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Heparan sulfate (HS) is involved in many biological activities, including the biogenesis and uptake of exosomes, which are related to the occurrence and development of tumors. This study investigated the role of HS analogues (heparin, low molecular weight heparin, and 6-O-desulfated heparin) in modulating exosome secretion, composition and functions. Exosomes derived from B16F10 cells exposed to different HS analogues were isolated and characterized by TEM, western blotting and Nanosight analyses. The number, size and protein cargo of exosomes secreted by HS analogues-induced B16F10 cells were detected. The findings indicated the reduced tumor-derived exosome secretion and protein cargo as reflected by lower levels of CD63, TSG101, heparinase and IL-6 in exosomes derived from heparin-induced B16F10 cells as compared with 6-O-desulfated heparin-induced tumor cells. Further functional assays demonstrated that exosomes from tumor cells exposed to heparin weakened tumor proliferation, migration and invasion most significantly among various exosomes derived from B16F10 cells treated with different HS analogues. Moreover, the sulfate group at 6-O position of heparan sulfate has been proved to play an important role in tumor-derived exosome formation and functions. This study suggested a vital view to develop more specific and efficient HS-based strategies in cancer treatment for targeting tumor-derived exosomes., (Copyright © 2021. Published by Elsevier B.V.)

Nanotechnology-based fabricated wound dressings are known as appropriate substrates to enhance healing in both acute and chronic wounds. These types of materials have the ability to deliver therapeutic agents. In this study, a wound dressing including heparinized zinc oxide nanoparticles in combination with chitosan and poly(vinyl alcohol) was developed to investigate its antibacterial and regenerative properties in a rat model of full thickness skin wounds. By adding nanoparticles, the mechanical strength increased up to twice as compared to the sample without nanoparticles. In addition, heparin release profile follows the Hixson-Crowell release kinetic. Protein adsorption enhanced by adding nanoparticles in hydrogels and the prepared wound dressings were completely biocompatible. In terms of antibacterial activity, the minimum inhibitory concentration decreased by conjugation of heparin on the surface of zinc oxide nanoparticles compared to the non-functionalized nanoparticles, and, this shows the increased antibacterial synergistic effect by adding heparin to nanoparticles. Furthermore, it was found that the heparinized zinc oxide nanoparticles effectively accelerate wound closure, re-epithelialization and decrease collagen deposition compared to other groups after implantation. Hence, the prepared wound dressings have the capacity to significantly enhance healing of acute wounds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The β-galactoside-binding protein, galectin-3, is extensively involved in cancer development, progression and metastasis through multiple mechanisms. Inhibition of the galectin-3-mediated actions is increasingly considered as a promising therapeutic approach for cancer treatment. Our early studies have identified several novel galectin-3 binding inhibitors from chemical modification of the anticoagulant drug heparin. These heparin-derived galectin-3 binding inhibitors, which show no anticoagulant activity and bind to the galectin-3 canonical carbohydrate-binding site, induce galectin-3 conformational changes and inhibit galectin-3-mediated cancer cell adhesion, invasion and angiogenesis in vitro and reduce metastasis in mice. In this study, we determined the binding affinities of these heparin-derived ligands to galectin-3 using an isothermal titration calorimetry (ITC) ligand displacement approach. Such ITC experiments showed that the 2-de-O-sulphated, N-acetylated (compound E) and 6-de-O-sulphated, N-acetylated (F) heparin-derived ligands and their ultra-low molecular weight sub-fractions (E3 and F3) bind to galectin-3 with K D ranging from 0.96 to 1.32 mM.Differential scanning fluorimetry analysis revealed that, in contrast to the disaccharide ligand, N-acetyl-lactosamine, which binds to the fully folded form of galectin-3 and promotes galectin-3 thermal stability, the heparin-derived ligands preferentially bind to the unfolded state of galectin-3 and cause destabilization of the galectin-3 protein structure. These results provide molecular insights into the interaction of galectin-3 with the heparin-derived ligands and explain the previously demonstrated in vitro and in vivo effects of these binding inhibitors on galectin-3-mediated cancer cell behaviours., Competing Interests: Declaration of competing interest EAY, JET and LGY are named as inventors on a patent covering the use of the heparin derivative inhibitors as anti-metastatic agents. JET and EAY hold shares in IntelliHep Ltd which currently holds an option to licence this patent. The other authors declare no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Heparin and heparin-like molecules have shown some promise in the treatment of several cancers. These molecules have roles in angiogenesis, cell proliferation, immune system modulation, cell migration, and cellular invasion. The pathways and mechanisms used by these molecules to inhibit the proliferation of cancer cells aid in understanding the utilization of these molecules in potential treatments. Our aim is to review the use of heparin and heparin-like molecules in cancer treatment, explore the results, and discuss their potential downfalls., Methods: Publications on heparin and heparin-like molecules and compounds were collected from the PubMed and EMBASE databases. Boolean operators and MeSH terms related to heparin, heparin-like molecules, and cancer were used to conduct this search. The articles were reviewed by the authors., Results: Several heparin mimetics are showing promise in cancer treatment. Various studies using mimetics alone or in combination with chemotherapy have been conducted and have yielded mixed results. They work on multiple target molecules, mostly receptors such as fibroblast growth factor and endothelial growth factor. The main types of cancers targeted by these drugs are multiple myeloma, pancreatic cancer, hepatocellular carcinoma (HCC), and other solid tumors., Conclusion: Although limited clinical evidence of efficacy and potential pitfalls are present, heparin and heparin-like molecules have shown potential in the management of cancer patients. Additional research is required to fully understand the biological mechanisms utilized by these molecules in cancer treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Numerous studies have proved that heparin, a sub-group of glycosaminoglycan, possesses great potential as anti-metastasis agent. However, the native strong anti-coagulant activity which causes serious side effects, such as bleeding, has limited its clinical applications for safety concern. To overcome this problem, we synthesized a panel of novel glycopolymers that mimic heparin structure with substantially reduced anti-coagulant activity by a simple grafted-on strategy. The influence of molecular weight & distribution, substituting degree and sulfonic density on cytotoxicity were determined by systematic MTT analysis to select the candidates with highly bio-compatibility. Among these glycopolymers, a sulfated poly‑2‑aminoethyl methacrylate grafted with heparin disaccharide (abbreviated as SGPHD) has shown potent efficacy in inhibition of heparanase activity and microvascular endothelial cell proliferation in vitro. Further experiments demonstrated that SGPHD inhibited B16 murine melanoma cell migration, invasion and adhesion to platelets or microvascular endothelial cells, thus, presented as a possible anti-metastatic agent by providing a whole course protection during tumor metastasis. The results will have significant impacts for the further rational design of glycopolymer medicine., (Copyright © 2018. Published by Elsevier B.V.)

