Pentasaccharide Sequence Research Articles

Treatment of heparin-induced thrombocytopenia after cardiac surgery: Preliminary experience with fondaparinux

Heparin-induced thrombocytopenia (HIT) is an immunemediated prothrombotic disorder that is often encountered after cardiac surgery. The appropriate alternative anticoagulant to be used in this setting is not univocal, especially for the coexistence of renal failure and high bleeding risk. Fondaparinux is a factor Xa inhibitor via the action of antithrombin but devoid of antifactor II (thrombin) activity. It is modeled after a critical heparin pentasaccharide sequence; although the drug is identical in structure to the pentasaccharide domain found in heparin, it is too small to be recognized by the majority of heparin-reactive antibodies. We describe our early experience with fondaparinux in the management of HIT in critically ill patients after cardiac surgery.

The Critical Role of Hinge-Region Expulsion in the Induced-Fit Heparin Binding Mechanism of Antithrombin

Antithrombin (AT) is the most important inhibitor of coagulation proteases. Its activity is stimulated by glycosaminoglycans, such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and in the presence of pentasaccharide. Pentasaccharide binding ultimately results in a conformational change that improves affinity by about 1000-fold. Crystal structures show several differences, including the expulsion of the hinge region of the reactive centre loop from beta-sheet A, which is known to be critical for the allosteric activation of AT. Here, we present data that reveal an energetically distinct intermediate on the path to full activation where the majority of conformational changes have already occurred. A crystal structure of this intermediate shows that the hinge region is in a native-like state in spite of having the pentasaccharide bound in the normal fashion. We engineered a disulfide bond to lock the hinge in its native position to determine the energetic contributions of the initial and final conformational events. Approximately 60% of the free-energy contribution of conformational change is provided by the final step of hinge-region expulsion and subsequent closure of the main beta-sheet A. A new analysis of the individual structural changes provides a plausible mechanism for propagation of conformational change from the heparin binding site to the remote hinge region in beta-sheet A.

The repertoire of glycan determinants in the human glycome

The number of glycan determinants that comprise the human glycome is not known. This uncertainty arises from limited knowledge of the total number of distinct glycans and glycan structures in the human glycome, as well as limited information about the glycan determinants recognized by glycan-binding proteins (GBPs), which include lectins, receptors, toxins, microbial adhesins, antibodies, and enzymes. Available evidence indicates that GBP binding sites may accommodate glycan determinants made up of 2 to 6 linear monosaccharides, together with their potential side chains containing other sugars and modifications, such as sulfation, phosphorylation, and acetylation. Glycosaminoglycans, including heparin and heparan sulfate, comprise repeating disaccharide motifs, where a linear sequence of 5 to 6 monosaccharides may be required for recognition. Based on our current knowledge of the composition of the glycome and the size of GBP binding sites, glycoproteins and glycolipids may contain approximately 3000 glycan determinants with an additional approximately 4000 theoretical pentasaccharide sequences in glycosaminoglycans. These numbers provide an achievable target for new chemical and/or enzymatic syntheses, and raise new challenges for defining the total glycome and the determinants recognized by GBPs.

Antiangiogenic Forms of Antithrombin Specifically Bind to the Anticoagulant Heparin Sequence

A specific pentasaccharide sequence of heparin binds with high affinity to native antithrombin and induces a conformational change in the inhibitor by a previously described two-step interaction mechanism. In this work, the interactions of heparin with the antiangiogenic latent and cleaved antithrombin forms were studied. Binding of heparin to these antithrombin forms was specific for the same pentasaccharide sequence as native antithrombin. Rapid kinetic studies demonstrated that this pentasaccharide induced a conformational change also in latent and cleaved antithrombin. The binding affinities of these antithrombin forms for the pentasaccharide, as compared to native antithrombin, were approximately 30-fold lower due to two to three fewer ionic interactions, resulting in less stable conformationally altered states. Affinities of latent and cleaved antithrombin for longer heparin chains, containing the pentasaccharide sequence, were 2-fold lower than for the pentasaccharide itself. This contrasts the interaction with native antithrombin and demonstrates that residues flanking the pentasaccharide sequence of heparin are repelled by the latent and cleaved forms. These findings contribute to delineating the mechanism by which heparin or heparan sulfate mediates antiangiogenic activity of antithrombin.

Molecular weight-Dependent Mechanism of Heparin Neutralization by Platelet factor 4.

