Antithrombin Binding Site Research Articles

A quantitative method to detect non-antithrombin-binding 3-O-sulfated units in heparan sulfate

Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of 13 units was prepared. Using reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system, the separation conditions were optimized to quantify the 13 units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units and establishes a new approach to explore the physiological functions of 3-O-sulfate.

Clinical Utilization of Non-Anticoagulant - A Review

Heparin is used as anticoagulant for long period and also other activities both experimentally and clinically. The heparin is prepared by many methods namely enzymatic hydrolysis, chemical hydrolysis by removing the antithrombin binding sites or inactivating functional groups or units of this sequence. In non-anticoagulant treatments the therapeutic spectrum helps to the structural variations to treat different diseases. Sulfated nonanticoagulant heparins (S-NACHs) might be preferred for potential clinical use in cancer patients without affecting hemostasis. Among still some of the drugs or its derivatives are undergoing the clinical trials for various non-anticoagulant diseases. This review analyses the effects of this group of drugs on non-anticoagulant heparin, acute pulmonary embolism, oral pulmonary embolism, Non anticoagulant in cancer, Antitumor activities of LMWHs, dermatology, Effects of non-anticoagulant on Fractures such as bone metabolism, bone mineral density, Anti-inflammatory, asthma, Malaria Parasites, Anti-Human Immunodeficiency Virus, Non-anticoagulant activities of LMWHs with their mechanism, Non-anticoagulant pharmacological activities in clinical trials are discussed in this review.

In vitro fermentation and isolation of heparin-degrading bacteria from human gut microbiota

Heparin and its derivative are commonly used as injectable anticoagulants in clinical procedures, but possess poor oral bioavailability. To explore the role of gut microbiota in the poor oral effect of heparin, the degradation profiles of heparin on six human gut microbiota were investigated. The heparin-degradation ability varied significantly among individuals. Furthermore, two strains of heparin-degrading bacteria, Bacteroides ovatus A2 and Bacteroides cellulosilyticus B19, were isolated from the gut microbiota of different individuals and the degradation products of the isolates were profiled. The ΔUA2S-GlcNS6S was the major end product with almost no desulfation. 3-O-sulfo group-containing tetrasaccharides were detected, which indicated that the antithrombin binding site was broken and this explained the lost anticoagulant activity of heparin. Collectively, the present study assessed the degradation profiles of heparin by human gut microbiota and provided references for the development of oral administration of heparin from a gut microbiota perspective.

Pentosan polysulfate to control hepcidin expression in vitro and in vivo

Hepcidin peptide is crucial in the regulation of systemic iron availability controlling its uptake from the diet and its release from the body storage tissues. Hepcidin dysregulation causes different human disorders ranging from iron overload (e.g. hemochromatosis) to iron deficiency (e.g. anemia). Hepcidin excess is common in the Anemia of Chronic Diseases or Anemia of Inflammation and in the genetic form of anemia named IRIDA; the pharmacological downregulation of hepcidin in these disorders could improve the anemia. Commercial heparins were shown to be strong inhibitors of hepcidin expression, by interfering with BMP6/SMAD pathway. The non-anti-coagulant heparins, modified to abolish the anti-thrombin binding site, were equally potent and could be used to improve iron status. To perform its anti-hepcidin activity heparin needs 2O- and 6O-sulfation and an average molecular weight (MW) up to 4000–8000 Dalton, depending on the sulfation level. The pentosane polysulfate (PPS), which shares with heparin a high degree of sulfation, is a compound with low anti-coagulant activity that is already in use for pharmaceutical treatment. In the present work we analyzed the anti-hepcidin activity of PPS in vitro and in vivo. We found that it acts as a strong inhibitor of hepcidin expression in HepG2 cells with an effect already visible after 2–3 h of treatment. It also suppressed hepcidin in mice in a dose dependent manner after 3 h and with a significant redistribution of systemic iron without evident side effects. PPS is also able to abolish the LPS dependent hepcidin upregulation similarly to that showed for heparin derivatives. These results suggest PPS as an interesting compound to control hepcidin in vivo.

Structural Analysis of Heparin-Derived 3- O -Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Structural Analysis of Heparin-Derived 3- O -Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Structural Analysis of Heparin-Derived 3-O-Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin’s anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described.