It is now well recognized that heparanase, an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, promotes tumorigenesis by diverse mechanisms. Compelling evidence strongly implies that heparanase is a viable target for cancer therapy, thus encouraging the development of heparanase inhibitors as anti-cancer therapeutics. Here, we examined the efficacy and mode of action of PG545, an HS-mimetic heparanase inhibitor, in human lymphoma. We found that PG545 exhibits a strong anti-lymphoma effect, eliciting lymphoma cell apoptosis. Notably, this anti-lymphoma effect involves ER stress response that was accompanied by increased autophagy. The persistent ER stress evoked by PG545 is held responsible for cell apoptosis because apoptotic cell death was attenuated by an inhibitor of PERK, a molecular effector of ER stress. Importantly, PG545 had no such apoptotic effect on naïve splenocytes, further encouraging the development of this compound as anti-lymphoma drug. Surprisingly, we found that PG545 also elicits apoptosis in lymphoma cells that are devoid of heparanase activity (i.e., Raji), indicating that the drug also exerts heparanase-independent function(s) that together underlie the high potency of PG545 in preclinical cancer models., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: In systemic lupus erythematosus, hemostasis disorders are mainly thrombotic, but more rarely hemorrhagic., Case Report: A 25-year-old man presented with a macrophagic activation syndrome revealing a systemic lupus erythematosus, secondarily complicated by a hemorrhagic syndrome ; biological investigations revealed an increase thrombin time and an activated partial thromboplastin time, normalized by protamin neutralization in vitro, thus confirming the presence of a heparin-like anticoagulant. The hemostasis balance normalized after the specific treatment of lupus., Conclusion: This rare anomaly of hemostasis balance has been described in blood cancers and solid cancers. This is the first description of a case associated with an autoimmune connective tissue disorder such as lupus. After one year of follow-up, no diagnosis of blood or solid cancer was made., (Copyright © 2018 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier Masson SAS. All rights reserved.)

Background and Aims: Leukocytosis, particularly monocytosis, has been shown to promote atherosclerosis in both diabetic and non-diabetic mouse models. We previously showed that hyperglycemia independently promotes monocytosis and impairs the resolution of atherosclerosis. Since patients with chronic diabetes often develop dyslipidemia and also have increased risk for atherosclerosis, we sought to examine how controlling blood glucose affects atherosclerosis development in the presence of severe hyperlipidemia., Methods: Diabetes was induced using streptozotocin (STZ) in low density lipoprotein receptor (Ldlr) knockout (Ldlr -/- ) mice after which they were fed a high-cholesterol diet for 4 weeks. Control and diabetic mice were treated with vehicle or sodium glucose cotransporter inhibitor (SGLT2i, Phlorizin or Dapagliflozin) for the duration of the diet., Results: Induction of diabetes resulted in a dramatic increase in plasma cholesterol (TC) and triglyceride (TG) levels. These mice also exhibited an increased number of circulating monocytes and neutrophils. Monocytosis was driven by increased proliferation of progenitor cells in the bone marrow. Tighter glycemic control by SGLT2i treatment not only reduced monocytosis and atherosclerosis but also improved plasma lipoprotein profile. Interestingly, improved lipoprotein profile was not due to decreased TG synthesis or clearance via low density lipoprotein receptor-related protein (Lrp) 1 or scavenger receptor class B member (Scarb1) pathways, but likely mediated by heparin sulfate proteoglycans (HSPG)-dependent clearance mechanisms in the liver. Further examination of the liver revealed an important role for bile acid transporters (Abcg5, Abcg8) and cytochrome P450 enzymes in the clearance of hepatic cholesterol., Conclusions: These data suggest that tighter glycemic control in diabetes can improve lipoprotein clearance exclusive of Ldlr, likely via HSPG and bile acid pathways, and has an overall net positive effect on atherosclerosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Water soluble matrix metalloproteinases (MMPs) have been regarded as diffusing freely in the extracellular matrix. Yet multiple MMPs are also observed at cell surfaces. Their membrane-proximal activities include sheddase activities, collagenolysis, bacterial killing, and intracellular trafficking reaching as far as the nucleus. The catalytic domains of MMP-7 and MMP-12 bind bilayers peripherally, each in two different orientations, by presenting positive charges and a few hydrophobic groups to the surface. Related peripheral membrane associations are predicted for other soluble MMPs. The peripheral membrane associations may support pericellular proteolysis and endocytosis. The isolated soluble domains of MT1-MMP can also associate with membranes. NMR assays suggest transient association of the hemopexin-like domains of MT1-MMP and MMP-12 with lipid bilayers. Peripheral association of soluble MMP domains with bilayers or heparin sulfate proteoglycans probably concentrates them near the membrane. This could increase the probability of forming complexes with membrane-associated proteins, such as those targeted for proteolysis. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Heparin sulfate proteoglycans (HSPGs) are responsible for the storage and stabilization of numerous growth factors in the extracellular matrix. In this complex native environment, the efficient binding of the growth factors is determined by multivalent, specific and reversible electrostatic interactions between the sulfate groups of HSPGs and the positively charged amino acids of the growth factor. Inspired by this naturally occurring stabilization process, we propose the use of diblock copolymers of heparin and polyethylene glycol (Hep-b-PEG) for protection and delivery of FGF-2. We describe the encapsulation of FGF-2 into spontaneously assembling polyelectrolyte complexes (PECs) with Hep-b-PEG in which the Hep block ensures the formation of the PECs, while the PEG moiety confers stability of the generated complex by a stealth corona. Our results demonstrate that by this method we can generate homogeneous complexes (ca. 400nm diameter, PDI 0.29±0.07) with a very high encapsulation efficiency (about 99% encapsulated FGF-2). The release of the growth factor in response to different stimuli such as pH, ionic strength or presence of heparinase was also studied. We report a sustained release of up to 80% during 28days which is not influenced by the presence of heparinase - a result that clearly demonstrates the protective effect of the stealth corona. We also show that FGF-2 remains bioactive as it influences the morphology of bone marrow mesenchymal stem cells., Statement of Significance: We describe a biopolymer that uses the way the cells shield a type of proteins (growth factors) to simultaneously assemble, slowly deliver and shield the protein in a "nanocarrier". Growth factors are essential for the regeneration of cartilage, bones by stem cell therapies but have a short life time as when added directly to tissues. Our design makes use of the heparin bioactivity towards such proteins in combination with a polyethylene glycol moiety (PEG) that makes a protecting shell. PEG, is biocompatible and used in approved medicines and countless cosmetic products. The highest novelty is the reaction (oxime click) used to bound these molecules that does not require modification of heparin and allows preservation of its bioactivity., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles., Methods: ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted., Results: Physiological levels of secreted ApoE (10-60µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10-60µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs)., Conclusions: Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies. Lay summary: This study shows that HCV particle infectivity is remodeled by secreted ApoE after particle release from cells. Fluctuation of the availability of ApoE likely influences HCV infectivity, antibody escape and transmission., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log 10  PFU viral reduction with IC 50 value of ∼2.5-5 μg/ml (1.93 μM-3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Crystalline acetylated ethyl 2-azido-2-deoxy-1-thio-α-d-cellobioside has been prepared on a multigram scale from cellobiose in an overall yield of 23% with no chromatography required and converted after deacetylation into the 4',6'-O-benzylidene and 4',6'-O-benzylidene-6-O-TBDMS protected derivatives. Applying a number of regioselective benzylation methods on these gave access to a variety of regioselectively protected derivatives, both mono-ols (2'- and 3-OH), diols (2',6-, 2',3-, and 3,6-di-OH), and triols (2',3,6- and 2',3',3-tri-OH). A number of these derivatives were further processed by benzoylation followed by removal or opening of the benzylidene acetal and selective oxidation of the exposed primary alcohol to give heparin building block intermediates comprising a range of possible sulfation patterns., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI-MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Synthesis of an array of differentially sulfated GlcN-IdoA disaccharides, accessible on good scale, directly from l-iduronate components is described. These are specifically directed to provide the sulfation variability at the key most common biologically relevant sulfation-variable l-IdoA O-2 and d-GlcN O-6 and amino sites of this heparin disaccharide. This sulfation-varied matrix has allowed the first evaluation of using Raman/ROA spectroscopy to characterize changes in spectra as a function of both site and level of sulfation with pure, defined heparin-related disaccharide species. This provides analysis of both similarities and differences to digest native heparin and this shows evidence of different types of changes in conformations and conformational freedom as a function of some specific sulfation changes at the disaccharide level. It is anticipated that this data set will open the way for applications to further site-specific sulfated saccharides and demonstrates the capability offered by Raman-ROA towards fingerprinting sulfation in heparin fragments., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this issue of Blood, Poli et al demonstrate that heparin analogs engineered to minimize their anticoagulant properties can potently downregulate hepcidin production in vitro and in vivo, and may potentially be used to treat the anemia of inflammation.