Heparins which contain both a specific pentasaccharide sequence and a protease binding site accelerate antithrombin inactivation of both thrombin and factor Xa (fXa). The bridging effect due to binding of AT and protease to heparin-which is dominating for thrombin- also participates in fXa inhibition. To address the hypothesis that the calcium-dependent bridging effect by heparin is an essential contributor of protease inhibition, we compared the ability of the heparin-neutralizing agent Platelet Factor 4 (PF4) to inhibit various therapeutic low-molecular-weight heparins (LMWH) in a kinetic assay. LMWH were in order of increasing chain-length; Bemiparin, Enoxaparin, Dalteparin and Tinzaparin. As a reference, the activity of the pentasaccharide Fondaparinux was included. Upon neutralization by PF4, the second-order rate constant describing AT inactivation of fXa by LMWH was 4-fold higher for the pentasaccharide (k2 = 3.8 x 105 M−1 s−1) vs the longer chain LMWH Tinzaparin (k2 = 1.0 x 105 M−1 s−1) and about 2-fold higher for the short-chain Bemiparin ( k2 = 2.4 x 105 M−1 s−1) vs Tinzaparin, in agreement with reports showing higher anti-fXa peak for administered short-chain LMWH. These results could be explained by neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH which increased with molecular weight, indicating a relationship between the affinity of PF4 for heparin and its neutralizing capacity. The molecular weight-enhanced neutralization was also reminiscent of earlier observations that PF4 binding to heparin followed the same chain length requirement as binding of protease. In contrast to PF4, protamine sulphate fully neutralized LMWH in a non-specific manner and EDTA, abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed to LMWH activity. In fact, as LMWH (Dalteparin) concentration was increased (0.05-0.8 U/ml), neutralization by PF4 decreased from 100 % to 35 %, a consequence of the template cofactor mechanism by heparin. Within a low range of LMWH concentration (<0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to LMWH were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin-bound AT on neutralization. Indeed, neutralization by PF4 of the fXa-AT reaction increased from 5 % to 55 % using fXa concentrations varying from 1 to 10 nM while neutralization of the thrombin-AT reaction required thrombin concentrations varying from 0.2 nM to 1.5 nM to obtain a similar level of neutralization than in the fXa-AT reaction. This was corroborated by saturation plots by PF4 which showed an increased apparent affinity of PF4 for LMWH as the enzyme concentration was increased in both fXa-AT and thrombin- AT reactions, although lower concentrations of thrombin were required to enhance PF4 affinity. Altogether the results suggested that heparin mediated an interaction of PF4 with protease which was of higher affinity in the presence of thrombin. Therefore, PF4 contributes to LMWH pharmacokinetics by specifically targeting the bridging function of heparin (via enzyme-PF4 interference of enzyme-AT interaction) and participates in the antifXa dependence of LMWH that remains after a major effect on the antithrombin activity.

Antithrombin-binding Octasaccharides and Role of Extensions of the Active Pentasaccharide Sequence in the Specificity and Strength of Interaction: EVIDENCE FOR VERY HIGH AFFINITY INDUCED BY AN UNUSUAL GLUCURONIC ACID RESIDUE

The antithrombotic activity of low molecular weight heparins (LMWHs) is largely associated with the antithrombin (AT)-binding pentasaccharide sequence AGA(*)IA (GlcN(NAc/NS,6S)-GlcA-GlcN(NS,3,6S)-IdoUA(2S)-GlcN(NS,6S)). The location of the AGA(*)IA sequences along the LMWH chains is also expected to influence binding to AT. This study was aimed at investigating the role of the structure and molecular conformation of different disaccharide extensions on both sides of the AGA(*)IA sequence in modulating the affinity for AT. Four high purity octasaccharides isolated by size exclusion chromatography, high pressure liquid chromatography, and AT-affinity chromatography from the LMWH enoxaparin were selected for the study. All the four octasaccharides terminate at their nonreducing end with 4,5-unsaturated uronic acid residues (DeltaU). In two octasaccharides, AGA(*)IA was elongated at the reducing end by units IdoUA(2S)-GlcN(NS,6S) (OCTA-1) or IdoUA-GlcN(NAc,6S) (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA(*)IA was elongated at the nonreducing side by units GlcN(NS,6S)-IdoUA and GlcN(NS,6S)-GlcA, respectively. Extensions increased the affinity for AT of octasaccharides with respect to pentasaccharide AGA(*)IA, as also confirmed by fluorescence titration. Two-dimensional NMR and docking studies clearly indicated that, although elongation of the AGA(*)IA sequence does not substantially modify the bound conformation of the AGA(*)IA segment, extensions promote additional contacts with the protein. It should be noted that, as not previously reported, the unusual GlcA residue that precedes the AGA(*)IA sequence in OCTA-4 induced an unexpected 1 order of magnitude increase in the affinity to AT with respect to its IdoUA-containing homolog OCTA-3. Such a residue was found to orientate its two hydroxyl groups at close distance to residues of the protein. Besides the well established ionic interactions, nonionic interactions may thus contribute to strengthen oligosaccharide-AT complexes.

