Pentasaccharide Sequence Research Articles

Pharmacology of Heparin and Related Drugs: An Update.

Heparin has been used extensively as an antithrombotic and anticoagulant for close to 100 years. This anticoagulant activity is attributed mainly to the pentasaccharide sequence, which potentiates the inhibitory action of antithrombin, a major inhibitor of the coagulation cascade. More recently it has been elucidated that heparin exhibits anti-inflammatory effect via interference of the formation of neutrophil extracellular traps and this may also contribute to heparin's antithrombotic activity. This illustrates that heparin interacts with a broad range of biomolecules, exerting both anticoagulant and nonanticoagulant actions. Since our previous review, there has been an increased interest in these nonanticoagulant effects of heparin, with the beneficial role in patients infected with SARS2-coronavirus a highly topical example. This article provides an update on our previous review with more recent developments and observations made for these novel uses of heparin and an overview of the development status of heparin-based drugs. SIGNIFICANCE STATEMENT: This state-of-the-art review covers recent developments in the use of heparin and heparin-like materials as anticoagulant, now including immunothrombosis observations, and as nonanticoagulant including a role in the treatment of SARS-coronavirus and inflammatory conditions.

A Cluster Sequencing Strategy To Determine the Consensus Affinity Domains in Heparin for Its Binding to Specific Proteins.

Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG-protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.

Implications of Heparanase on Heparin Synthesis and Metabolism in Mast Cells.

Heparin is a polysaccharide expressed in animal connective tissue-type mast cells. Owing to the special pentasaccharide sequence, heparin specifically binds to antithrombin (AT) and increases the inhibitory activity of AT towards coagulation enzymes. Heparin isolated from porcine intestinal mucosa has an average molecular weight of 15 kDa, while heparins recovered from rat skin and the peritoneal cavity were 60–100 kDa and can be fragmented by the endo-glucuronidase heparanase in vitro. In this study, we have examined heparin isolated from in vitro matured fetal skin mast cells (FSMC) and peritoneal cavity mast cells (PCMC) collected from wildtype (WT), heparanase knockout (Hpa-KO), and heparanase overexpressing (Hpa-tg) mice. The metabolically 35S-labeled heparin products from the mast cells of WT, Hpa-KO, and Hpa-tg mice were compared and analyzed for molecular size and AT-binding activity. The results show that PCMC produced heparins with a size similar to heparin from porcine intestinal mast cells, whilst FSMC produced much longer chains. As expected, heparanase overexpression resulted in the generation of smaller fragments in both cell types, while heparins recovered from heparanase knockout cells were slightly longer than heparin from WT cells. Unexpectedly, we found that heparanase expression affected the production of total glycosaminoglycans (GAGs) and the proportion between heparin and other GAGs but essentially had no effect on heparin catabolism.

Heparinase Digestion of 3-O-Sulfated Sequences: Selective Heparinase II Digestion for Separation and Identification of Binding Sequences Present in ATIII Affinity Fractions of Bovine Intestinal Heparins

Binding to antithrombin-III (ATIII) determines the anticoagulant activity of heparin. The complexes formed between heparin and ATIII result from a specific pentasaccharide sequence containing a 3-O-sulfated glucosamine in medium position. Building block analysis of heparins, following heparinase digestion, is a critical method in quality control that provides a simple structural characterization of a complex product. Hence, in these applications, study of the digestion of 3-O-sulfated moieties merits special attention. With heparinase II, specific inhibition of cleavage of the non-reducing bond of 3-O-sulfated units is observed. This specificity was erroneously generalized to other heparinases when it was observed that in exhaustive digests of heparins with the heparinase mixture, resistant 3-O-sulfated tetrasaccharides were also obtained from the specific ATIII-binding pentasaccharides. In fact, the detection of unsaturated 3-O-sulfated disaccharides in digests of heparin by heparinases I+II+III, resulting from the cleavage of the 3-O sulfated unit by heparinase I in non-conventional sequences, shows that this inhibition has exceptions. Thus, in experiments where heparinase II is selectively applied, these sequences can only be digested into tetra- or hexasaccharides where the 3-O-sulfated glucosamine is shifted on the reducing end. Heparinase I+II+III and heparinase II digests with additional tagging by reductive amination with sulfanilic acid were used to study the structural neighborhood of 3-O-sulfated disaccharides in bovine mucosal heparin fractions with increasing affinity for ATIII. The 3-O-sulfated disaccharides detected in heparinase I+II+III digests turn into numerous specific 3-O-sulfated tetrasaccharides in heparinase II digests. Additionally, ATIII-binding pentasaccharides with an extra 3-O-sulfate at the reducing glucosamine are detected in fractions of highest affinity as heparinase II-resistant hexasaccharides with two consecutive 3-O-sulfated units.