Poly(ethylene glycol acrylate)-functionalized hydrogels for heparan sulfate oligosaccharide recognition.

Heparan sulfates are complex polysaccharides belonging to the family of glycosaminoglycans that participate to the regulation of cell behavior and tissue homeostasis. The biological activities conferred to heparan sulfates are largely dependent on the content and positioning of the sulfate groups along their saccharidic units. At present, identification of particular sulfation patterns in biologically relevant heparan sulfate sequences remains challenging. Although several approaches for structure analysis exist, the complexity of heparan sulfates makes new and original approaches still required. Here, we used molecular imprinting technologies to prepare a library of polyethylene glycol acrylate functionalized hydrogels with the aim to investigate their applicability as specific recognizing systems for fondaparinux, a synthetic pentasaccharide analog to the antithrombin binding site of heparin. Adequate choice of the hydrogel composition and controlling rebinding conditions were important determinants for improving the sulfated oligosaccharide recognition specificity and selectivity. Our results suggest that molecular imprinting approaches could be a possibility for the specific recognition of biologically active sequences in heparan sulfates.

Detection of Factor IXa Activity By the Enhanced Thrombin Generation Assay

Introduction – Factor Xa generation by the factor IXa-factor VIIIa (FIXa-FVIIIa) complex is the rate-limiting step for plasma thrombin generation (TG). Modeling of blood coagulation suggests that subnanomolar concentrations of FIXa are sufficient to support plasma TG, however, the isolated enzyme is poorly reactive with both substrate and inhibitors. Thus, most detection methods for FIXa require at least nanomolar protease concentrations, which does not allow characterization of physiologically relevant enzyme levels. Our laboratory has developed a novel, highly sensitive assay to detect FIXa activity based on TG in FIX-deficient plasma.Method – The enhanced thrombin generation assay (ETGA) detects FIXa activity in test samples by dilution into citrated FIX-deficient plasma. Briefly, a standard curve was established by adding 10 µl of test plasma containing 0-80 pM plasma-derived FIXa (pFIXa) to 50 µl of FIX-deficient plasma. Simultaneously, human FVIII (19.2 nM) was activated with 12.8 nM thrombin for 30 sec, neutralized with a 1.25-fold molar excess of hirudin, and the resulting thrombin-activated FVIIIa was added to plasma (final plasma concentration 1.3 nM) immediately after addition of calcium and the fluorogenic substrate. Plasma TG was detected by cleavage of Z-Gly-Gly-Arg-AMC in a Biotek Synergy HT plate reader, and data exported to TECHNOTHROMBIN TGA evaluation software to generate TG curve parameters including lag time, peak thrombin concentration, time to thrombin peak and velocity index. FIXa concentration was plotted versus mean peak thrombin ± SEM (n=3) and the data fit to a parabolic function. Sample FIXa activity was obtained from this standard curve using mean peak thrombin concentration. The specificity of the TG response was confirmed by pre-incubation of test plasma with inhibitory antibodies. To prevent contact pathway-dependent FIXa generation, the monoclonal antibody (MoAb) O1A6 was added, which directly blocks FIX activation by factor XIa[PT1] . Plasma TG was ̴20-50% lower when the assay was performed in the presence of MoAb O1A6. The dependence of the remaining TG activity on FIXa was confirmed by pre-incubation of test plasma with an inhibitory anti-FIX Gla domain antibody. An inhibitory anti-TF antibody had no effect on plasma activity in this assay.Results – The ETGA was employed to: 1) measure FIXa activity in plasma and ascites samples from patients with gynecologic malignancies (ovarian and uterine) and healthy controls, and 2) determine the half-life of FIXa in human plasma. Plasma FIXa activity from 14 patients with gynecologic malignancies, and 12 healthy controls were analyzed by the ETGA. The mean plasma FIXa activity level in the cancer group was 7.6 ± 2.1 pM, and in the control group was 8.2 ± 2.5 pM, confirmed by preincubation with MoAb as above. Coagulant activity in 6 malignant ascites samples from these patients was similarly evaluated with the ETGA assay. The citrated ascites samples demonstrated substantial TG activity, however, preincubation with inhibitory MoAb controls demonstrated that this activity was strictly TF-dependent and FIX-independent. The EGTA was also employed to detect the half-life of FIXa in FIX-deficient citrated human plasma. pFIXa (35 pM) and recombinant FIXa (50pM) was incubated in FIX-deficient plasma for 0-120 min at 37°C, and the residual FIXa activity was determined at varying timepoints. pFIXa demonstrated a remarkably long half-life in FIX-deficient plasma (36.0 ± 3.6 min). rFIXa WT demonstrated a similar half-life (40.9 ± 1.4 min), while mutation in the antithrombin (AT) binding site (R150) substantially prolonged protease half-lives (> 2 hr).Conclusion – These results demonstrate that the ETGA is capable of detecting physiologically relevant levels of FIXa activity. Initial evaluation of plasma FIXa activity suggests no significant differences between controls and patients with gynecologic malignancies, and no significant FIXa activity in malignant ascites. While rFIXa WT and pFIXa have comparable plasma half-lives, the R150A mutation in the AT-binding exosite markedly prolonged protease half-life, consistent with the role of AT as the primary plasma FIXa inhibitor. This novel assay may have applications in the detection of physiologically relevant FIXa activity in plasma and other blood derived products, recombinant FIX preparations, and other body fluids. DisclosuresNo relevant conflicts of interest to declare.

