Thrombin cleaves IL-33 and modulates IL-33-activated allergic lung inflammation.

BACKGROUND: Organisms have orchestrated coagulation and immune systems. Although a link between inflammation and haemostasis has been reported in asthma, the interaction mechanism has not been completely elucidated. Here, we investigated the direct link between the mammalian immune and coagulation systems. METHODS: Mice were administered protease or antigens intranasally to induce airway inflammation with or without thrombin inhibitors treatment. The effects of thrombin and its inhibitors on interleukin (IL)-33 were investigated both in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) and plasma from asthma patients are collected to verify the correlation between thrombin and group 2 innate lymphocytes (ILC2s). RESULTS: Low-molecular-weight heparin (LMWH, an indirect inhibitor of thrombin) restrained both papain- and fungus-induced type 2 immune responses in mice by inhibiting IL-33 cleavage. Upon examining the potential thrombin protease consensus sites, we found that IL-33 was directly cleaved by thrombin at specific amino acids (R48 and R106) to generate a mature form of IL-33 with potent biological activity. In addition, we found that bivalirudin TFA (a direct inhibitor of thrombin) inhibited a variety of type 2 inflammatory responses, such as those in house dust mite (HDM)- and ovalbumin (OVA)-mediated pulmonary inflammation models. We found that plasma thrombin-antithrombin complex (TATc) levels in asthma patients were positively associated with the number and function of IL-33-responder group 2 innate lymphocytes (ILC2s) among peripheral blood mononuclear cells (PBMCs) from asthma patients. CONCLUSION: The data suggested that thrombin inhibitors administration could be effective in treating lung inflammation by regulating ILC2s via IL-33 maturation, indicating that targeting thrombin is a potential way to treat allergic diseases.

A synthetic heparinoid blocks Tau aggregate cell uptake and amplification.

Tau aggregation underlies neurodegeneration in Alzheimer's disease and related tauopathies. We and others have proposed that transcellular propagation of pathology is mediated by Tau prions, which are ordered protein assemblies that faithfully replicate in vivo and cause specific biological effects. The prion model predicts the release of aggregates from a first-order cell and subsequent uptake into a second-order cell. The assemblies then serve as templates for their own replication, a process termed "seeding." We have previously observed that heparan sulfate proteoglycans on the cell surface mediate the cellular uptake of Tau aggregates. This interaction is blocked by heparin, a sulfated glycosaminoglycan. Indeed, heparin-like molecules, or heparinoids, have previously been proposed as a treatment for PrP prion disorders. However, heparin is not ideal for managing chronic neurodegeneration, because it is difficult to synthesize in defined sizes, may have poor brain penetration because of its negative charge, and is a powerful anticoagulant. Therefore, we sought to generate an oligosaccharide that would bind Tau and block its cellular uptake and seeding, without exhibiting anticoagulation activity. We created a compound, SN7-13, from pentasaccharide units and tested it in a range of assays that measured direct binding of Tau to glycosaminoglycans and inhibition of Tau uptake and seeding in cells. SN7-13 does not inhibit coagulation, binds Tau with low nanomolar affinity, and inhibits cellular Tau aggregate propagation similarly to standard porcine heparin. This synthetic heparinoid could facilitate the development of agents to treat tauopathy.

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence.

Although glycosaminoglycan (GAG)-protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.

Heparan Sulfate Proteoglycans in Viral Infection and Treatment: A Special Focus on SARS-CoV-2.

Heparan sulfate proteoglycans (HSPGs) encompass a group of glycoproteins composed of unbranched negatively charged heparan sulfate (HS) chains covalently attached to a core protein. The complex HSPG biosynthetic machinery generates an extraordinary structural variety of HS chains that enable them to bind a plethora of ligands, including growth factors, morphogens, cytokines, chemokines, enzymes, matrix proteins, and bacterial and viral pathogens. These interactions translate into key regulatory activity of HSPGs on a wide range of cellular processes such as receptor activation and signaling, cytoskeleton assembly, extracellular matrix remodeling, endocytosis, cell-cell crosstalk, and others. Due to their ubiquitous expression within tissues and their large functional repertoire, HSPGs are involved in many physiopathological processes; thus, they have emerged as valuable targets for the therapy of many human diseases. Among their functions, HSPGs assist many viruses in invading host cells at various steps of their life cycle. Viruses utilize HSPGs for the attachment to the host cell, internalization, intracellular trafficking, egress, and spread. Recently, HSPG involvement in the pathogenesis of SARS-CoV-2 infection has been established. Here, we summarize the current knowledge on the molecular mechanisms underlying HSPG/SARS-CoV-2 interaction and downstream effects, and we provide an overview of the HSPG-based therapeutic strategies that could be used to combat such a fearsome virus.

