species the lower the MS response is. This explains the apparent discrepancy between the LC-MS profile displayed in Figure 2 and the oligomeric composition profiles obtained by size exclusion chromatography (Section 2.5, Figures S3–S5).

The main species detected are associated to anhydromannitol derivative structures, most of them having a high degree of sulfation and no acetyl group. Almost all the oligosaccharides have a high degree of sulfation, but a few monoacetylated species were also observed. Odd species (penta- and heptasaccharides) were detected as minor components. Since they have a glucosamine residue at the non-reducing end, they represent original terminal chains of the parent heparin which dalteparin was derived from.

Interestingly, a number of fully sulfated oligosaccharides were detected, such as U8,11,0-aM.ol, U10,14,0-aM.ol, U12,17,0-aM.ol and U14,20,0-aM.ol. They are expected to correspond to regular sequences made up of trisulfated disaccharides (I2S-GlcNS,6S) and terminating with the typical anhydromannitol moiety, i.e., (I2S-GlcNS,6S)n-I2S-aM.ol6S, where n range from 3 to 6. Nevertheless, for each of the above oligomers at least two isomers were found: for U8,11,0-aM.ol in particular, two isomers were recorded as main components under the peaks 7 and 9, and a third one as minor species in the peak 10. Different isomers could contain a G-A* sequence in different positions (positional isomers).

The molecular mass of dalteparin oligosaccharides terminating with 2,5-anhydro-D-mannitol shows a loss of 15 Da with respect to the MW of unmodified heparin oligosaccharides, corresponding to the loss of an amino group produced by the depolymerization process.

Mass signals corresponding to a further loss of 15 Da can be often observed, suggesting an additional deamination, most probably occurred on glucosamine residues within the chain, with no subsequent depolymerization. In agreement with NMR data, such signals were associated to a ring contracted (Rc) unit, derived from a glucosamine residue [21]. Structures with the Rc unit were observed in nearly all peaks of the chromatogram. As reported in Figure 2, the presence of all these structures has been verified in peaks 6, 16, 17, 20 and 21. Minor mass signals corresponding to the expected molecular mass with 2 Daltons less were detected and identified as an aldehyde form (aM) produced by an incomplete reduction to the 2,5-anhydro-D-mannitol terminal residue.

2.3. Building Blocks Analysis by Heparinases I, II, III Digestion

Under exhaustive digestion conditions, 23 major entities were typically observed (Figure 3). Mass spectra details of the main signals detected in all dalteparin samples are reported in Table S2. Regular unsaturated disaccharides with different sulfation degree, but also monoacetylated and saturated disaccharides were observed. In particular, two isomers of the saturated disaccharide U2,3,0 were detected under the peak of the main trisulfated disaccharide ∆U2,3,0, suggesting the presence of the following isomers I2S-ANS,6S and G-A* (Figure 3). Accordingly, a number of dalteparin chains starting with G-A* sequence at the non-reducing end are expected.

Several anhydromannitol residues were identified in different chains ranging from disaccharides to hexasaccharides. Minor signals were assigned to disaccharide ∆U2,1,0-aM.ol, ∆U2,2,0-aM.ol and to hexasaccharide ∆U6,6,1-aM.ol. The highest component in the tetrasaccharides region was attributed to ∆U4,5,0-aM.ol with five sulfate groups and the anhydro derivative at the reducing end glucosamine, followed by ∆U4,4,1 with four sulfates and an acetyl group. The former could be interpreted as ∆U2S-ANS,6S-I2S-aM.ol6S and its survival can be explained by the presence of anhydromannitol derivative; the latter can be explained as ∆U-ANAc,6S-G-A*. In fact, the presence of 3-O-sulfate glucosamine renders the glycosidic bond between N-acetylated, 6-O-sulfated glucosamine and the unsulfated glucuronic acid impervious to the action of heparinases [22]. This effect can be used to obtain interesting structural information, mainly in the tetra-hexasaccharides region. Actually, among the minor tetrasaccharide species, two interesting structures were detected such as ∆U4,5,0 and ∆U4,5,1, both deriving from the possible fragmentation of two structural variants of the binding site. Their most probable sequence interpretations are as follows: ∆U-ANS,6S-G-A* and ∆U2S-ANAc,6S-G-A*, respectively.

2.4. Bottom up Analysis by Heparinases III Digestion

Heparinase III cleaves the linkage between N-acetylated glucosamine ANAc, with or without 6-O-sulfate, and glucuronic or iduronic acid, with preference for the former. As expected, all the fully sulfated oligosaccharides and their isomers, identified by the previous chain mapping analysis, were not recognized by this enzyme. The LC-MS profile and mass spectra assignment (Figure 4) show several oligosaccharides arising from the intact parent Fragmin® (with m/z values accounting for oligomers ranging from hexa- to hexadecasaccharides). Mass spectra details of the main signals detected in all dalteparin samples are reported in Table S3.