A heparin-like glycosaminoglycan from shrimp containing high levels of 3-O-sulfated d-glucosamine groups in an unusual trisaccharide sequence

The detailed characterization of a novel heparin-like glycosaminoglycan purified from the viscera (heads) of the shrimp Litopenaeus vannamei is reported. Structural analysis performed by mono- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed it to be rich in both glucuronic acid and N,6-sulfated glucosamine residues. The key peculiarities were its high 3-O-sulfated glucosamine content compared to mammalian heparins; a residue which is usually associated with the antithrombin (AT) binding site, and the location of these residues within 2-O-sulfated iduronate and glucuronate-containing sequences (I2S–A∗–G), a situation not found in mammalian heparin. It also exhibited higher molecular weight (∼36kDa) than conventional heparin (∼16kDa) but, negligible anticoagulant activity (∼5IU/mg compared to heparin ∼190IU/mg) and stabilization of AT, which has been linked directly to anticoagulation activity. A high affinity fraction, eluting at a similar salt concentration (0.75–1.5M NaCl) from an antithrombin affinity column, to the high affinity fraction of heparin, also showed only weak thermal stabilization of AT (+∼2°C). These structural peculiarities may help elucidate more clearly the relationship between structure and function of sulfated polysaccharides, and provide useful model compounds with which to better understand interactions of biological significance.

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.

Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.

Unravelling Structural Information from Complex Mixtures Utilizing Correlation Spectroscopy Applied to HSQC Spectra

The first use of statistical correlation spectroscopy to extract chemical information from 2D-HSQC spectra, termed HSQC correlation spectroscopy (HSQCcos), is reported. HSQCcos is illustrated using heparin, a heterogeneous polysaccharide, whose diverse composition causes signals in HSQC spectra to disperse. HSQCcos has been used to probe the chain modifications that cause this effect and reveals hitherto unreported structural details. An interesting finding was that the signal for position 2 of trisulfated glucosamine [N-, 3-O-, and 6-O-sulfated] (A*) is bifurcated, owing to the presence of A* residues in both the "normal" antithrombin binding site and also at the nonreducing end of the molecule, which is reported in intact heparin for the first time. The method was also applied to investigating the environment around other rare sequences/disaccharides, suggesting that the disaccharide; 2-O-sulfated iduronic acid linked to 6-O-sulfated N-glucosamine, which contains a free amine at position 2, is adjacent to the heparin linkage region. HSQCcos can extract chemically related signals from information-rich spectra obtained from complex mixtures such as heparin.