Heparin-Binding Copolymer as a Complete Antidote for Low-Molecular-Weight Heparins in Rats.

Bleeding resulting from the application of low-molecular-weight heparins (LMWHs) may be treated with protamine sulfate, but this treatment lacks efficiency; its action against antifactor Xa activity is limited to ∼60%. Moreover, protamine sulfate can cause life-threatening hypersensitivity reactions. We developed diblock heparin-binding copolymer (HBC), which can neutralize the anticoagulant activity of parenteral anticoagulants. In the present study, we explored the safety profile of HBC and its potential to reverse enoxaparin, nadroparin, dalteparin, and tinzaparin in human plasma and at in vivo conditions. HBC-LMWH complexes were characterized using zeta potential, isothermal titration calorimetry, and dynamic light scattering. The rat cardiomyocytes and human endothelial cells were used for the assessment of in vitro toxicity. Male Wistar rats were observed for up to 4 days after HBC administration for clinical evaluation, gross necropsy, and biochemistry and histopathological analysis. Rats were treated with LMWHs alone or followed by short-time intravenous infusion of HBC, and bleeding time and antifactor Xa activity were measured. HBC completely reversed antifactor Xa activity prolonged in vitro by all LMWHs with an optimal weight ratio of 2.5:1. The complexes of HBC-LMWHs were below 5 µm. We observed no effects on the viability of cardiovascular cells treated with HBC at concentrations up to 0.05 mg/ml. Single doses up to 20 mg/kg of HBC were well tolerated by rats. HBC completely reversed the effects of LMWHs on bleeding time and antifactor Xa activity in vivo after 20 minutes and retained ∼80% and ∼60% of reversal activity after 1 and 2 hours, respectively. Well-documented efficacy and safety of HBC both in vitro and in vivo make this polymer a promising candidate for LMWHs reversal. SIGNIFICANCE STATEMENT: Over the last decade, there has been significant progress in developing antidotes for the reversal of anticoagulants. Until now, there has been no effective and safe treatment for patients with severe bleeding under low-molecular-weight heparin therapy. Based on our in vitro and in vivo studies, heparin-binding copolymer seems to be a promising candidate for neutralizing all clinically relevant low-molecular-weight heparins.

Radiolabeled 111In-FGF-2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging.

Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an 111In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). 111In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as 125I-FGF-2. Furthermore, 111In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. 111In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (Kd: 0.6 ± 0.07 µM and 0.33 ± 0.03 µM, respectively) as assessed by isothermal titration calorimetry. The binding of 111In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC50 values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of 111In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, 111In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.

Comprehensive Identification and Quantitation of Basic Building Blocks for Low-Molecular Weight Heparin.

Low-molecular weight heparins (LMWHs) are widely used anticoagulant drugs. They inherit the heterogeneous backbone sequences of the parent heparin, while the chemical depolymerization process modifies the nonreducing end (NRE) and reducing end (RE) of their sugar chains. Some side reactions may also occur and increase the structural complexity of LMWHs. It is important to precisely characterize the structures of LMWHs, especially their chemical modifications, to ensure drug quality and safety. Compositional analysis provides a powerful approach to reveal the building blocks that make up the LMWHs, which are the mutual consequence of the heparin starting materials and the manufacturing process. Here, we introduce a comprehensive analytical method to recover the most basic building blocks of LMWHs. A strategy of combining both enzymatic digestion and oxidative degradation of LMWH was used to make the NRE, RE, and backbone structures differentiable from one another. Satisfactory separation, identification, and quantitation were achieved by coupling hydrophilic interaction chromatography with a triple quadrupole mass spectrometer operating under the multiple reaction monitoring mode. After enzymatic digestion, over 30 species were detected, with both natural and chemically modified heparin basic building blocks. Two novel structures, including a trisaccharide containing two glucosamine residues and a tetrasaccharide containing a 3-O-sulfated uronic acid residue, were discovered. Reduced and oxidatively degraded samples were analyzed to provide the complementary information on both termini of LMWHs. The reproducibility of this method was evaluated, and enoxaparin injections were analyzed to demonstrate the application of this method for evaluating the sameness of LMWH products.

Role of parnaparin in atherosclerosis.