Hepcidin controls systemic iron availability, and its excess contributes to the anemia of chronic diseases, the most prevalent anemia in hospitalized patients. We previously reported that heparins are efficient hepcidin inhibitors both in vitro and in vivo, but their anticoagulant activity limits therapeutic use. We studied nonanticoagulant heparins produced by N-acetylation and oxidation/reduction (glycol-split) that lost antithrombin-binding affinity. Four nonanticoagulant heparins inhibited hepcidin expression in hepatic HepG2 cells and primary hepatocytes. The 2 most potent ones used in mice suppressed liver hepcidin expression and serum hepcidin in 6 hours, with a significant decrease of spleen iron. This occurred also in lipopolysaccharide (LPS)-treated animals that mimic inflammation, as well as after chronic 1-week treatments, without evident adverse effects on coagulation. Heparin injections increased iron mobilization and facilitated the recovery from the anemia induced by heat-killed Brucella abortus, a model of inflammatory anemia. The heparins were used also in Bmp6(-/-) mice. A single dose of heparin reduced the already low level of hepcidin of these mice and prevented its induction by LPS. These nonanticoagulant compounds impair bone morphogenetic protein /sons of mothers against decapentaplegic signaling with no evident adverse effect in vivo, even when administered chronically. They may offer a strategy for the treatment of diseases with high hepcidin levels.

Durable and blood-compatible coating of metallic biomaterials remains a major issue in biomedical fields despite its long history of development. In this study, in situ forming, metal-adhesive heparin hydrogels were developed to coat metallic substrates to enhance blood compatibility. The hydrogels are composed of metal-adhesive and enzyme-reactive amphiphilic block copolymer (Tetronic-tyramine/dopamine; TTD) and enzyme-reactive heparin derivatives (heparin-tyramine or heparin-polyethylene glycol-tyramine), which are cross-linkable in situ via an enzyme reaction. The combinations of heparin and Tetronic formed hydrogels with relatively high mechanical strengths of 300-5000 Pa within several tens of seconds; this was also confirmed by observing a dried porous structure as coated on a metal surface. The introduction of dopamine to the hydrogel network enhanced the durability of the hydrogel layers coated on metal, such that more than 60% heparin remained for 7 days. Compared to bare metal surfaces, hydrogel-coated metal surfaces exhibited significantly enhanced blood compatibility. Reduced fibrinogen adsorption and platelet adhesion showed that blood compatibility was 3-5-fold-enhanced on coated hydrogel layers than on the bare metal surface. In conclusion, hydrogels containing heparin and dopamine prepared by enzyme reaction have the potential to be an alternative coating method for enhancing blood compatibility of metallic biomaterials., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis., (Copyright © 2012 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Traditional strategies for oligosaccharide synthesis often require extensive protecting and/or leaving group manipulations between each glycosylation step, thereby increasing the total number of synthetic steps while decreasing the efficiency of the synthesis. In contrast, expeditious strategies allow for the rapid chemical synthesis of complex carbohydrates by minimizing extraneous chemical manipulations. Oligosaccharide synthesis by selective activation of one leaving group over another is one such expeditious strategy. Herein, the significant improvements that have recently emerged in the area of the selective activation are discussed. The development of orthogonal strategy further expands the scope of the selective activation methodology. Surveyed in this article, are representative examples wherein these excellent innovations have been applied to the synthesis of various oligosaccharide sequences., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hepcidin is a major regulator of iron homeostasis, and its expression in liver is regulated by iron, inflammation, and erythropoietic activity with mechanisms that involve bone morphogenetic proteins (BMPs) binding their receptors and coreceptors. Here we show that exogenous heparin strongly inhibited hepcidin expression in hepatic HepG2 cells at pharmacologic concentrations, with a mechanism that probably involves bone morphogenetic protein 6 sequestering and the blocking of SMAD signaling. Treatment of mice with pharmacologic doses of heparin inhibited liver hepcidin mRNA expression and SMAD phosphorylation, reduced spleen iron concentration, and increased serum iron. Moreover, we observed a strong reduction of serum hepcidin in 5 patients treated with heparin to prevent deep vein thrombosis, which was accompanied by an increase of serum iron and a reduction of C-reactive protein levels. The data show an unrecognized role for heparin in regulating iron homeostasis and indicate novel approaches to the treatment of iron-restricted iron deficiency anemia.