Hepcidin Inhibition by Modified Heparins without Anticoagulant Activity

AbstractAbstract 483 Background.There is an increasing interest in the development of pharmacological agents able to modulate hepcidin, the peptide hormone that critically regulates iron metabolism. In particular, hepcidin antagonist may have a therapeutic role in the anemia of chronic diseases, where hepcidin levels are often increased by pro-inflammatory cytokines. We previously demonstrated that heparin is a potent inhibitor of hepcidin expression in hepatic cell lines, probably by interfering with BMP/HJV/SMAD signalling, and that it was also effective in reducing hepcidin expression in mice (Poli M, Blood 2011; 117:997–1004). Since the therapeutic use of heparin for hepcidin modulation is hampered by its strong anticoagulant activity, we were interested in evaluating modified heparins without such activity. Methods.Heparins modified to inactivate the antithrombin binding site, with different molecular weight and degree of sulfation, were supplied to hepatic cell lines and mice to evaluate their potential modulation of hepcidin expression. We analysed their interference with the BMP/SMAD signalling, as well as serum hepcidin levels in mice by mass spectrometry. Results.Over 20 modified heparins were initially screened by evaluating their dose-dependent suppression of hepcidin expression before and after BMP induction. All of them showed a certain degree of anti-hepcidin activity, with two glycol-split molecules being as potent as classical unfractionated heparin. These two molecules suppressed BMP/SMAD signalling in HepG2 cells at pharmacological concentrations with maximum inhibition after 6 hours. In mice, treatments with 20 or 60 mg/Kg did not affect coagulation but strongly reduced liver pSMAD, hepcidin mRNA and serum hepcidin. Again, the maximum effect on liver hepcidin expression was observed 6 hours after the injection. This effect was observed also in conditions of high hepcidin caused by experimental inflammation or iron overload. Conclusions.Some non-anticoagulant heparins have strong anti-hepcidin activity both in vitro and in vivo, and may represent promising hepcidin antagonist with potential therapeutic applications. Disclosures:No relevant conflicts of interest to declare.

1697P - Semuloparin Efficiently Inhibits Thrombin Generation Triggered by Pancreas Adenocarcinoma Cells BXPC3. Distinct Roles of Anti-XA and Anti-IIA Activity

ABSTRACT Background Venous thromboembolism (VTE) is a common complication in patients with cancer receiving chemotherapy. Currently no anticoagulant is approved for VTE prophylaxis in this setting. Semuloparin is an ultra-LMWH generated through a highly selective depolymerization of heparin which protects the antithrombin (AT) binding site in order to improve the benefit/risk ratio compared to existing anitcoagulants. Aims We studied in vitro the mechanism of action of semuloparin on the inhibition of thrombin generation (TG) of human platelet poor plasma (PPP) triggered by human pancreatic cancer cells BXPC3. We compared the antithrombotic efficiency of semuloparin to that of enoxaparin and the specific AT-dependent factor Xa inhibitor fondaparinux. Materials and methods BXPC3 cells were suspended in PPP spiked with clinically relevant concentrations of semuloparin, enoxaparin and fondaparinux. The endogenous thrombin potential (ETP) and the mean rate index (MRI) of the propagation phase of TG were monitored with the CAT assay (Stago France) as described previously (Gerotziafas et al Thromb Res 2011). Anti-Xa and anti-IIa specific activities were assessed assays obtained from Diagnostica Stago. Results Both semuloparin and enoxaparin, at the concentration of 0.4 anti-Xa IU/ml, completely abrogated TG. Total inhibition of TG occured in the presence of 0.002 anti-IIa IU/ml of semuloparin and 0.05 anti-IIa IU/ml of enoxaparin. Fondaparinux, even at concentrations higher than 2 µg/ml, reduced the MRI but did not significantly affect the ETP and did not completely inhibited TG,. The IC50 for the ETP of the anti-IIa activity of enoxaparin was 37-fold higher as compared to that of semuloparin. Conclusion In a cancer cell model of hypercoagulability, semuloparin reduced efficiently thrombin generation. The unique anticoagulant profile of semuloparin based on its high AT-affinity differentiates it from enoxaparin and fondaparinux. The residual anti-IIa activity of semuloparin amplifies its antithrombotic efficacy. This profile is expected to translate into an improved benefit/risk ratio. Disclosure All authors have declared no conflicts of interest.