Highly sulphated odd intact structures were also observed and identified as pentasaccharide A5,8,0-aM.ol and heptasaccharide A7,11,0-aM.ol. Confirmation of the sum formula of both oligosaccharides was provided by Ion Cyclotron Resonance-FT-MS (ICR-FT-MS) analysis (Figures S2 and S3, Supplementary Material). These unusual oligosaccharide composition can be explained by the presence of I2S-A* sequence inside their chain, (ANS,6S-I2S)1or2-A*-I2S-aM.ol6OS. Such interpretation would be in agreement with the previous building blocks results where the disaccharide ∆U2,4,0, compatible with the structure ∆U2S-A*, was detected.

2.5. Isolation of Octasaccharide and Decasaccharide Fractions

Fractionation of dalteparin by size exclusion chromatography into size homogeneous oligomeric fractions can be achieved both by Biogel P6 and Biogel P10 [20]. For the present work, all the analysed dalteparin samples underwent chromatographic fractionation on Biogel P6, to obtain a fingerprint of their overall oligomeric composition: their highly similar chromatographic profiles are compared in Figures S3–S5 (Supplementary Material). A series of peaks were resolved with each peak corresponding to a size-homogeneous oligomeric family. In particular, octa- and decasaccharide fractions were isolated from Frag-11 by implementing three chromatographic runs which yielded, after desalting, 27.6 mg of octasaccharides and 60.5 mg of decasaccharides, corresponding to 3.1% and 7.8%, respectively, of the whole sample.

2.6. Affinity Chromatography Separation of NA and HA Components

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Review

New Applications of Heparin and Other Glycosaminoglycans

Marcelo Lima 1,2,*, Timothy Rudd 2,3,* and Edwin Yates 1,2,*

1

Department of Biochemistry, Federal University of São Paulo (UNIFESP), Vila Clementino, São Paulo, S.P. 04044-020, Brazil

2

Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK

3

National Institute of Biological Standards and Controls (NIBSC), Blanche Lane, Potters Bar, Herts EN6 3QG, UK

Academic Editor: Giangiacomo Torri

Received: 5 April 2017 / Accepted: 28 April 2017 / Published: 6 May 2017

Abstract

:

Heparin, the widely used pharmaceutical anticoagulant, has been in clinical use for well over half a century. Its introduction reduced clotting risks substantially and subsequent developments, including the introduction of low-molecular-weight heparin, made possible many major surgical interventions that today make heparin an indispensable drug. There has been a recent burgeoning of interest in heparin and related glycosaminoglycan (GAG) polysaccharides, such as chondroitin sulfates, heparan sulfate, and hyaluronate, as potential agents in various applications. This ability arises mainly from the ability of GAGs to interact with, and alter the activity of, a wide range of proteins. Here, we review new developments (since 2010) in the application of heparin and related GAGs across diverse fields ranging from thrombosis and neurodegenerative disorders to microbiology and biotechnology.

*

Correspondence: Tel.: +55-11-992725274 (M.L.); +44-1707-641120 (T.R.); +44-151-795-4429 (E.Y.)

Keywords:

heparin; glycosaminoglycans; chondroitin sulfate

1. Introduction

Heparin, a member of the glycosaminoglycan (GAG) family of sulfated polysaccharides, is one of the most widely used pharmaceuticals, whose major role is in the inhibition of clot formation and thrombi, especially during surgery or following trauma. Beyond referring to some important historical literature, this review will attempt to survey recent (by which we mean from ca. 2010 to 2017) applications for this important and widely used drug, and will include other GAG members, which are being explored increasingly for other potential uses.