Degeneracy of the Antithrombin Binding Sequence in Heparin: 2-O-Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid.

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this "canonical" GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1 C4 -2 S0 equilibrium in the unbound saccharide, shift to full 2 S0 and full 1 C4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.

A non-anticoagulant heparin-like snail glycosaminoglycan promotes healing of diabetic wound

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

Combinatorial virtual library screening analysis of antithrombin binding oligosaccharide motif generation by heparan sulfate 3-O-Sulfotransferase 1

Pharmaceutical heparin’s activity arises from a key high affinity and high selectivity antithrombin binding motif, which forms the basis for its use as an anticoagulant. The current problems with the supply of pig heparin raises the emphasis of understanding heparin biosynthesis so as to control and advance recombinantly expressed agent that could bypass the need for animals. Unfortunately, much remains to be understood about the generation of the antithrombin-binding motif by the key enzyme involved in its biosynthesis, 3-O-sulfotransferase–1 (3OST-1). In this work, we present a novel computational approach to understand recognition of oligosaccharide sequences by 3OST-1. Application of combinatorial virtual library screening (CVLS) algorithm on hundreds of tetrasaccharide and hexasaccharide sequences shows that 3OST-1 belongs to the growing number of proteins that recognize glycosaminoglycans with very high selectivity. It prefers very well defined pentasaccharide sequences carrying distinct groups in each of the five residues to generate the antithrombin binding motif. CVLS also identifies key residues including His271, Arg72, Arg197 and Lys173, which interact with 6-sulfate, 5-COO¯, 2-/6-sulfates and 2-sulfate at the −2, −1, +2, and +1 positions of the precursor pentasaccharide, respectively. Additionally, uncharged residues, especially Gln163 and Asn167, were also identified as playing important roles in recognition. Overall, the success of CVLS in predicting 3OST-1 recognition characteristics that help engineer selectivity lead to the expectation that recombinant enzymes could be designed to help resolve the current problems in the supply of anticoagulant heparin.