Parenteral Anticoagulants: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)

This chapter describes the pharmacology of approved parenteral anticoagulants, including the indirect anticoagulants, unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and danaparoid as well as the direct thrombin inhibitors hirudin, bivalirudin, and argatroban. UFH is a heterogeneous mixture of glycosaminoglycans that bind to antithrombin via a unique pentasaccharide sequence and catalyze the inactivation of thrombin factor Xa and other clotting factors. Heparin also binds to cells and other plasma proteins, endowing it with unpredictable pharmacokinetic and pharmacodynamic properties, and can lead to nonhemorrhagic side effects, such as heparin-induced thrombocytopenia (HIT) and osteoporosis. LMWHs have greater inhibitory activity against factor Xa than thrombin and exhibit less binding to cells and proteins than heparin. Consequently, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties, have a longer half-life than heparin, and have a lower risk of nonhemorrhagic side effects. LMWHs can be administered once or twice daily by subcutaneous injection, without anticoagulant monitoring. Based on their greater convenience, LMWHs have replaced UFH for many clinical indications. Fondaparinux, a synthetic pentasaccharide, catalyzes the inhibition of factor Xa, but not thrombin, in an antithrombin-dependent fashion. Fondaparinux binds only to antithrombin; therefore, HIT and osteoporosis are unlikely to occur. Fondaparinux has excellent bioavailability when administered subcutaneously, has a longer half-life than LMWHs, and is given once daily by subcutaneous injection in fixed doses, without anticoagulant monitoring. Three parenteral direct thrombin inhibitors and danaparoid are approved as alternatives to heparin in HIT patients.

Sequential One-Pot Glycosidations Catalytically Promoted: Unprecedented Strategy in Oligosaccharide Synthesis for the Straightforward Assemblage of the Antitumor PI-88 Pentasaccharide

The pentasaccharide sequence of the most active components of the antitumor drug PI-88, currently in phase II clinical trial, has been rapidly assembled in high overall yield and in only three steps starting from three monosaccharide building blocks. The procedure takes advantage of the first reported strategy of sequential one-pot glycosidations conducted exclusively under catalytic activation. In addition, the procedure relies only on shelf-stable and mild promoters such as Yb(OTf)(3) and Bi(OTf)(3).

Heparin is procoagulant in the absence of antithrombin

21Department of Internal Medicine, College of Medicine, University of Illinois at Urbana-Champaign,Urbana, Illinois, USA2Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana,Illinois, USADear Sir,Antithrombin is a serine protease inhibitor that inactivates thrombin, factor Xa (FXa) and othercoagulation proteases. The inhibitory activity of AT is enhanced many fold by heparin orendothelial cell surface heparan sulfate proteoglycans (HSPGs) ( 1) and a variety of non-humanorigin proteoglycans exhibit anticoagulant activity (2,3). Approximately 30% of heparinmolecules in pharmaceutical preparations of unfractionated heparin (UFH) contain a specificsulfated pentasaccharide sequence that confers high affinity binding to AT (1), but less than5% of endothelial HSPG molecules bind AT (5,6). Heparins and HSPGs are highly anionicand they bind to many other proteins, including growth factors, extracellular matrix proteins,selectins, lipoproteins, cytokines and chemokines. As a result, HPSGs have a wide variety ofnonanticoagulant effects, including influences on platelet function, cell adhesion,inflammation, apotosis, angiogenesis, and development (1,4).A recent report by Liu et al. (8) described the procoagulant activity and ability to abrogatetissue factor pathway inhibitor (TFPI) function by AV513, a non-anticoagulant sulfatedproteoglycan (NASP) of plant origin. Studies in our laboratory suggest that AV513 acceleratesthrombin generation through promoting the activation of factor V (FV) (9). We also recentlyreported that polyphosphate (polyP), a linear polymer of inorganic phosphate ( 10), is a potentprocoagulant (11). PolyP accelerates blood coagulation by promoting FV activation. PolyPalso profoundly abrogates the function of tissue factor pathway inhibitor (TFPI). This appearsto be secondary to accelerated FVa generation, since FXa is protected from inhibition by TFPIwhen incorporated into the prothrombinase complex ( 12). The combined effects of polyP resultin an earlier peak in thrombin generation without changing the total amount of thrombingenerated (11). We now report that, in the absence of AT, heparins demonstrate procoagulantactivity similar of that of polyP.Tissue factor-induced clotting in plasma immunodepleted of AT was slightly accelerated whenUFH was added, but heparin markedly accelerated clot formation triggered by FXa. (Figure1A). Maximal procoagulant activity occurred at a concentration of about 1 U/mL UFH. On theother hand, clotting time triggered by thrombin was prolonged by UFH in AT-deficient plasma(Figure 1A), presumably due to heparin-enhanced thrombin inhibition by heparin cofactor II(HCII). In plasma deficient in both AT and HCII, the addition of UFH resulted in very mildprolongation of clotting times initiated by thrombin (Figure1B), but when clotting was initiatedby FXa, addition of UFH shortened the time to clot formation (Figure 1B). These findings