Heparin has been an established anticoagulant drug for more than 60 years for the prevention and control of thrombotic events owing to its interaction with a number of proteins of the blood clotting cascade, notably antithrombin and thrombin [1]. It consists of a linear, highly sulfated polysaccharide chain of various lengths varying from 2000 to 40,000 Da [2,3,4], composed of repeating disaccharide units of 1,4 linked α-l-iduronic or β-d-glucuronic acid (d-GlcA), and α-d-glucosamine (d-GlcN). The predominant substitution pattern comprises 2-O-sulfation of the iduronate residues and N- and 6-O-sulfation of the glucosamine residues [5]. Heparin is also closely related structurally to heparan sulfate (HS), which is present on cell surfaces and in the extracellular matrix. HS is generally less sulfated than heparin and is often considered to have a defined domain structure [6,7,8] and a lower proportion of l-iduronate residues. There are also d-GlcA and N-acetylated glucosamine (d-GlcNAc) residues, as well as variations in sulfation, including a small proportion of 3-O-sulfates on glucosamine residues. Hyaluronic acid (HA) is the only member of the GAG family that is not sulfated, being a homo-polymer consisting of beta (1→4) linked disaccharides of d-GlcA β (1→3) d-GlcNAc. Chondroitin sulfate (CS) encompasses various structures, based on repeating β (1-4) linked disaccharides of GlcA β (1→3) GalNAc containing 6-(Chondroitin sulfate-C: CS-C) and 4-sulfates (CS-A), sulfation at position-2 of the GlcA residues (GlcA2S β (1→3) GalNAc6S) (CS-D), and 4,6-di-sulfated GalNAc (GlcA β (1→3) GalNAc4, 6diS (in CS-E). The polysaccharide, dermatan sulfate (DS), which was formerly known as CS-B, contains repeating →4) l-IdoA β (1→3) d-GalNAc β (1→ units with 4-O-sulfation on the GalNAc moiety. GAG-like structures from non-mammalian sources, often marine, have become the focus of attention recently, and these, while possessing similar backbone structures, often include branches of fucose units [9], which can include non-reducing terminal fucose with sulfation at positions-2 and -3 [10].

The discovery and development of heparin as an anticoagulant agent had a tremendous impact on health because major surgical procedures became possible, especially those in which cardiopulmonary bypass (CPB), which was first demonstrated in animals in 1939 [11], were necessary. Another important medical procedure that is only possible due to the anticoagulant properties of heparin is dialysis. Indeed, unfractionated (full-length) heparin (UFH) named heparin sodium, its antidote, protamine sulfate [12], as well as a form of low-molecular-weight heparin (LMWH) [13] all appear in the World Health Organization’s (WHO) List of Essential Medicines. The pharmacological activity of heparin results mainly from its ability to bind and accelerate the AT activity, thereby considerably enhancing the inhibition of coagulation factors Xa and IIa, although it interacts in concert with AT and other members of the blood clotting cascade as well, including factor IXa, XIa, and XIIa [14]. In a biological rather than a clinical context though, heparin action is more closely linked to defense against exogenous pathogens [15] and responses following tissue damage. Heparin alters cytokine levels [16], and one of its biological roles may be to dampen the effects of the sudden release of large numbers of cytokines following infection or sudden trauma.

In recent times, the majority of pharmaceutical heparin has been sourced from the intestine of pigs, and detailed studies of the sequence of heparin have involved laborious separation techniques. Recent advances in the biosynthesis of heparin-related structures employing recombinant enzymes and synthetic uridine-diphosphate-monosaccharide donors have been developed by Liu and Linhardt, allowing not only for the production of heparin sequences but also for the systematic and controlled preparation of heparin (and HS) analogues, which will be suitable for a wide-range of experimental purposes and applications [17].

2. Applications in Anticoagulation and Cancer Treatments

In current clinical use, one of the most important forms of heparin is low-molecular-weight heparin (LMWH) which consists of a complex mixture of fragments ranging from tetra to hexadecasaccharides and somewhat higher oligosaccharides [18,19,20] obtained by various chemical and enzymatic depolymerization processes. The main advantages of LMWHs over UFH are improved bioavailability and higher anti-factor Xa/anti-factor IIa activity ratios, with decreased hemorrhagic risk during prolonged treatments [21]. While UFH can be monitored effectively and reversed in patients undergoing surgery with extracorporeal circulation, LMWHs cannot, because neutralization with protamine sulfate is ineffective. The development of new anticoagulant agents with the beneficial properties of both UFH and LMWH is therefore still pursued and is an important area in which biosynthetic structures are being applied [22,23].

Aside from the well-known anticoagulant activities of heparin and related GAGs or GAG mixtures, such as sulodexide (a mixture of LMWH and DS in the ratio 80:20), heparin is also active in a wide range of activities in which the naturally occurring cell surface polysaccharide, heparan sulfate (HS), is a participant. Many of the proteins with which heparin interacts originate in the extracellular matrix, the best studied of which belong to the fibroblast growth factor (FGF) family. The potential use of heparin to interfere with aberrant cell–cell signaling by this route has been investigated and the relationship between sequence and activity sought [24,25,26,27].