Atherosclerosis is characterized by a proliferation of vascular smooth muscle cells (VSMCs) and their migration to the intima, which induces thickening of the intima itself, but the mechanism remains poorly understood. Low molecular weight heparin (LMWH) inhibits the proliferation of VSMCs. Previous studies have shown that a LMWH, parnaparin (PNP), acts on the processes of atherogenesis and atheroprogression in experimental animal models. The aim of this study was to investigate the involvement of oxidative stress, inflammation and VSMCs in the regulation of vascular wall homeostasis. We also considered the possibility of restoring vascular pathological changes using PNP treatment. In order to evaluate vascular remodelling in this study we have analysed the morphological changes in aortas of an animal model of atherosclerosis, apolipoprotein E-deficient mice (ApoE-/-) fed with a normal or a western diet without treatment or treated with PNP. We also analysed, by immunohistochemistry, the expression of proteins linked to atherogenesis and atheroprogression - an enzyme involved in oxidative stress, iNOS, examples of inflammatory mediators, such as tumour necrosis factor alpha (TNF-α), interleukins 1 and 6 (IL-1 and IL-6), and markers of VSMC changes, in particular plasminogen activator inhibitor-1 and thrombospondin-1 (PAI-1 and TSP-1). Our results could suggest that PNP downregulates VSMC proliferation and migration, mediated by PAI-1 and TSP-1, and reduces inflammation and oxidative stress in vessels. These data suggested that LMWH, in particular PNP, could be a theoretically practical tool in the prevention of atherosclerotic vascular modification.

Heparin binding confers prion stability and impairs its aggregation.

The conversion of the prion protein (PrP) into scrapie PrP (PrP(Sc)) is a central event in prion diseases. Several molecules work as cofactors in the conversion process, including glycosaminoglycans (GAGs). GAGs exhibit a paradoxical effect, as they convert PrP into protease-resistant PrP (PrP-res) but also exert protective activity. We compared the stability and aggregation propensity of PrP and the heparin-PrP complex through the application of different in vitro aggregation approaches, including real-time quaking-induced conversion (RT-QuIC). Transmissible spongiform encephalopathy-associated forms from mouse and hamster brain homogenates were used to seed RT-QuIC-induced fibrillization. In our study, interaction between heparin and cellular PrP (PrP(C)) increased thermal PrP stability, leading to an 8-fold decrease in temperature-induced aggregation. The interaction of low-molecular-weight heparin (LMWHep) with the PrP N- or C-terminal domain affected not only the extent of PrP fibrillization but also its kinetics, lowering the reaction rate constant from 1.04 to 0.29 s(-1) and increasing the lag phase from 12 to 19 h in RT-QuIC experiments. Our findings explain the protective effect of heparin in different models of prion and prion-like neurodegenerative diseases and establish the groundwork for the development of therapeutic strategies based on GAGs.

Polyanion binding accelerates the formation of stable and low-toxic aggregates of ALS-linked SOD1 mutant A4V.

The toxic property thus far shared by both ALS-linked SOD1 variants and wild-type SOD1 is an increased propensity to aggregation. However, whether SOD1 oligomers or aggregates are toxic to cells remains to be well defined. Moreover, how the toxic SOD1 species are removed from intra- and extracellular environments also needs to be further explored. The DNA binding has been shown to be capable of accelerating the aggregatio\n of wild-type and oxidized SOD1 forms under acidic and neutral conditions. In this study, we explore the binding of DNA and heparin, two types of essential life polyanions, to A4V, an ALS-linked SOD1 mutant, under acidic conditions, and its consequences. The polyanion binding alters the A4V conformation, neutralizes its local positive charges, and increases its local concentrations along the polyanion chain, which are sufficient to lead to acceleration of the pH-dependent A4V aggregation. The accelerated aggregation, which is ascribed to the polyanion binding-mediated removal or shortening of the lag phase in aggregation, contributes to the formation of amorphous A4V nanoparticles. The prolonged incubation with polyanions not only results in the complete conversion of likely soluble toxic A4V oligomers into non- and low-toxic SDS-resistant aggregates, but also increases their stability. Although this is only an initial step toward reducing the toxicity of SOD1 mutants, the accelerating role of polyanions in protein aggregation might become one of the rapid pathways that remove toxic forms of SOD1 mutants from intra- and extracellular environments.

Bottom-up low molecular weight heparin analysis using liquid chromatography-Fourier transform mass spectrometry for extensive characterization.

Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-O-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production.

Glycosaminoglycans: anticoagulant and nonanticoagulant actions: a short history of symposia held at villa vigoni.