Chemistry and Biology of Heparin Mimetics that Bind to Fibroblast Growth Factors

We aim from this review to stimulate further research in this area by providing a description of the different types of inhibitors containing heparin mimetic molecules that have recently been reported and data on their biological activity. Molecules that mimic heparin and bind to heparin-binding growth factors are important building blocks for synthetic biomaterials. Different types of synthetic mimics of the biological properties of heparin have been prepared inclu-ding high molecular weight compounds or small molecule mimics. Peptide-based mimics of heparin functionality are limited and because of their low degree of sulfation, they are natural targets as heparin mimics. Aromatic sulfonamide derivatives exhibit a range of bioactivities and a novel angiogenesis inhibitor (E 7820) is used as a TF model for screening assay. The anticoagulant activity of the known heparin pentasaccharide sequence prompted synthetic efforts aimed at the procurement of this structure as well as a host of related sequences. Chemical modification of the natural or synthetic heparin increased factor activation of AT III Xa affinity. A variety of non-peptide non-saccharides inhibitors as anti-angiogenesis therapies directed against the VEGFR kinase are a promising and well-validated therapeutic approach under active evaluation of their safety and efficacy in multiple clinical trials. These low molecular weight modulators could be useful tools for biologists and may have potential as drugs or as leads for drug development.

Fondiparinux Worsens Leukocyte Endothelial Interactions Improved by Antithrombin and Increases Leukocyte Extravasation.