Towards computational prediction of the heparan sulfate interactome

Glycosaminoglycans (GAGs), are linear polysaccharides with repeating disaccharide units. GAGs interact with numerous proteins to regulate various physiological and pathological processes such as hemostasis, cell adhesion, growth factor signaling, coagulation, viral invasion and protease regulation. Heparin (H) and heparan sulfate (HS), members of the GAG superfamily, are composed of alternating glucosamine (GlcNp) and uronic acid (UAp) residues (either glucuronic acid (GlcAp) or iduronic acid (IdoAp)) linked by 1→4 linkage that are incompletely modified through sulfation, acetylation and epimerization reactions. These modifications can produce 48 distinct disaccharides, of which 23 have been found in nature to date. Further, the IdoAp residue can exist in multiple conformations, especially 1C4 and 2SO, which can interconvert easily in solution to enhance structural possibilities. Thus, combinatorial arrangements of the several configurational and conformational variations possible at the monosaccharide level generate millions of distinct HS sequences. Yet, only one sequence, i.e., the heparin pentasaccharide sequence that binds to antithrombin with high affinity, has found therapeutic application. Surely, a number of HS sequences bind to proteins and modulate their functions. To date, these interactions have been studied in silos, which has led to rather poor understanding of HS interactome.Elucidating the HS interactome is challenging. An analysis of gene sequences has led to prediction that 435 human proteins could interact with heparin. Yet, whether these are specific, and therefore physiologically relevant, remains to be understood. We have developed a computational strategy called Combinatorial Virtual Library Screening (CVLS) to identify sequences that display a high level of specificity in binding to protein targets. The CVLS algorithm uses a two‐step approach in which in silico ‘affinity’ and in silico ‘specificity’ of interaction are key drivers of analysis. The CVLS algorithm utilizes all possible GAG sequences binding to the predicted site of GAG binding on target proteins to identify the sequence with highest specificity for each target protein. We have now validated the application of the CVLS algorithm for understanding GAG recognition of several proteins including antithrombin (AT), heparin cofactor II (HCII), fibroblast growth factor‐2 & its receptor (FGF‐2/FGFR1), transforming growth factor β2 (TGFβ2), thrombin, histone acetyltransferase p300, chemokine CXCL13 and human neutrophil elastase. We expect that our CVLS tool, which can help experimental biologists in identifying the key GAG sequence(s) to study, will be especially useful in elucidating the HS interactome.Support or Funding InformationNIH grant HL107152 to URD. Computational facility provided by National Center for Research Resources to Virginia Commonwealth University through grant S10 RR027411.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heparin: An essential drug for modern medicine.

Heparin is a life-saving drug, which belongs to few clinically used drugs without defined molecular structures in modern medicine. Heparin is the mostly negatively charged biopolymer with a broad distributions in molecular weight, charge density, and biological activities. Heparin is mainly composed of repeating trisulfated disaccharide units, which is made by mast cells that are enriched in the intestines, lungs or livers of animals. Porcine intestines and bovine lungs are two mostly used sources for heparin isolation. Heparin is well known for its anticoagulant and antithrombotic pharmacological effects. The anticoagulant activity of heparin is attributable to a 3-O-sulfate and 6-O-sulfate containing pentasaccharide sequence or a minimum eight-repeating disaccharide units containing the pentasaccharide sequence that catalyzes the suicidal inactivation of factor Xa or thrombin by a serpin or serine protease inhibitor named antithrombin III, respectively. Thus, heparin is responsible for the simultaneous inhibition of both thrombin generation and thrombin activity in the blood circulation. Moreover, heparin has many pharmacological properties such as anti-inflammatory, anti-viral, anti-angiogenesis, anti-neoplastic, and anti-metastatic effects though high affinity interactions with a variety of proteases, protease inhibitors, chemokines, cytokines, growth factors, and their respective receptors. The one drug multiple molecular targeting properties make heparin a very special drug in that various clinical trials are still conducting worldwide even 100 years after its discovery. In this review, we will summarize the structure-function relationship and the molecular mechanisms of heparin. We will also provide an overview of different clinical and potential clinical applications of heparin.

Recognition and Conformational Properties of an Alternative Antithrombin Binding Sequence Obtained by Chemoenzymatic Synthesis.

Heparin is a highly sulfated glycosaminoglycan (GAG) of natural origin used as an anticoagulant and antithrombotic drug. These properties are principally based on the binding and activation of antithrombin (AT) through the pentasaccharide sequence GlcNAc/NS,6S-GlcA-GlcNS,3,6S-IdoA2S-GlcNS,6S (AGA*IA). Literature data show that the population of the 2 S0 ring conformation of the 2-O-sulfo-α-l-iduronic acid (IdoA2S) motif correlates with the affinity and activation of AT. It was recently demonstrated that two synthetic AGA*IA-containing hexasaccharides (one G unit added at the reducing end), differing in the degree of sulfation of the IdoA unit, show comparable affinity and ability to activate AT, despite a different conformation of the IdoA residue. In this paper, the binding of these two glycans to AT was studied by isothermal titration microcalorimetry (ITC), transferred (tr-) NOESY, saturation transfer difference (STD) NMR spectroscopy and molecular dynamics (MD) simulations. Results indicated that both the IdoA2S and the IdoA units assume a 2 S0 conformation when bound with AT, and so present a common binding epitope for the two glycans, centred on the AGA*IA sequence.

