Synthetic Heparin Research Articles

Abstract 845: Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis

Abstract Transforming growth factor β (TGF-β) is a key regulator of several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparin sulfate which is a member of the glycosaminoglycan family of carbohydrates. Our subsequent studies with highly sulfated heparin-like glycosaminoglycans (HLGAGs) with low anti-coagulant activity indicated that a high-molecular-weight E. coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. We then evaluated the therapeutic potential of K5-NSOS and a low-molecular-weight synthetic heparin (fragmin) in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were then administered with K5-NSOS, fragmin, or vehicle once daily for 4 weeks. Both HLGAGs increased body weight, decreased osteolytic lesion area, and reduced tumor burden in bone. Our in vitro studies showed that both K5-NSOS and fragmin inhibited adhesion of breast cancer cells to ICAM-1, and K5-NSOS also inhibited adhesion to E- and P-selectin. Furthermore, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. Our data support and further clarify the critical role of heparan sulfate glycosaminoglycans in breast cancer metastasis and indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent with low anticoagulant activity that could be further optimized as an anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 845. doi:10.1158/1538-7445.AM2011-845

Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate

Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.

Heparin Mimicking Polymer Promotes Myogenic Differentiation of Muscle Progenitor Cells

Heparin and heparan sulfate mediated basic fibroblast growth factor (bFGF) signaling plays an important role in skeletal muscle homeostasis by maintaining a balance between proliferation and differentiation of muscle progenitor cells. In this study we investigate the role of a synthetic mimic of heparin, poly(sodium-4-styrenesulfonate) (PSS), on myogenic differentiation of C2C12 cells. Exogenous supplementation of PSS increased the differentiation of C2C12 cells in a dose-dependent manner, while the formation of multinucleated myotubes exhibited a nonmonotonic dependence with the concentration of PSS. Our results further suggest that one possible mechanism by which PSS promotes myogenic differentiation is by downregulating the mitogen activated extracellular regulated signaling kinase (MAPK/ERK) pathway. The binding ability of PSS to bFGF was found to be comparable to heparin through molecular docking calculations and by native PAGE. Such synthetic heparin mimics could offer a cost-effective alternative to heparin and also reduce the risk associated with batch-to-batch variation and contamination of heparin.

Desorption Electrospray Ionization Mass Spectrometry of Glycosaminoglycans and Their Protein Noncovalent Complex

Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiring thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous determination of the protein-bound sulfated oligosaccharide.

HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides

Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO(3) moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate-derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na](-) were easily observed on mass spectra. MALDI-MS characterization of challenging analytes such as heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide was successfully achieved.

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a K m of 46 μM and a k cat of 3.5 s −1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a K i of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

Conformational Dynamics of Antithrombin III With its Allosteric Activator Heparin

Antithrombin III (ATIII) is a serpin that is involved in the regulation of blood coagulation through the inhibition of blood clotting enzymes. Heparin is an allosteric activator of ATIII that binds to helix-D and causes a conformational change in the reactive center loop (RCL), expelling it from its position partially inserted into beta-sheet A. RCL expulsion in turn increases ATIII activity toward fXa several hundred-fold. Hydrogen/deuterium exchange and mass spectrometry were used to probe the dynamics of ATIII in the presence and absence of a synthetic heparin pentasaccharide (Fondaparinux). Results of our initial hydrogen/deuterium exchange mass spectrometry experiments provide direct, solution phase evidence that heparin cofactor binding alters conformational dynamics in four specific regions of the antithrombin molecule. (1) Helix D -- Heparin binding reduced H/D, consistent with hD extension upon cofactor binding. (2) Breach region -- Beta strands 3A and 5A, flanking the site for RCL insertion into sheet A, showed reduced H/D exchange, consistent with increased rigidity of the breach region and stabilization of a loop-expelled form. (3) Proximal RCL and hinge region -- H/D exchange for residues 376-387, which includes the N-terminal hinge region of the RCL, increased in the presence of heparin, indicating greater solvent exposure and expulsion from beta sheet A. (4) Distal RCL -- Deuterium exchange for 388-402, which includes s1C and the distal side of the RCL, decreased significantly in the presence of heparin, suggesting that s1C extension-mediated stabilization on the C-terminal side of RCL contributes to exposure of the proximal its end upon cofactor binding. Thus, dynamic H/D exchange studies of free and heparin-bound antithrombin molecules in solution will be useful for validating and refining models of ATIII heparin activation inferred from crystal structures.

ANTICOAGULANTS IN ACUTE CORONARY SYNDROME

Clinical efficacy of unfractionated and low molecular heparins in acute coronary syndrome is discussed. New synthetic heparin derivative fondaparinux (Arixtra) is focused. Author’s brief experience of fondaparinux clinical implementation is presented.