The importance of the FGF signaling system to development and regulation, and hence when its function is impaired or altered, also to disease is one obvious area in which heparins, acting in this case as analogues for HS, can be applied. One area in which modulating FGF signaling would be desirable is cancer treatment. However, it is noteworthy that cancer-related thrombotic disorders are also well known and were first described as long ago as 1865, when the clinical association between thrombosis and an initially undiagnosed cancer was noted. Unfractionated heparin and LMWHs have been used successfully for the prophylaxis and treatment of cancer-related hemostatic disorders; however, since cancer patients have an increased risk of bleeding [28], LMWHs have gained significant ground since they produce a more predictable anticoagulant response, display improved subcutaneous bioavailability, and exhibit an extended half-life. Furthermore, heparin-induced thrombocytopenia (HIT) incidence is lower when LMWHs and the synthetic pentasaccharide, Fondaparinux, are used. Novel oral anticoagulants are under development, but their efficacy and safety in cancer-patients has yet to be proved. Despite being well recognized now, the pathogenesis is complex and results from the combination of several factors, but the expression of tumor cell-associated clotting factors is a shared characteristic. The prevention and treatment of these conditions extends beyond the ease of symptoms, it has a direct impact in cancer patient survival [29], and there are some known beneficial effects of heparin, but elucidating these is hampered by the complexity of the systems involved. The widespread use of heparin as a treatment is hindered by unwanted anticoagulant side-effects [30] (see also review by Afratis et al. [31]); however, the possibility of correcting signaling defects caused by altered HS structure (e.g., by sulf enzyme activity) in cancer [32,33] or the direct inhibition of the sulf enzymes with heparin or heparin analogues is an area of potential interest. Of direct relevance to the progression of the disease is the inhibition of a mammalian heparanase enzyme [34], while other routes are thought to influence metastasis, acting via P- and L-selections [35,36,37], inhibiting galectins [38], inhibiting tumorogenesis and angiogenesis through cellular receptors such as CD44 and growth factor receptors [39], or, in the case of the use of heparin as a means of ameliorating resistance to cisplatin treatment, acting through as-yet unidentified mechanisms [40], all of which are affected by heparin or heparin analogues. Among other GAGs, an example of the treatment of cancer is the use of the non-sulfated GAG, hyaluronate (HA) in pancreatic ductal adenocarcinoma [41].

3. Recovery from Nervous System Damage

Recent work shows that, while CS inhibits the regeneration of neurites in the CNS, CS-bound HB-GAM (pleiotrophin) activates them, inducing dendrite regeneration in adult cerebral cortex and axonal regeneration in adult spinal cord [42]. Chondroitin sulfate forms a barrier following nerve injury [43], but can be digested by enzymes to improve repair [44,45,46,47]. Chondroitin sulfate-E (CS-E), containing unusual 4,6 di-sulfated GalNAc residues and 4-sulfated CS in aggrecan, have been shown to be inhibitory to neurite outgrowth [48], and CS-E mediates estrogen-induced osteoanabolism [49], suggesting several potential future applications for CS and its derivatives.

4. Respiratory Diseases

GAGs and heparin in particular have been suggested as playing a protective roles in the inflammation response [50], which has been interpreted as involving (among other things) the inhibition of elastase and the interaction with several cytokines [51]. Heparin and its analogues have been proposed for the application to elastase inhibition for cystic fibrosis treatment (or other conditions, such as acute respiratory distress syndrome (ARDS). Although they do not originate from a conventional mammalian GAG, oligosaccharide fragments of the fucosylated GAG structures from the sea cucumber (Holothuria forskali) [9] were shown to lower neutrophil infiltration by reducing selectin interactions.

5. Neurodegenerative Diseases

There is an emerging role in Parkinson's Disease for heparin acting via cathepsin-d activity affecting α-synuclein accumulation [52], while, in Alzheimer’s Disease, the inhibition of BACE-1, the key protease responsible for the generation of toxic Aβ fragments (1–42) is inhibited by heparin and its derivatives [53,54], some of which have been engineered to possess very low anticoagulant activity [55,56]. Furthermore, it is known that heparin modulates fibril formation [57], and understanding the mechanisms underlying such events may open new routes for the prevention, for instance, of toxic fibril formation and deposition.