Background: External reporting of medical errors a adverse events enables learning from the errors of others in the pursuit of systems-level improvements that can prevent future errors. It is logical to presume that medication errors involving the use of anticoagulants, among the most frequently cited product classes involved in harmful medication errors, would be captured in a variety of patient safety reporting programs., Methods: Data on reported errors involving the anticoagulant heparin were reviewed, compared, and aggregated from the databases of three large patient safety reporting programs-MEDMARX, the Pennsylvania Patient Safety Authority's Patient Safety Reporting System, and the University Health System Consortium, together representing more than 1,000 reporting organizations for 2005, Results: Approximately 300,000 medication errors and near misses were reported to the programs, and 10,359-a mean of 3.6% (range, 3.1%-5.5%)-involved heparin products. The proportion of heparin-related reports that involved patient harm ranged from 1.4% to 4.9%. The phase of the medication use process cited most frequently in harmful events was the administration phase (56% of errors leading to harm), followed by the prescribing phase (19% of errors leading to harm)., Discussion: This study represents the first attempt by these three large reporting systems to combine data on a single clinical process. The consistent patterns evident in the reports, such as the percentage of all medication errors that involved heparin, suggests that reporting programs, at least for common events such as medication errors, may reach a point of diminishing returns in which aggregating more reports of a certain type yields no additional insight once a large volume of similar events is captured and analyzed.

Heparin (H) and heparan sulfate (HS) play major roles in a number of biological processes. Yet, H/HS-based pharmaceutical agents are also associated with multiple adverse effects. This has led to the concept of designing noncarbohydrate, aromatic mimetics that modulate H/HS function. In this work, we study a library of synthetic, aromatic H/HS mimetics for their capillary electrophoretic profiles, the acid and base stability, and aqueous-organic partitioning property. The nonsugar H/HS mimetics exhibit electrophoretic properties similar to sulfated oligosaccharides suggesting that the mimetics can be rapidly and quantitatively analyzed. Stability studies show that the mimetics are essentially stable under neutral and basic conditions in a manner similar to the heparins, but are considerably unstable under acidic conditions in contrast to heparins. The measurement of partition coefficients show major differences within the sulfated mimetics as well as between the measured and calculated log P values. Understanding these physico-chemical properties is expected to have significant implications in the pharmaceutical development of this growing class of molecules., (2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Background and Objectives: AVE5026 is a novel, hemisynthetic, ultra-low-molecular-weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)-binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low-molecular-weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models., Method and Results: AVE5026 has a higher anti-factor Xa (anti-FXa) activity (approximately 160 U mg(-1)) along with a catalytic anti-thrombin (anti-FIIa) activity (approximately 2 U mg(-1)) as a result of its structure being strongly enriched in specific AT-binding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti-FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose-dependent antithrombotic activity comparable to that of enoxaparin (ED50-AVE5026 = 1.6 mg kg(-1), ED50-enoxaparin = 2.8 mg kg(-1)). Interestingly, non-occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg(-1) AVE5026, whereas 0.316 mg kg(-1) enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg(-1)), AVE5026 dose-dependently inhibited arterial thrombosis (ED50 = 2.0 mg kg(-1)). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs., Conclusion: These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in well-established arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit-risk profile as compared to that of enoxaparin.

Background: AVE5026 is a new hemisynthetic ultra-low-molecular-weight heparin, with a novel anti-thrombotic profile resulting from high anti-factor (F)Xa activity and residual anti-FIIa activity. AVE5026 is in clinical development for venous thromboembolism (VTE) prevention, a frequent complication after total knee replacement (TKR) surgery., Objectives: This study evaluated the dose-response of AVE5026 for the prevention of VTE in patients undergoing TKR surgery., Patients/methods: In this parallel-group, double-blind, double-dummy study, 690 patients were randomized, and 678 treated with once-daily doses of AVE5026 (5, 10, 20, 40, or 60 mg) or enoxaparin 40 mg in the calibrator arm. The primary efficacy end point was VTE until post-operative day 11, defined as deep vein thrombosis (DVT) detected by bilateral venography, symptomatic DVT, non-fatal pulmonary embolism (PE) and VTE-related death. The primary safety outcome was the incidence of major bleeding., Results: The primary efficacy outcome was assessed in 464 patients. There was a significant dose-response across the five AVE5026 groups for VTE prevention (P<0.0001), with the incidence of VTE ranging from 5.3% to 44.1% compared with 35.8% in the enoxaparin group and for proximal DVT (P=0.0002). Also, a significant dose-response for AVE5026 was seen for major bleeding (P=0.0231) and any bleeding (P=0.0003). Six patients in the AVE5026 groups, four in the 60 mg group, experienced major bleeding; none did in the enoxaparin group., Conclusions: The safety and efficacy results of this study suggest that a AVE5026 dose of between 20 and 40 mg presents an adequate benefit-to-risk ratio.