Background: A phase III trial of antithrombin (AT) in sepsis showed benefit only in patients who did not receive concurrent heparin. We have shown that some of the beneficial effects of AT on leukocyte-endothelial (LE) interactions in sepsis models requires the activity of heparan sulfate 3-O-sulfotransferase-1 (3-OST-1), the enzyme that is responsible for the rate-limiting step in the biosynthesis of anticoagulant heparan sulfate. Fondiparinux (FOND) is a pharmacological agent that mimics the anticoagulant heparan sulfate pentasaccharide sequence synthesized by 3-OST-1.Objectives: We hypothesized that FOND treatment would alter LE interactions in sepsis models in mice, and AT effects in these models.Methods: Using intravital microscopy, we evaluated the effects of proinflammatory stimuli on leukocyte rolling and firm adhesion in post-capillary venules, and extravasation, into the cremaster muscle in live C57BL/6 mice. Human AT (0.25 U/g Thrombate III), and/or heparanoid or vehicle was infused intravenously prior to proinflammatory stimuli. Venules were chosen such that in all comparisons, the average diameter (range 20 to 50 μm) and shear rate (range 200 to 800 s−1) was not different between treatment groups. The number of leukocytes rolling past a defined vessel point was expressed as leukocyte rolling flux, which normalizes for leukocyte count and flow rate. The number of firmly adherent leukocytes (stationary for 30 s) was normalized for the area of vessel wall analyzed. The number of extravasated leukocytes was normalized for the area of tissue analyzed.Results: AT treatment decreased leukocyte rolling flux (23 vs. 15%, P=0.01) induced by an injection of lipopolysaccharide (LPS at 1 μg i.p.) 2 h before observation. However, in this model, AT did not inhibit the number of firmly adherent leukocytes. When mice were pretreated with FOND (0.1 μg/g), the leukocyte rolling flux was increased compared to without heparanoid (22 vs. 25%, P<0.05) and the number of firmly adherent leukocytes was decreased (294 vs. 127 mm−2, P<0.05), implying an inhibitory effect of FOND. However when mice were treated with AT and FOND, LE interactions were worsened: although the rolling flux decreased from 25 to 17% with AT treatment, the number of firmly adherent leukocytes increased from 127 to 305 mm−2. Mice pretreated with danaparoid (0.025 U/g), where the pentasaccharide is rare, were not different from untreated mice, both with and without AT-pretreatment. Thus although AT improves LE interaction induced by LPS, it worsens these in the presence of FOND, as it did in 3-OST-1 deficient mice. Because we had previously shown that 3-OST-1 deficient mice had poorer survival to an LPS challenge, we wondered whether the effect of FOND was to increase leukocyte trafficking toward extravasation. Five hours after injection of IL1β (30 ng i.p.), the tissue surrounding flowing vessels in the cremaster muscle was observed for one hour for interstitial leukocytes. FOND increased the number of extravasated leukocytes in untreated mice (from 0.34 to 0.47 mm−2, P<0.05) and IL1β-pretreated mice (from 0.32 to 0.64 mm−2, P<0.005). The effect of AT on extravasation is currently being evaluated.Conclusions: These studies further our knowledge of how the anticoagulant pentasaccharide sequence, mimicked by fondiparinux and lacking in 3-OST-1 deficient mice, is important in leukocyte recruitment that occurs in response to inflammation. This will allow for more rational use of mediators of this pathway such as AT in sepsis.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.

An unexpected role for anticoagulant heparan sulfate proteoglycans in reproduction.

Major tissue remodelling occurs in hormone responsive tissues of the female genital tract, at ovulation and during gestation, involving proteolysis and inflammation. Disorders of tissue remodelling events are associated with infertility in women with luteinized unruptured follicle syndrome and with gestational pathologies as preeclampsia. Ovulation impairment is an important factor of infertility and a major concern in reproductive medicine. The gonadotrophin discharge inducing ovulation triggers proteolytic activities involved in the breakdown of the follicular wall and elicits an acute inflammatory reaction in the ovary. Tight control of these reactions is required to allow successful ovulation while avoiding excessive tissue damage. Anticoagulant heparan sulfate proteoglycans (aHSPG), like heparin, possess a pentasaccharide sequence which binds and activates antithrombin III. These proteoglycans are produced by endothelial cells and are thought to endow the vascular wall with antithrombotic properties. aHSPG are also present in the reproductive tract; in the ovary they are strongly expressed in granulosa cells of preovulatory follicles and they are co-localised with serine protease inhibitors involved in the control of proteolytic activities at ovulation. The presence of aHSPG in the oviduct, in the uterus and in human follicular fluid, suggests that they could play additional distal roles in gestation. The females exhibited impaired ovarian function as well as intrauterine growth restriction linked to delayed placenta development. In these mice, the placenta is challenged by inflammation at mid-gestation, occasionally resulting in miscarriage and maternal death. Collectively, these observations suggest that aHSPG are involved in the control of inflammatory events occurring during tissue remodelling in hormone-responsive tissues. Further studies are needed to identify the inflammation mediators involved in this process.

An unexpected role for anticoagulant heparan sulfate proteoglycans in reproduction.

Major tissue remodelling occurs in hormone responsive tissues of the female genital tract, at ovulation and during gestation, involving proteolysis and inflammation. Disorders of tissue remodelling events are associated with infertility in women with luteinized unruptured follicle syndrome and with gestational pathologies as preeclampsia. Ovulation impairment is an important factor of infertility and a major concern in reproductive medicine. The gonadotrophin discharge inducing ovulation triggers proteolytic activities involved in the breakdown of the follicular wall and elicits an acute inflammatory reaction in the ovary. Tight control of these reactions is required to allow successful ovulation while avoiding excessive tissue damage. Anticoagulant heparan sulfate proteoglycans (aHSPG), like heparin, possess a pentasaccharide sequence which binds and activates antithrombin III. These proteoglycans are produced by endothelial cells and are thought to endow the vascular wall with antithrombotic properties. aHSPG are also present in the reproductive tract; in the ovary they are strongly expressed in granulosa cells of preovulatory follicles and they are co-localised with serine protease inhibitors involved in the control of proteolytic activities at ovulation. The presence of aHSPG in the oviduct, in the uterus and in human follicular fluid, suggests that they could play additional distal roles in gestation. The females exhibited impaired ovarian function as well as intrauterine growth restriction linked to delayed placenta development. In these mice, the placenta is challenged by inflammation at mid-gestation, occasionally resulting in miscarriage and maternal death. Collectively, these observations suggest that aHSPG are involved in the control of inflammatory events occurring during tissue remodelling in hormone-responsive tissues. Further studies are needed to identify the inflammation mediators involved in this process.