6. Roles as Antimicrobial Agents

6.1. Viruses

GAGs might be expected, on the basis of their universal presence on cell surfaces, to serve as a broad spectrum and relatively non-specific receptors for virus binding [58]; [see also an earlier review: [59]]. Such interactions are not unexpected, since virus envelope proteins present patches of positively charged amino acids. While GAGs have been shown to attach to filoviruses [60] and CS-E, but not CS-D, and inhibit dengue virus infection [61], it is clear that GAGs are indeed of relevance to the mechanisms of viral attachment and invasion. The ability of viruses to bind cell surface HSs and, by extension, bind heparin under experimental conditions has long been known in the case of herpes simplex (HSV) and dengue (DENV) viruses. Heparin as a potential inhibitor of viral attachment follows naturally from these observations and has been demonstrated for DENV [62]. What is less clear is whether and how this knowledge can be exploited to enable GAGs to be applied as inhibitors of attachment, a process that may be able to be exploited for the well-known property of multivalency to increase the avidity of the binding of the viral particles to the GAG, perhaps immobilized in some device, or attached to multidentate ligands in a nanotechnology format (see also a review of this field: [63]). The potential importance of such applications is difficult to over-emphasize, however. Recently, viral attachment to GAGs was reported for IFNα/βBP from variola (smallpox virus), monkeypox viruses [64], and GAGs have been shown to prevent measles virus infection in cell lines via hemagglutinin protein [65]. Other viruses, including hepatitis B, have been found to bind to heparin [66] as has Japanese encephalitis virus (JEV) [67]. Thus, the interest in GAG-based intervention seems set to increase.

Heparin and derivatives have been shown to be effective in preventing the infection of cells by the influenza virus, strain H5N1 [68], and to have effects on ZIKV-induced cell death that are independent of adhesion and invasion [69]. In this case, heparin has only a modest ability to protect infection by analogy with dengue virus, which is also of the flavivirus family, and which interacts with heparin through the envelope glycoproteins [70]. Rather, heparin may be acting to protect infected cells from cytotoxic effects and cell death via the activation of cell survival signaling pathways. The ability of heparin to protect cells from programmed cell death had already been observed in human cells (non-infected) [71], and this may form an interesting new avenue for the application of GAG derivatives in itself.

6.2. Parasites

The involvement of GAGs in a parasitic disease has been studied relatively little. However, the potential for the application of GAGs remains high in cases where GAGs form a major part of the means of attachment and invasion, especially where there is the possibility of topical application. The ability of heparin and derivatives to inhibit rosetting of parasite infected erythrocytes has been noted [72] and, recently, effective inhibitors (comprising fucosylated CS (FucCS)) of cytoadherence (the process of adhesion of infected red blood cells to vascular epithelia) derived from sea cucumber have been reported [73]. Heparin is known to alter the activity of a cysteine protease in the parasite, L. mexicana, that causes Leishmaniasis [74] and interacts with a metalloprotease from L.(v). braziliensis [75]. Heparin has also been shown to accelerate protein degradation by human neutrophil elastase [76]. Several heparin-binding proteins have also been identified in T. cruzi, where some of them are implicated in parasite adhesion to midgut epithelial cells [77].

6.3. Bacteria

Binding of GAGs facilitates Streptococcal entry into the CNS of Drosophila [78] and determines disease progression [79]. In relation to the phenomenon of shock, GAGs seem to be elevated overall, perhaps originating from damaged tissue, but they also seem to neutralize antimicrobial peptides [80]. Glycosaminoglycans were studied en masse, however, without paying attention to the individual types involved. Heparin use in sepsis patients, where crosstalk between thrombosis and inflammation plays a critical role, has also been recognized. The use of heparin prevents the development of microthromboembolic disease during sepsis, impairing tissue hypoxygenation and further organ damage and dysfunction [81]. There is also literature pointing to the use of heparin-coated stents for the prevention of bacterial biofilm formation and subsequent microbe attachment [82].

7. Panceatitis

Acute pancreatitis has been reported independently by several groups to benefit from treatment with heparin [83] in rats and in patients [84,85,86]. Furthermore, LMW heparin has also been shown to be effective [87].

8. Roles in Rheumatoid Arthritis (RA)

Naturally occurring autoantibodies to GAGs are markers in rheumatoid arthritis [88]. The level of IgM type Ab for CS-C in serum correlates with RA, although GAG concentration alone is not a good indicator in knee injury [89] in agreement with earlier findings in horses [90].

9. Inflammation Reduction

Many of the applications of heparin mentioned in this review may stem, at least in part, from the ability of heparin to moderate inflammation. This topic has itself been reviewed in 2013 [91] and, in general, heparin is perceived as acting via several routes, which include interaction with cytokines and as an inhibitor of heparanase, to achieve reduced leukocyte recruitment.

Applications in which these properties may be important include the treatment of burns [92] and the proposed use of heparin in asthma treatment [93]. The potential, based on a projection of the ability of heparin to inhibit bacterial infection and reduce inflammation, to improve the symptoms of cystic fibrosis has also been investigated [94] but no marked improvement at low doses (50,000 IU) has been shown.

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