Heparin inhibits the growth of several cell types in vitro, including bovine pulmonary artery smooth muscle cells (BPASMCs). To understand more about the heparin structure required for endogenous activity, chemically modified derivatives of native heparin and glycol-split heparin, namely, 2-O-desulfonated iduronic/glucuronic acid residues in heparin, and 2-O-desulfonated iduronic residues in glycol-split heparin were prepared. These were assayed for their antiproliferative potency on cultured BPASMCs. All of the 2-O-desulfonated heparin derivatives had significantly decreased less antiproliferative activity on BPASMCs. These results suggest that the 2-O-sulfo group of iduronic acid residues in heparin's major sequence is essential for the antiproliferative properties of heparin. The size of heparin does not affect the growth-inhibitory properties of heparin on BPASMCs at the three dose levels examined.

Investigations on extracellular matrix (ECM) and growth factor (GF) complexes have revealed an underappreciated phenomenon: they can either negate GF activity or generate synergistic signals for cell function, in particular mitogenesis. ECM and pericellular matrix molecules were first recognized to complex with GFs and regulate GF activity by the seminal observations that basic fibroblast growth factor (bFGF or FGF-2) required binding to a cell-surface heparin sulfate proteoglycan and to its authentic cell-surface receptor for biological activity (Klagsbrun and Baird, 1991; Yayon et al., 1991). Subsequently, numerous ECM-GF interactions that modulate GF activity were discovered; we have reviewed many of these findings (Macri et al., 2007).

The carbazole assay is commonly employed to quantify heparin and other uronic acid-containing polysaccharides. Heparin-derived standard curves are often employed to quantify solutions of various natural and unnatural heparin structures that have different levels of sulfate substitution, different levels of N-sulfo and N-acetyl groups, and other structural changes as a consequence of reducing molecular weight. Recent studies in our laboratory have focused on chemically modified heparin derivatives comprised of structurally diverse N-acyl moieties substituted into heparin in place of N-sulfo groups. We report here that although differing degrees of 2-N-sulfo-, 2-N-acetyl- or 2-amino-d-glucosamine residues within heparin do not affect signal intensity in the carbazole assay, replacing N-sulfo groups in heparin with structurally diverse N-acyl moieties affords products that display significant variation in the assay. The structure of different N-acyl groups, and to a lesser extent the degree of N-acylation by individual N-acyl groups, is shown to variably alter signal intensity in the carbazole assay even though content and structure of uronic acid residues is unaltered.

Background: Puromycin aminonucleoside-induced nephrosis is characterized by increased renal excretion of plasma proteins. We employed this experimental model to study the urinary clearance of dextran sulfate., Methods: The dextran sulfate eliminated by the urine was determined using a metachromatic assay. Polysaccharide fragments were analyzed by chromatographic and electrophoretic procedures. Disaccharide composition of the glomerular heparan sulfate was assessed using digestion with specific lyases., Results: In normal rats dextran sulfate is partially degraded to lower molecular weight fragments and only then eliminated by the urine. Surprisingly, in puromycin aminonucleoside-induced glomerulosclerosis the molecular size of the fragments of dextran sulfate found in the urine is the same or even lower than in control animals in spite of the marked proteinuria. Furthermore, urinary excretion of dextran sulfate decreases in the experimentally induced nephrosis. This observation cannot be totally attributed to a reduced number of physiologically active nephrons since the glomerular filtration rate decreases approximately 32% after puromycin aminonucleoside administration while the urinary excretion of 8 kDa-dextran sulfate decreases 3-fold. The glomerular heparan sulfate shows reduced sulfation when compared with normal animals. Possibly puromycin aminonucleoside decreases the activity of kidney endoglycosidases, which reduce the molecular size of the sulfated polysaccharide, leading to a decrease in its renal clearance. Reduced sulfation of the glomerular heparan sulfate in the puromycin aminonucleoside-induced nephrosis does not alter the size of the dextran sulfate eliminated by the kidney, as suggested for protein., Conclusions: Each pathological process induces a particular modification in the kidney, which in turn can affect the renal selectivity to specific macromolecules in different ways.

Structures of heparin disaccharide have been analyzed by DFT using the B3LYP/6-311++G( * *) method. The optimized geometries of two forms of this disaccharide, differing in the conformation ((1)C(4) and (2)S(0)) of the IdoA2S residue, confirmed considerable influences of the sulfate and the carboxylate groups upon the pyranose ring geometries. The computed energies showed that disaccharide having the (1)C(4) form of the IdoA2S residue is more stable than that with the (2)S(0) form. Interatomic distances, bond and torsion angles showed that interconversion of the IdoA2S residue results in geometry changes in the GlcN,6S residue as well. Three-bond proton-proton and proton-carbon spin-spin coupling constants computed for both forms agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium in disaccharide. Influences of the charged groups upon the magnitudes of spin-spin coupling constants are also discussed.

Modified heparin disaccharides were obtained by the alkaline treatment of a solution containing the disulfated heparin disaccharide DeltaHexA-alpha-(1-->4)-D-GlcNSO(3),6SO(3). Their structures were characterized by one- and two-dimensional NMR spectroscopy: DeltaHexA-alpha-(1-->4)-1,6-anhydro-GlcNSO(3), DeltaHexA-alpha-(1-->4)-1,6-anhydro-ManNSO(3) and DeltaHexA-alpha-(1-->4)-ManNSO(3),6OSO(3). NMR spectroscopy, in combination with HPLC, provided the composition of the mixture. Characteristic NMR signals of the disaccharides were identified, even at low levels, in a high field of (1)H-(13)C correlation NMR spectra (HSQC) of a low molecular weight heparin (LMWH) obtained by beta-elimination (alkaline hydrolysis) of heparin benzyl ester, providing a more complete structural profile of this class of compounds.