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.

Electrochemical Recognition of Synthetic Heparin Mimetic at Liquid/Liquid Microinterfaces

Electrochemically controlled molecular recognition of a synthetic heparin mimetic, Arixtra, at nitrobenzene/water microinterfaces was investigated to obtain a greater understanding of interfacial recognition and sensing of heparin and its analogues with biomedical importance. In contrast to unfractionated heparin, this synthetic pentasaccharide that mimics the unique Antithrombin III binding domain of heparin possesses well-defined structure and ionic charge to enable quantitative interpretation of cyclic voltammetric/chronoamperometric responses based on the interfacial recognition at micropipet electrodes. Arixtra is electrochemically extracted from the water phase into the bulk nitrobenzene phase containing highly lipophilic ionophores, methyltridodecylammonium or dimethyldioctadecylammonium. Numerical analysis of the kinetically controlled cyclic voltammograms demonstrates for the first time that formal potentials and standard rate constants of polyion transfer at liquid/liquid interfaces are ionophore dependent. Moreover, octadecylammonium and octadecylguanidinium are introduced as new, simple ionophores to model recognition sites of heparin-binding proteins at liquid/liquid interfaces. In comparison to octadecyltrimethylammonium, the best ionophore for heparin recognition at liquid/liquid interfaces reported so far, these new ionophores dramatically facilitate Arixtra adsorption at the interfaces. With a saline solution at physiological pH, an Arixtra molecule is selectively and cooperatively bound to 5 molecules of the guanidinium ionophore, suggesting hydrogen-bond-directed interactions of each guanidinium with a few of 10 negatively charged sulfo or carboxyl groups of Arixtra at the interfaces.

Heparin-induced cis- and trans-Dimerization Modes of the Thrombospondin-1 N-terminal Domain

Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by x-ray crystallography. We have found that dp8 and dp10 bind to TSPN-1 in a manner similar to Arixtra and that dp8 and dp10 induce the formation of trans and cis TSPN-1 dimers, respectively. In silico docking calculations partnered with our crystal structures support the importance of arginine residues in positions 29, 42, and 77 in binding sulfate groups of the dp8 and dp10 forms of heparin. The ability of several TSPN-1 domains to bind to glycosaminoglycans simultaneously probably increases the affinity of binding through multivalent interactions. The formation of cis and trans dimers of the TSPN-1 domain with relatively short segments of heparin further enhances the ability of TSP-1 to participate in high affinity binding to glycosaminoglycans. Dimer formation may also involve TSPN-1 domains from two separate TSP-1 molecules. This association would enable glycosaminoglycans to cluster TSP-1.

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

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

Profiling Heparin–Chemokine Interactions Using Synthetic Tools

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.

Potentiation of Fibroblast Growth Factor Activity by Synthetic Heparin Oligosaccharide Glycodendrimers

Heparin is a highly sulfated polysaccharide that regulates a variety of cellular processes by interaction with a host of proteins. We report the preparation of synthetic heparin oligosaccharide glycodendrimers and their use as heparin mimetics to regulate heparin-protein interactions. The multivalent display of sugar epitopes mimics the naturally occurring glycans found on cell surfaces and enhances their binding capacity. Binding of the heparin dendrimers to basic fibroblast growth factor (FGF-2) was analyzed using heparin microarray experiments and surface plasmon resonance measurements on gold chips. Heparin-coated dendrimers bind FGF-2 significantly more effectively than monovalent heparin oligosaccharides. Dendrimer 1, which displays multiple copies of the sulfated hexasaccharide (GlcNSO(3)[6-OSO(3)]-IdoA[2-OSO(3)])3, was employed to promote FGF-2-mediated mitogen-activated kinase activation, demonstrating the utility of glycodendrimers to modulate heparin-protein interactions.

Characterization of the surface immobilized synthetic heparin binding domain derived from human fibroblast growth factor‐2 and its effect on osteoblast differentiation

Fibroblast growth factor (FGF)-2 regulates a variety of cellular functions, such as proliferation and differentiation, by binding to cell surface FGF receptors (FGFRs) in the presence of heparin proteoglycans. FGF-2 is known as a heparin-binding growth factor, but the localization of the heparin binding site has not been fully investigated until now. We used two potential heparin binding domains of FGF-2, the residues 105-111 (F105, YKRSRYT) and 119-135 (F119, KRTGQYKLGSKTGPGQK). Peptides could be stably immobilized onto the surface of tissue culture plates. Using solid phase binding assays, we demonstrated that both peptides had higher binding affinity toward heparin compared with nonbinding control sequence. The biological significance of these sites was tested by cell attachment and osteoblast differentiation studies. Cell attachment to the peptides F105 and F119 increased in a dose-dependent manner. Heparin and heparinase treatments decreased cell adhesion to both F105 and F119. This demonstrates that both F105 and F119 interact with cell-surface heparan sulfate proteoglycans, suggesting that FGF-2 has two heparin binding sites. In addition, osteoblast differentiation, confirmed by ALPase activity and mineralization, was increased by surface immobilized peptide F105 and F119. Taken together, these heparin binding peptides could be applied as biological agents enhancing osteoblast differentiation as well as surface modification tools in the tissue regeneration area, especially for bone regeneration.