Understanding Specificity of Glycosaminoglycan Interactions with Proteins

Glycosaminoglycans (GAGs) play key roles in virtually all biologic responses through their interaction with proteins. Generally, GAGs are believed to interact non‐specifically with proteins. The primary reason for this belief is the highly electrostatic nature of their interactions and the fact that only one homogeneous GAG sequence has reached the clinic – the anticoagulant heparin pentasaccharide sequence. Are highly specific GAG–protein interactions so rare? Does nature rely primarily on non‐specificity to mediate the biological roles of GAGs? These questions are of major significance and insights into specificity of GAG–protein interactions may lead to many more GAG sequences reaching the clinic. Yet, a major challenge in understanding these interactions is the massive structural complexity of GAGs. Computational approaches are extremely useful in navigating this challenge and, in some cases, the only avenue to gain comprehensive insight. Computationally, most basic and many advanced questions on GAG–protein interactions can be answered including 1) does my protein bind to GAGs?, 2) where does the GAG bind?, 3) does the protein preferentially recognize a particular GAG type?, 4) what is the most optimal GAG chain length?, 5) what is the structure of the most favored GAG sequence?, 6) is the GAG–protein system ‘specific’, ‘non‐specific’, or a combination of both?, etc. Recent advances in the areas of thrombosis and cancer from our laboratories have led to discoveries of GAG sequences that are highly specific for their protein targets. The results raise the promise that many proteins may recognize GAGs with high specificity and clinically relevant GAG sequences are waiting to be identified.Support or Funding InformationP01 HL107152R01 HL090586R25 HL128639This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Computational Study of Glycosaminoglycan Specificity for Growth Factor and Chemokine Family Members

Glycosaminoglycans (GAGs), linear polysaccharides with repeating disaccharide units, bind to proteins through primarily electrostatic interactions, which tend to induce considerable non‐specificity of recognition. Yet, GAG–protein interactions are known to regulate various physiological and pathological functions. To date, only one sequence the heparin pentasaccharide sequence that binds to antithrombin has been identified as a specific sequence with therapeutic promise. Are specific GAG sequences so rare despite the millions of natural occurring GAG sequences? Our hypothesis has been that GAG sequences that bind to proteins are not rare but it has been difficult to identify them due to the phenomenal heterogeneity of GAGs and the near impossibility of synthesizing all possible natural (and unnatural) sequences. For example, a natural hexasaccharide sequence belonging to the heparan sulfate family can be of 46,656 types. To overcome this, our lab has developed a computational strategy called as Combinatorial Virtual Library Screening (CVLS), which is exhibit impressive application in identifying sequences displaying a high level of specificity against chosen targets. Here, we extend the applicability of CVLS by adding molecular dynamics (MD) simulations as a powerful approach to identify specific and non‐specific GAG sequences for a particular target. Application of CVLS‐MD approach to well‐known GAG target, fibroblast growth factor 2 (FGF2) and chemokine CXCL13 led to unique understanding on the nature of GAG recognition and origin of specificity for therapeutic targeting. Libraries of 2592 tetrasaccharides (HS04) and 46,656 hexasaccharides (HS06) were screened against both protein targets. CVLS identified 14 HS04 sequences as highly promising sequences for CXCL13, whereas FGF2 was found to be better recognized by 9 HS06 sequences. The co‐complexes of these sequences were studied using MD in the presence of explicit water and analyzed to understand specificity of interaction through analysis of direct and indirect hydrogen bonding, single residue energy decomposition and binding free energy. There was a strong correlation of CVLS‐MD predictions with experimental data. Interesting, FGF2–HS06 co‐complex exhibited differences in hydrogen bonding pattern and binding free energy due to IdoA2S ring puckering (both 1C4 to 2SO), whereas CXCL13 showed preferential recognition of one HS04 binding site of the two possible on protein surface. Most importantly, the CVLS–MD approach identified HS sequences showing specific as well as plastic (non‐specific) hydrogen bonding interactions in solution. The in silico predictions corresponded very well with solution NMR chemical shift perturbation (CSP) data resulting in identification of a specific HS04 sequence that stabilizes the chemokine dimer. Overall, this combined CVLS–MD approach is likely to serve as a promising strategy for the identification of specific and non‐specific GAG sequences against various target proteins.Support or Funding InformationThis work was supported in part by grants from the NIH including HL107152, HL090586 and HL128639. We thank Lortat‐Jacob H, University of Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France hugues.lortat-jacob@ibs.fr. for experimental dataThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.

Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline β-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.

Pharmacology of Heparin and Related Drugs.

Heparin has been recognized as a valuable anticoagulant and antithrombotic for several decades and is still widely used in clinical practice for a variety of indications. The anticoagulant activity of heparin is mainly attributable to the action of a specific pentasaccharide sequence that acts in concert with antithrombin, a plasma coagulation factor inhibitor. This observation has led to the development of synthetic heparin mimetics for clinical use. However, it is increasingly recognized that heparin has many other pharmacological properties, including but not limited to antiviral, anti-inflammatory, and antimetastatic actions. Many of these activities are independent of its anticoagulant activity, although the mechanisms of these other activities are currently less well defined. Nonetheless, heparin is being exploited for clinical uses beyond anticoagulation and developed for a wide range of clinical disorders. This article provides a "state of the art" review of our current understanding of the pharmacology of heparin and related drugs and an overview of the status of development of such drugs.

The Impact of Chain Length and Flexibility in the Interaction between Sulfated Alginates and HGF and FGF-2.

Alginate is a promising polysaccharide for use in biomaterials as it is biologically inert. One way to functionalize alginate is by chemical sulfation to emulate sulfated glycosaminoglycans, which interact with a variety of proteins critical for tissue development and homeostasis. In the present work we studied the impact of chain length and flexibility of sulfated alginates for interactions with FGF-2 and HGF. Both growth factors interact with defined sequences of heparan sulfate (HS) at the cell surface or in the extracellular matrix. Whereas FGF-2 interacts with a pentasaccharide sequence containing a critical 2-O-sulfated iduronic acid, HGF has been suggested to require a highly sulfated HS/heparin octasaccharide. Here, oligosaccharides of alternating mannuronic and guluronic acid (MG) were sulfated and assessed by their relative efficacy at releasing growth factor bound to the surface of myeloma cells. 8-mers of sulfated MG (SMG) alginate showed significant HGF release compared to shorter fragments, while the maximum efficacy was achieved at a chain length average of 14 monosaccharides. FGF-2 release required a higher concentration of the SMG fragments, and the 14-mer was less potent compared to an equally sulfated high-molecular weight SMG. Sulfated mannuronan (SM) was subjected to periodate oxidation to increase chain flexibility. To assess the change in flexibility, the persistence length was estimated by SEC-MALLS analysis and the Bohdanecky approach to the worm-like chain model. A high degree of oxidation of SM resulted in approximately twice as potent HGF release compared to the nonoxidized SM alginate. The release of FGF-2 also increased with the degree of oxidation, but to a lower degree compared to that of HGF. It was found that the SM alginates were more efficient at releasing FGF-2 than the SMG alginates, indicating a greater dependence on monosaccharide identity and charge orientation over chain flexibility and charge density.

Production of size-defined heparosan, heparan sulfate, and heparin oligosaccharides by enzymatic depolymerization.