Background: Patients receiving heparin for thromboprophylaxis or treatment may have new or recurrent venous thromboembolism (VTE) if immune-mediated heparin-induced thrombocytopenia (HIT) occurs or for other reasons, eg, if anticoagulation fails. We estimated from the literature how frequently a patient presenting with VTE during or following heparin therapy has HIT-associated VTE., Methods: A comprehensive, systematic literature search was conducted to identify studies using unfractionated or low-molecular-weight heparin (LMWH) for thromboprophylaxis or treatment in which new or recurrent VTE and serologically confirmed HIT were reported. From extracted study data, the proportion of patients with HIT-associated VTE relative to any VTE was calculated by heparin type and mode of administration., Results: We identified 10 studies, some with multiple arms, that used unfractionated heparin (IV administration, 5 studies; subcutaneous administration, 3 studies) or subcutaneous LMWH (5 studies) and met analysis criteria. Across these studies, 386 of 6,219 heparin-treated patients had VTE, including 32 patients who also had HIT. The frequency of HIT-associated VTE among heparin-treated patients with VTE was comparable between IV and subcutaneous unfractionated heparin therapy (13.2% [17 of 129 patients] vs 12.4% [14 of 113 patients]; odds ratio, 1.07; 95% confidence interval, 0.50 to 2.3; p > 0.99) yet significantly different between unfractionated heparin and LMWH therapy (12.8% [31 of 242 patients] vs 0.7% [1 of 144 patients]; odds ratio, 21.0; 95% confidence interval, 2.8 to 156; p < 0.001)., Conclusions: VTE is associated with HIT infrequently (< 1%) in LMWH-treated patients, yet often (approximately one in eight cases) in unfractionated heparin-treated patients. Physicians should suspect the possibility of HIT if VTE develops during or soon after unfractionated heparin use; if thrombocytopenia is present, alternative anticoagulation should be used until HIT is excluded.

The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide-protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS-protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.

Background: Bivalirudin is replacing heparin as the anticoagulant agent of choice for elective percutaneous coronary intervention (PCI). This study aimed to assess the safety and clinical outcomes of bivalirudin versus unfractionated heparin (UFH) in patients undergoing PCI for acute myocardial infarction (AMI)., Methods: A cohort of 672 consecutive patients presenting with AMI without prior thrombolytic therapy were treated with either bivalirudin (216 patients) or UFH (456 patients). Platelet glycoprotein IIb/IIIa inhibitors were administered at the operator's discretion. The in-hospital, 30-day, and 6-month outcomes of the two groups were compared., Results: Baseline clinical and angiographic characteristics were similar between the groups. In-hospital complications were similar, although there was a trend of a less major hematocrit drop in the bivalirudin group (0.9% vs. 3.1%, P=.09). All clinical outcomes were similar between the groups at 30-day and 6-month follow-ups. There was no statistical significance for acute thrombosis and subacute thrombosis between the groups, and there was no late thrombosis from either group. The event-free survival rate was similar between the groups (P=.41)., Conclusion: The use of bivalirudin in patients undergoing PCI after AMI is safe and feasible. Bivalirudin should be considered as an alternative anticoagulant agent during PCI to treat patients presenting with AMI.

O-Desulfated heparin (ODSH) is a promising new anti-inflammatory agent for the prevention of reperfusion injury following myocardial infarction or stroke. This partially desulfated heparin derivative has less anticoagulant activity than unfractionated heparin but retains the inherent anti-inflammatory properties of heparin. Thus, ODSH could be administered at the high doses needed to achieve desired anti-inflammatory function without risk of hemorrhage. However, given the very low anticoagulant activity of this species, traditional methods for heparin determination in clinical samples might not be well suited for ODSH measurements. In this article, a novel titrimetric method for detection of ODSH in buffer and plasma is described using a protamine-sensitive polymer membrane electrode as the detector. Titrations of ODSH with the heparin antagonist protamine yield sharp endpoints with sensitivity to ODSH in the micrograms per milliliter range for plasma samples. The stoichiometry for protamine interaction with ODSH is determined to average 1.39 microg protamine/microg ODSH in plasma. This technology is further applied to a toxicokinetic study of ODSH in an animal model, demonstrating the ability to detect the changes in ODSH concentrations in biological samples.

Fibroblast growth factors (FGF) are secreted molecules which function through the activation of specific tyrosine kinases receptors, the FGF receptors that transduce the signal by activating different pathways including the Ras/MAP kinase and the phospholipase-C gamma pathways. FGFs are involved in the regulation of many developmental processes including patterning, morphogenesis, differentiation, cell proliferation or migration. Such a diverse set of activities requires a tight control of the transduction signal which is achieved through the induction of different feedback inhibitors such as the Sproutys, Sef and MAP kinase phosphatase 3 which are responsible for the attenuation of FGF signals, limiting FGF activities in time and space.

The presence and integrity of amniotic fluid is fundamental for the normal development of the human fetus during pregnancy. Its production rate changes throughout pregnancy and is mainly related to the functions of the different fetal, placental and amniotic compartments. Premature rupture of the membranes (PROM) occurs in about 5% of deliveries, with complications such as infection and preterm birth. The management of patients with PROM, regardless of gestational age, remains controversial, and it is therefore important to develop new biological tests in order to achieve accurate diagnoses by identifying the presence of specific amniotic fluid markers in vaginal environment. We recently showed the usefulness of amniotic fluid proteomics in identifying a series of peptides that were absent from the corresponding maternal plasma. Several peptides corresponded to fragments of plasma proteins. Two peptides, absent from plasma samples of pregnant women, were identified in amniotic fluid. They corresponded to the COOH-terminal parts of perlecan (SwissProt: P98160) and of agrin (SwissProt: O00468) protein cores, two major heparan sulfate proteoglycans of basement membranes. In this review we will discuss modern proteomic strategies that may improve the laboratory assessment of PROM, and will focus on some of the biochemical characteristics of agrin and perlecan fragments identified in amniotic fluid.