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, was discovered in the beginning of the 20th century and was initially identified as a procoagulant isolated from liver tissue. After the first application in patients approximately 30 years later, further purification identified the major as well as minor, but important, component units of the complex chain mixtures constituting heparin and the multiplex actions became a scientific challenge recently. A series of "Glycosaminoglycan symposium-anticoagulant and nonanticoagulant actions" developed over the past 20 years and focused on this topic has published research data in three issues of Seminars in Thrombosis & Hemostasis and in several other international scientific journals. The latest developments on the methods of analysis, the synthesis, the degradation by heparanases and the nonanticoagulant effects in tumor growth, in anti-inflammatory diseases, and in Alzheimer diseases as presented in the 21st symposium are summarized in the present overview on the occasion of the 40th anniversary of the journal with special reference to the journal's founding Editor in Chief, Eberhard F. Mammen.

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

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

Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography-mass spectrometry.

Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography-mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3wt% of heparin and from 6.2 to 8.3wt% of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin's antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group-containing tetrasaccharides in a heparin correlated positively (r=0.8294) to heparin's anticoagulant activity.

Micellar nanoparticles inhibit breast cancer and pulmonary metastasis by modulating the recruitment and depletion of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are notorious for their pathological characteristics of immunosuppression and their promoting effect on cancers. They can induce the formation of pre-metastatic niche (PMN) characterized by inflammation, immunosuppression and vascular leakage, and promote pulmonary metastasis of breast cancer. Herein, a tumor targeting c(RGDfk) peptide modified low molecular-weight-heparin-all-trans-retinoic-acid (LMWH-ATRA) micellar nanoparticle loaded with chemotherapeutic drug doxorubicin (DOX) and immune adjuvant α-galactosylceramide (αGC) (RLA/DOX/αGC NP) was developed. The hydrophilic segment LMWH inhibited the recruitment of MDSCs by competitively binding with P-selectin on the surface of vascular endothelial cells (VECs), while the hydrophobic segment ATRA promoted the depletion of MDSCs by inducing their differentiation. Through the modulation of MDSCs, micelles can significantly improve the inflammatory and immunosuppressive microenvironment of the lung and tumor sites, and inhibit the formation of PMN. Not only this, the micelles also produced a synergistic effect with αGC, which effectively improved the anti-tumor immunity of tumor bearing mice and provided a promising therapeutic strategy for breast cancer and pulmonary metastasis.

Therapeutic effects of adipose-derived mesenchymal stem/stromal cells with enhanced migration ability and hepatocyte growth factor secretion by low-molecular-weight heparin treatment in bleomycin-induced mouse models of systemic sclerosis.

BACKGROUND: Adipose-derived mesenchymal stem cells (ASCs) have gained attention as a new treatment for systemic sclerosis (SSc). Low-molecular-weight heparin (LMWH) enhances cell function and stimulates the production of hepatocyte growth factor (HGF) in a variety of cells. This study investigated the effects of LMWH on the functions of mouse ASCs (mASCs), and the therapeutic effects of mASCs activated with LMWH (hep-mASCs) in mouse models of SSc. METHODS: The cellular functions of mASCs cultured with different concentrations of LMWH were determined. Mice were divided into four groups: bleomycin (BLM)-induced SSc (BLM-alone), BLM-induced SSc administered with mASCs (BLM-mASC), and BLM-induced SSc administered with mASCs activated with 10 or 100 µg/mL LMWH (BLM-hep-mASC); there were 9 mice per group (n = 9). Skin inflammation and fibrosis were evaluated using histological and biochemical examinations and gene expression levels. RESULTS: In vitro assays showed that migration ability and HGF production were significantly higher in hep-mASCs than in mASCs alone. The mRNA expression levels of cell migration factors were significantly upregulated in hep-mASCs compared to those in mASCs alone. The hep-mASCs accumulated in the skin tissues more than mASCs alone. The thickness of skin and hydroxyproline content in BLM-hep-mASC groups were significantly decreased, and the skin mRNA expression levels of interleukin-2, α-smooth muscle actin, transforming growth factor ß1, collagen type 1 alpha 1, and tissue inhibitor of metalloproteinase 2 were significantly downregulated compared to those in the BLM-alone group. CONCLUSIONS: hep-mASCs showed higher anti-inflammatory and anti-fibrotic effects than mASCs alone and may be a promising candidate for SSc treatment.

Generation of an anticoagulant aptamer that targets factor V/Va and disrupts the FVa-membrane interaction in normal and COVID-19 patient samples.

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.

Bioactivity screening of partially desulfated low-molecular-weight heparins: a structure/activity relationship study.

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns.

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M(w)) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A(2)) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M(w) of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M(w) values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M(w) results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

Simple fluorescence assay for quantification of OSCS in heparin.

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ ((em)) 330 nm, λ ((ex)) 510 nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with (1)H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.