3-O-Sulfated Oligosaccharide Structures Are Recognized by Anti-heparan Sulfate Antibody HS4C3

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O- and 6-O-sulfated hexa- to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mm. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mm salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the anti-thrombin binding pentasaccharide sequence.

Cover Picture: The Design and Evaluation of Heparin‐Binding Foldamers (Angew. Chem. Int. Ed. 41/2005)

Small inhibitors for low‐molecular‐weight heparin, namely short aryl amide foldamers, were designed with the aid of molecular‐dynamics calculations. The cover picture shows that the binding is primarily dominated by electrostatic interactions between a pentasaccharide sequence of heparin and the antagonist. In their Communication on page 6685 ff., W. F. DeGrado and co‐workers show that heparin binds and modulates the biological properties of a number of proteins, including antithrombin II and factor Xa.

New anticoagulants.

The limitations of heparin and warfarin have prompted the development of new anticoagulant drugs for prevention and treatment of venous and arterial thromboembolism. Novel parenteral agents include synthetic analogs of the pentasaccharide sequence of heparin that mediates its interaction with antithrombin. Fondaparinux, the first synthetic pentasaccharide, is licensed for prevention of venous thromboembolism (VTE) after major orthopedic surgery and for initial treatment of patients with VTE. Idraparinux, a long-acting pentasaccharide that is administered subcutaneously once-weekly, is being compared with warfarin for treatment of VTE and for prevention of cardioembolic events in patients with atrial fibrillation. New oral anticoagulants include direct inhibitors of thrombin, factor Xa and factor IXa. Designed to provide more streamlined anticoagulation than warfarin, these agents can be given without routine coagulation monitoring. Ximelagatran, the first oral direct thrombin inhibitor, is as effective and safe as warfarin for prevention of cardioembolic events in patients with atrial fibrillation. However, ximelagatran produces a three-fold elevation in alanine transaminase levels in 7.9% of patients treated for more than a month, the long-term significance of which is uncertain. Whether other direct thrombin inhibitors or inhibitors of factors Xa or IXa also have this problem is under investigation. After a brief review of coagulation pathways, this paper focuses on new anticoagulants in advanced stages of clinical testing.

In vivo rabbit acute model tests of polyurethane catheters coated with a novel antithrombin-heparin covalent complex

Catheter use has been associated with an increased risk of thrombotic complications. The objective was to make catheters less thrombogenic with the use of antithrombin-heparin covalent complex (ATH). The antithrombotic activity of ATH-coated catheters was compared to uncoated (control) and heparincoated catheters in an acute rabbit model of accelerated occluding clot formation. Anaesthetized rabbits were pre-injected with rabbit (125)I-fibrinogen, followed by insertion of test catheters into the jugular vein. Blood was drawn and held in a syringe, reinjected, then flushed with saline. The experiment was terminated when blood could no longer be withdrawn (occluding clot). Efficacy was defined as the ability of catheters to remain unoccluded. Clot formation, determined by measuring deposition of radiolabeled fibrin, was a secondary endpoint. ATH-coated catheters were resistant to clotting for the full 240-minute duration, while uncoated and heparin-coated catheters had an average clotting time of 78 and 56 minutes, respectively. The patency of ATH coating was dependant on intact heparin pentasaccharide sequences, rather than the chemistries of the basecoat, the PEO spacer arm, or the antithrombin (AT) protein. The ATH coating was stable to ethylene oxide sterilization, modest abrasion, protease attack, and the coating did not appear to leach off the catheter. Surface tension measurements showed that the ATH modified surface was more hydrophilic than uncoated control catheters or heparin-coated catheters. Thus, ATH-coated catheters are better at preventing clots than uncoated or heparin-coated catheters and show promise as an alternative to the currently available catheters in reducing thrombotic complications associated with its use.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.