Objectives: Hospital administrative data were analyzed to assess treatment patterns, in-hospital mortality, rates of hemorrhagic events and thrombus propagation, utilization of health care resources, and hospital costs associated with various treatments during inpatient care for venous thromboembolism (VTE)., Study Design: Data from inpatient records were collected for deep venous thrombosis (DVT) and pulmonary embolism (PE) encounters at 132 US hospitals between January 1999 and December 2000. Patients receiving the most frequently employed treatments were compared with respect to demographics, related procedures and diagnostics, length of stay, adverse events, in-hospital mortality, and hospital costs., Results: A total of 953 primary DVT and 3933 primary PE admissions were identified. Most admissions involved treatment with unfractionated heparin and vitamin K antagonist (UFH/VKA, 64.2% of admissions), followed by UFH with VKA and low-molecular-weight heparin (UFH/LMWH/VKA, 14.4%), and LMWH/VKA (12.9%). Compared with those treated with UFH/VKA, patients treated with LMWH/VKA experienced higher anticoagulant costs (dollar 540 vs. dollar 106), but lower total hospital costs (dollar 5198 vs. dollar 5977) and shorter lengths of stay (4.4 vs. 5.7 days for those without PE and 5.7 vs. 6.7 days for those with PE)., Conclusions: UFH/VKA was the most common regimen used to treat VTE. In spite of its higher medication cost, however, treatment with LMWH/VKA was associated with significantly shorter hospital stays and lower total hospitalization costs, compared with UFH/VKA.

Granzyme B (GzmB), a serine protease of cytotoxic T lymphocytes and natural killer (NK) cells, induces apoptosis by caspase activation after crossing the plasma membrane of target cells. The mechanism of this translocation during killer cell attack, however, is not understood. Killer cells release GzmB and the membrane-disturbing perforin at the contact site after target recognition. Receptor-mediated import of glycosylated GzmB and release from endosomes were suggested, but the role of the cation-independent mannose 6-phosphate receptor was recently refuted. Using recombinant nonglycosylated GzmB, we observed binding of GzmB to cellular membranes in a cell type-dependent manner. The basis and functional impact of surface binding were clarified. GzmB binding was correlated with the surface density of heparan sulfate chains, was eliminated on treatment of target cells with heparinase III or sodium chlorate, and was completely blocked by an excess of catalytically inactive GzmB or GzmK. Although heparan sulfate-bound GzmB was taken up rapidly into intracellular lysosomal compartments, neither of the treatments had an inhibitory influence on apoptosis induced by externally added streptolysin O and GzmB or by natural killer cells. We conclude that membrane receptors for GzmB on target cells are not crucial for killer cell-mediated apoptosis.

Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identified by NMR spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six differentially protected monosaccharide building blocks (three l-iduronic acids and three d-glucosamines) were prepared. These include a l-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-alpha-l-idopyranosyluronate trichloroacetimidate for which a single-crystal X-ray structure was determined to have M(r)=576.79, a=9.3098(11)A alpha=90 degrees , b=10.3967(12)A beta=90 degrees , c=28.026(3)A gamma=90 degrees , V=2712.7(6)A(3), P2(1)2(1)2(1), Z=4, mu=0.71073A, and R=0.0378 for 7586 observed reflections. It shows that the molecular structure of the donor is in the (1)C(4) conformation with significant 1,3-diaxial interactions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the temporary protecting group to protect the hydroxyl for elongation.

Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (V(RBC)) in mice. We found faster V(RBC) of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease.

Background: The K5 polysaccharide obtained from Escherichia coli strain 010:K5:H4 is a polymer of the disaccharidic unit formed by D-glucuronic acid and N-acetylglucosamine. This structure is akin to N-acetylheparosan, the precursory polymer of heparin and of heparan sulfate. This structural affinity with N-acetylated heparin and with de-sulfated heparin makes the K5 polysaccharide extremely useful for the preparation of sulfated heparin-like semi-synthetic derivatives. It has been demonstrated that heparins are able to inhibit tissue factor and cytokine production and expression by human monocytes., Objective: The aim of this study was to evaluate the effects of four different heparin-like semi-synthetic derivatives on inflammatory cytokine production and expression by human mononuclear cells., Results: The simultaneous addition of lipopolysaccharide (LPS; 0.2 and 10 micro g mL(-1)) and the K5 polysaccharide did not inhibit interleukin (IL)-1beta, IL-6 or tumor necrosis factor (TNF)-alpha production by stimulated mononuclear cells. IL-1beta, IL-6 and TNF-alpha concentrations in supernatants of LPS-stimulated mononuclear cells were not influenced by the addition of N,O-sulfated K5 polysaccharide (K5-N, OS) and epimerized N-sulfated K5 polysaccharide (K5 NS epi) at 5 and 10 microg mL(-1), whereas the addition of epimerized N,O-sulfated K5 polysaccharide (K5-N, OS epi) (5 and 10 microg mL(-1)) and O-sulfated K5 polysaccharide (K5-OS) (5 and 10 microg mL(-1)) to LPS-stimulated cells caused a significant dose-dependent inhibition of IL-1beta, IL-6 and TNF-alpha. All sulfated heparin-like semi-synthetic derivatives did not influence the IL-10 production by LPS-stimulated mononuclear cells. In LPS-stimulated cells (0.2 and 10 microg mL(-1)), K5-OS or K5-N, OS epi at 5 and 10 microg mL(-1) markedly decreased TNF-alpha mRNA expression., Conclusions: These results indicate that the sulfated heparin-like semi-synthetic derivatives K5-OS and K5-N, OS epi are able to inhibit both expression and production of inflammatory cytokines, whereas they do not influence the anti-inflammatory cytokine IL-10, suggesting a potential role for these products as modulators of inflammatory reactions.