Evaluation of the Antithrombotic and Hemorrhagic Effects of a Novel Ultra Low Molecular Weight Heparin (ULMWH)

Semuloparin (S) (Sanofi‐Aventis, Paris, France) is an ULMWH exhibiting high anti‐Xa (aXa) activity due to preservation of antithrombin‐binding sites. Antithrombotic (jugular vein clamping model) and hemorrhagic (tail transection model) effects of S, the LWMH enoxaparin (E) and heparin (H) were compared in Sprague‐Dawley rats treated subcutaneously (SC) with doses of 1.0 and 2.5 mg/kg (n=5). Blood samples collected prior to euthanasia were analyzed using aPTT, aXa and Heptest (HT) assays. 1.0 mg/kg SC H significantly prolonged bleeding time compared to control (C). All agents had equivalent antithrombotic effects. S and E significantly increased HT prolongation and aXa activity compared to C and H (HT: p<0.001 A vs H; p<0.05 E vs H; Xa: p<0.001 A vs H; p<0.01 E vs H). At 2.5 mg/kg SC, only S did not significantly prolong bleeding time compared to C. Only H significantly increased aPTT compared to C. Prolongation of HT and aXa activity was observed with all agents. S exhibited a more potent aXa activity than H (p<0.001 S vs H). All agents demonstrated a dose‐dependent antithrombotic effect. Bleeding time was lower with S at both dosages, suggesting a better safety profile. AXa activity with S was closely related to its antithrombotic activity. These studies suggest that S represents a new alternative for the prevention of thrombotic disorders with an improved benefit/risk profile compared with standard H and E.

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

Heparin and HS (heparan sulfate) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS binding to VEGF (vascular endothelial growth factor) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that the VEGF binding affinity likely depends on the specific structural features of these oligosaccharides, including their degree of sulfation, sugar-ring stereochemistry and conformation. Notably, the unique 3-O-sulfo group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue-specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.

Heparin-like heparan sulfate from rabbit cartilage

Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.

New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate

Heparin (HE) and heparan sulfated glycosaminoglycans are well-known mediators of tissue development, maintenance and functions; the activities of these polysaccharides are depending mainly on their sulfate substitutions. The HE structure is also a very important feature in antithrombotic drug development, since the antithrombin binding site is composed by sequences of a specific sulfation pattern. The analysis of disaccharide composition is then a fundamental point of all the studies regarding HE/heparan sulfate glycosaminoglycan (and thereby proteoglycan) functions. The present work describes two analytical methods to quantify the disaccharides constituting HE and heparan sulfate chains. The use of PAGE of fluorophore-labeled saccharides and HPLC coupled with a fluorescence detector allowed in one run the identification of 90-95% of HE disaccharides and 74-100% of rat kidney purified heparan sulfate. Moreover, the protocol here reported avoid the N-sulfation disaccharides degradation, which may affect N-sulfated/N-acetylated disaccharides ratio evaluation. These methods could be also very important in clinical treatments since they are useful for monitoring the availability kinetics of antithrombotic drugs, such as low-molecular-weight HEs.

Neutralization of the Anticoagulant and Anti‐Xa Effects of Fondaparinux and Idraparinux by a Novel Synthetic Antagonist. Pharmacologic Implications

Synthetic analogues of the oligosaccharide sequence mimicking the antithrombin binding sites of heparin (UFH) have been developed as antithrombotic agents. Arixtra (Fondaparinux) (F) is currently approved for the management of post surgical DVT and ACS, while its methylated derivative, namely Idraparinux (I) is in clinical development for different clinical indications. Despite their clinical efficacy, bleeding complications have been reported with the use of these agents. Currently, there is no pharmacologic antagonist available to neutralize these agents. Recently, a series of synthetic salicylamide derived antagonists for (UFH) and low molecular weight heparins are developed (Polymedix, Radnor, PA). To further investigate the relative neutralization of F and I, each of these agents were added to citrated whole blood and citrated plasma at a concentration range of 0–10 ug/ml. PS and PMX 60056 were added at fixed concentrations of 6.2, 12.5 and 25 ug/ml. Heptest was used to measure the anticoagulant effects of these agents. In addition, FPA and thrombin generation was also measured. PMX 60056 effectively neutralized the heptest prolongation by both the F and I. However, PS did not produce any antagonism of the anticoagulant effects of these two agents. In the anti‐Xa assay as measured by using the amidolytic assays, neither the PMX 60056 nor the PS produced any neutralization of these effects at any concentrations. In the whole blood assays, PMX 60056 was effective in the neutralization of the fibrinopeptide A generation inhibition, however PS did not produce any inhibition. These results suggest that PMX 60056 is effective in neutralizing the anticoagulant effects of Fand I and warrant additional in vivo validation of these studies.