1450: Double J Stent with Synthetic Heparin Coating does not Prevent Biofilm Deposit and Mineral Incrustation in Comparison with Standard Double J Stent

You have accessJournal of UrologyModerated Poster 44, Tuesday, May 22, 2007, 1:00 - 3:00 pm1 Apr 20071450: Double J Stent with Synthetic Heparin Coating does not Prevent Biofilm Deposit and Mineral Incrustation in Comparison with Standard Double J Stent Frédéric Thibault, Michel Daudon, Bernard Gattegno, and Olivier Traxer Frédéric ThibaultFrédéric Thibault More articles by this author , Michel DaudonMichel Daudon More articles by this author , Bernard GattegnoBernard Gattegno More articles by this author , and Olivier TraxerOlivier Traxer More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)31651-3AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1450: Double J Stent with Synthetic Heparin Coating does not Prevent Biofilm Deposit and Mineral Incrustation in Comparison with Standard Double J Stent." The Journal of Urology, 177(4S), p. 479 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 479 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Frédéric Thibault More articles by this author Michel Daudon More articles by this author Bernard Gattegno More articles by this author Olivier Traxer More articles by this author Expand All Advertisement PDF DownloadLoading ...

Preparation and Use of Microarrays Containing Synthetic Heparin Oligosaccharides for the Rapid Analysis of Heparin—Protein Interactions.

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Why differentiate low molecular weight heparins for venous thromboembolism?

Why differentiate low molecular weight heparins for venous thromboembolism?

Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.

Liquid Heparin Assay: A Straightforward Approach for Monitoring the Clinical Use of Heparins.

There are number of commercial heparins available in the market. The choice of heparin may depend on the clinical condition of the patient. For example, patients with deep-vein thrombosis (DVT), low molecular weight heparins (LMWH) (such as enoxaparin) may be used; whereas for pulmonary embolism (PE) cases, unfractionated heparins (UFH) may be the heparin of choice. In clinical studies, the synthetic heparin pentasaccharide, Fondaparinux (commercially known as Arixtra) has been shown to be efficacious in the treatment of both types of patients. Monitoring heparin activity in patients under treatment is critical in reducing the patient risk of bleeding or thromboembolism. The majority of current available tests allow measurement of heparins in the range of 0.0 – 1.0 U/mL. In many clinical laboratories, the analysis of each type of heparin requires a separate calibration curve, which makes the assay more complex and time consuming. In most cases these calibrators are “home made” and are not related to a standard, which may contribute to variability in testing. The reagents are usually provided in lyophilized form and require prior reconstitution which can introduce additional variation and increase handling time. An assay developed here, based on ready to use liquid reagents and designed for use on ACL TOP™, ACL Futura®/, ACL Advance™ ACL Elite™/ACL Elite Pro™ and ACL™ 9000/10000, addresses these issues and minimizes testing time. The Liquid Heparin Assay is built on the one stage principle of heparin detection, utilizing endogenous antithrombin III. The assay kit consists of two reagents, a liquid Factor Xa and a chromogenic substrate for Factor Xa, S2732. The composition of the kit and assay parameters are optimized to achieve overlay of the calibration curves for UFH and LMWH in the range from 0.0 to 2.0 U/mL of heparin activity. Furthermore, the assay is evaluated with respect to the various commercial heparins including Enoxaparin, Fragmin and Arixtra. The calibration curves for WHO UFH standard, WHO LMWH standard, Enoxaparin, and Fragmin are observed to overlap, with a maximum difference of 10% at each calibration point. The recoveries of heparin activities in samples spiked with various heparins are also within 10% of the expected values. The weight concentration (μg/mL) of Arixtra in plasma samples and its corresponding heparin activity (U/mL) can also be measured using this assay. The potential utility of the Universal Calibrator, composed of the mixture of UFH and LMWH is also evaluated and shown to be promising. Thus, the Liquid Heparin Assay permits simultaneous determination of various commonly used heparins in patient samples using a single calibration curve, and ready to use reagents. This can simplify analysis, reduce time and improve assay reliability. Since one assay is sufficient to measure different heparins, this assay could also reduce the overall costs of heparin analysis in the clinical laboratory.