Objectives: Our purpose was to improve the performance of carbon-coated expanded polytetrafluoroethylene vascular grafts by bonding the grafts with silyl-heparin, a biologically active heparin analog, using polyethylene glycol as a cross-linking agent. Material and method Silyl-heparin-bonded carbon-coated expanded polytetrafluoroethylene vascular grafts (Bard Peripheral Vascular, Tempe, Ariz), were evaluated for patency and platelet deposition 2 hours, 7 days, and 30 days after graft implantation in a canine bilateral aortoiliac artery model. Platelet deposition was determined by injection of autologous, (111)Indium-radiolabeled platelets, followed by a 2-hour circulation period prior to graft explantation. Histologic studies were performed on a 2-mm longitudinal strip of each graft (7-day and 30-day groups). Heparin activity of the explanted silyl-heparin grafts was determined by using an antithrombin-III based thrombin binding assay., Results: Overall chronic graft patency (7-day and 30-day groups) was 100% for the silyl-heparin bonded (16/16) grafts versus 68.75% for control (11/16) grafts (P =.043). Acute 2-hour graft patency was 100% for the silyl-heparin bonded (6/6) grafts versus 83.3% for control (5/6) grafts. Radiolabeled platelet deposition studies revealed a significantly lower amount of platelets deposited on the silyl-heparin grafts as compared with control grafts in the 30-day group (13.8 +/- 7.18 vs 28.4 +/- 9.73, CPM per cm2 per million platelets, mean +/- SD, P =.0451, Wilcoxon rank sum test). In the 2-hour group of dogs, a trend towards a lower deposition of platelets on the silyl-heparin grafts was observed. There was no significant difference in platelet deposition between the two grafts in the 7-day group. Histologic studies revealed a significant reduction in intraluminal graft thrombus present on the silyl-heparin grafts as compared with control grafts in the 30-day group of animals. In contrast, there was no difference in amount of graft thrombus present on both graft types in the 7-day group of dogs. Pre-implant heparin activity on the silyl-heparin bonded grafts was 2.0 IU/cm(2) (international units[IU]/cm(2)). Heparin activity remained present on the silyl-heparin grafts after explantation at all 3 time points (2 hours: above upper limit of assay, upper limit = 0.57, n = 6; 7 days: 0.106 +/- 0.015, n = 5; 30 days: 0.007 +/- 0.001, n = 5; mean +/- SD, IU/cm(2))., Conclusion: Silyl-heparin bonding onto carbon-coated expanded polytetrafluoroethylene vascular grafts resulted in (1) improved graft patency, (2) increased in vivo graft thromboresistance, and (3) a significant reduction in intraluminal graft thrombus. This graft may prove to be useful in the clinical setting.

Keratinocyte growth factor (KGF) is a paracrine-acting, epithelial mitogen produced by cells of mesenchymal origin. It is a member of the fibroblast growth factor (FGF) family, and acts exclusively through a subset of FGF receptor isoforms (FGFR2b) expressed predominantly by epithelial cells. The upregulation of KGF after epithelial injury suggested it had an important role in tissue repair. This hypothesis was reinforced by evidence that intestinal damage was worse and healing impaired in KGF null mice. Preclinical data from several animal models demonstrated that recombinant human KGF could enhance the regenerative capacity of epithelial tissues and protect them from a variety of toxic exposures. These beneficial effects are attributed to multiple mechanisms that collectively act to strengthen the integrity of the epithelial barrier, and include the stimulation of cell proliferation, migration, differentiation, survival, DNA repair, and induction of enzymes involved in the detoxification of reactive oxygen species. KGF is currently being evaluated in clinical trials to test its ability to ameliorate severe oral mucositis (OM) that results from cancer chemoradiotherapy. In a phase 3 trial involving patients who were treated with myeloablative chemoradiotherapy before autologous peripheral blood progenitor cell transplantation for hematologic malignancies, KGF significantly reduced both the incidence and duration of severe OM. Similar investigations are underway in patients being treated for solid tumors. On the basis of its success in ameliorating chemoradiotherapy-induced OM in humans and tissue damage in a variety of animal models, additional clinical applications of KGF are worthy of investigation.

A novel, simple, and sensitive assay was developed to monitor, quantitatively, the hyaluronidase and heparinase I-catalyzed cleavage of fluoresceinamine-labeled hyaluronic acid and heparin, respectively. The fluoresceinamine-labeled substrates were hydrophobically absorbed onto 4-microm polystyrene beads. In the presence of enzyme, the change in fluorescence output of the substrate-absorbed beads was monitored in a noncontinuous manner using a flow cytometer. Our results show that hyaluronidase and heparinase I can cleave their respective substrates on the beads in a concentration- and time-dependent manner. The assay is suitable for detecting the presence of these glycosaminoglycan-degrading enzymes in cell lysates, extracts, or purified fractions, for quantifying their amounts, and for investigating the activity of potential inhibitors.

Background: Glomerulosclerosis is a severe complication of many immunologically-mediated kidney diseases, eventually resulting in loss of renal function. In chronic graft-versus-host disease (GvHD) in mice, a model for human lupus nephritis, the end-stage sclerotic lesions were previously shown to contain large amounts of fibronectin (FN). This study investigated a domain-specific accumulation process of circulating plasma FN (pFN) in sclerotic lesions., Methods: GvHD mice were injected with FITC-conjugated pFN or pFN-fragments, with or without heparin pre-incubation. pFN fragments were generated by digestion of pFN by cathepsin D, after which the fragments were separated on a heparin affinity column. Thus, two batches of fragments were obtained with either low or high affinity for heparin., Results: FN accumulation was accompanied by an up-regulated expression of integrin alpha5beta1, the FN receptor, in the periphery of sclerotic lesions. pFN-FITC injected into GvHD mice was trapped in sclerotic glomeruli within 24 hours. Both heparin and non-anti-coagulant heparin blocked the accumulation of pFN-FITC, indicating that the protective effect of heparin in the trapping of FN is independent of its anticoagulant properties, and probably results from preventing direct binding of FN in the sclerotic lesions. To investigate whether FN binds in the glomerulus via the heparin-binding regions, pFN fragments were generated and injected into GvHD mice. Whereas the fraction with high affinity for heparin did not accumulate in the sclerotic glomeruli, the fraction with low affinity for heparin did. Partial sequencing of the isolated peptides showed that in the glomerulus fibronectin does not bind via the heparin II binding region., Conclusions: We hypothesize that the protective effect of heparin treatment may be the result of steric hindrance of the specific binding sites, that is, the I1-5 and/or III1 self-assembly sites of FN.

It was previously reported that treatment with the sulfated polysaccharide fucoidan or the structurally similar dextran sulfate increased circulating mature white blood cells and hematopoietic progenitor/stem cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that the SDF-1 increase is due to its competitive displacement from heparan sulfate proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other tissues. Although moderately increased levels of interleukin-8, MCP1, or MMP9 were also present after fucoidan treatment, studies in gene-ablated mice (GCSFR(-/-), MCP1(-/-), or MMP9(-/-)) and the use of metalloprotease inhibitors do not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan-mobilizing treatments and not with several other mobilizing agents tested, are likely responsible. To the authors' knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and peripheral blood through a physiologically relevant mechanism, resulting in mobilization with kinetics similar to other mobilizing CXC chemokines. The study further underscores the importance of the biological roles of carbohydrates.