Glycosaminoglycans (GAG) are most commonly isolated as large polymers from various animal origins, the functional units of which are oligosaccharides, which bind their target proteins to induce conformational changes, compete with other ligands, or facilitate the formation of signaling complexes. One example, the extensively studied heparin pentasaccharide sequence-which binds antithrombin-III, inducing a conformational change that increases its serpin protease activity by 1,000-fold-is unique in that no other specific GAG-protein structure-function relations have been described to the same degree. Thus, production of heparan sulfate (HS) oligosaccharides is critical for obtaining specific structural information regarding the binding interactions of GAG and their ligands (typically proteins). Purely synthetic methods of oligosaccharide synthesis are possible, but the cost, time requirement, and difficulty of their preparation prohibit library synthesis in significant amounts. Herein, the use of bacterial heparin lyases for the production of HS oligosaccharides via enzymatic depolymerization of HS polymers is discussed. The separation and purification of these oligosaccharides by liquid chromatography are also described.

ChemInform Abstract: Synthetic Oligosaccharides as Active Pharmaceutical Ingredients: Lessons Learned from the Full Synthesis of One Heparin Derivative on a Large Scale

AbstractReview: 67 refs.

Antithrombin-binding oligosaccharides: structural diversities in a unique function?

Heparin-antithrombin interaction is one of the most documented examples of heparin/protein complexes. The specific heparin sequence responsible for the binding corresponds to a pentasaccharide sequence with an internal 3-O-sulfated glucosamine residue. Moreover, the position of the pentasaccharide along the chain as well as the structure of the neighbor units affects the affinity to antithrombin. The development of separation and purification techniques, in conjunction with physico-chemical approaches (mostly NMR), allowed to characterize several structural variants of antithrombin-binding oligosaccharides, both in the free state and in complex with antithrombin. The article provides an overview of the studies that lead to the elucidation of the mechanism of interaction as well as acquiring new knowledge in heparin biosynthesis.

Surface modification of polydimethylsiloxane with a covalent antithrombin–heparin complex to prevent thrombosis

To prevent coagulation in contact with blood, polydimethylsiloxane (PDMS) was modified with an antithrombin–heparin (ATH) covalent complex using polyethylene glycol (PEG) as a linker/spacer. Using NHS chemistry, ATH was attached covalently to the distal chain end of the immobilized PEG linker. Surfaces were characterized by contact angle and X-ray photoelectron spectroscopy; attachment was confirmed by decrease in contact angles and an increase in nitrogen content as determined by X-ray photoelectron spectroscopy. Protein interactions in plasma were investigated using radiolabeled proteins added to plasma as tracers, and by immunoblotting of eluted proteins. Modification of PDMS with PEG alone was effective in reducing non-specific protein adsorption; attachment of ATH at the distal end of the PEG chains did not significantly affect protein resistance. It was shown that surfaces modified with ATH bound antithrombin selectively from plasma through the pentasaccharide sequence on the heparin moiety of ATH, indicating the ability of the ATH-modified surfaces to inhibit coagulation. Using thromboelastography, the effect of ATH modification on plasma coagulation was evaluated directly. It was found that initiation of coagulation was delayed and the time to clot was prolonged on PDMS modified with ATH/PEG compared to controls. For comparison, surfaces modified in a similar way with heparin were prepared and investigated using the same methods. The data suggest that the ATH-modified surfaces have superior anticoagulant properties compared to those modified with heparin.

Affinity capillary electrophoresis for the determination of binding affinities for low molecular weight heparins and antithrombin‐III

The anticoagulant properties of heparin stem in part from high-affinity binding to antithrombin-III (AT-III) inducing a 300-fold increase in its inhibitory activity against the coagulation protease factor Xa. The minimal structural requirements for AT-III binding are contained in the rare heparin pentasaccharide sequence containing a 3,6-O-sulfated N-sulfoglucosamine residue. ACE is used in this work to measure the relative AT-III binding affinities of the low molecular weight heparins (LWMHs) dalteparin, enoxaparin, and tinzaparin and the synthetic pentasaccharide drug fondaparinux (Arixtra). Determination of the AT-III binding affinities of the LWMHs is complicated by their inherent structural heterogeneity and polydispersity. The fractional composition of 3-O-sulfo-N-sulfoglucosamine residues was determined for each drug substance using 2D NMR and used in the interpretation of the ACE results.