Oversulfated Chondroitin Sulfate Research Articles

Influence of Anticoagulants and Heparin Contaminants on the Suitability of MMP-9 as a Blood-Derived Biomarker.

In contrast to other common anticoagulants such as citrate and low-molecular-weight heparin (LMWH), high-molecular-weight heparin (HMWH) induces the expression of matrix metalloproteinase (MMP)-9, which is also measured as a biomarker for stroke in blood samples. Mechanistically, HMWH-stimulated T cells produce cytokines that induce monocytic MMP-9 expression. Here, the influence of further anticoagulants (Fondaparinux, Hirudin, and Alteplase) and the heparin-contaminating glycosaminoglycans (GAG) hyaluronic acid (HA), dermatan sulfate (DS), chondroitin sulfate (CS), and over-sulfated CS (OSCS) on MMP-9 was analyzed to assess its suitability as a biomarker under various conditions. Therefore, starved Jurkat T cells were stimulated with anticoagulants/contaminants. Subsequently, starved monocytic THP-1 cells were incubated with the conditioned Jurkat supernatant, and MMP-9 mRNA levels were monitored (quantitative (q)PCR). Jurkat-derived mediators secreted in response to anticoagulants/contaminants were also assessed (proteome profiler array). The supernatants of HMWH-, Hirudin-, CS-, and OSCS-treated Jurkat cells comprised combinations of activating mediators and led to a significant (in the case of OSCS, dramatic) MMP-9 induction in THP-1. HA induced MMP-9 only in high concentrations, while LMWH, Fondaparinux, Alteplase, and DS had no effect. This indicates that depending on molecular weight and charge (but independent of anticoagulant activity), anticoagulants/contaminants provoke the expression of T-cell-derived cytokines/chemokines that induce monocytic MMP-9 expression, thus potentially impairing the diagnostic validity of MMP-9.

A turn-on fluorescence sensor for detection of heparinase with heparin templated aggregation of tetracationic porphyrin derivative

Heparinase is the only mammalian endoglycosidase that breaks down the commonly used blood-anticoagulant heparin into therapeutically relevant low-molecular-weight-heparin. Importantly, heparinase has been considered a malignant disease diagnostic marker. Thus, it is essential to develop detection scheme for heparinase. However, optical methods for heparinase determination are limited. In the present work, we report a turn-on fluorescence sensor for detection of heparinase that utilizes heparin-templated aggregation of a tetra-cationic porphyrin derivative, TMPyP4+, as a sensing framework. Heparinase cleaves the glycosidic linkage between hexosamine and uronic acid in the structure of heparin to destroy its polyelectrolytic nature that originally causes the aggregation of TMPyP4+. Thus, heparinase leads to dissociation of TMPyP4+ aggregates and generates an optical signal. This system leads to a sensitive and selective response towards heparinase with a Limit of Detection (LOD) of 0.3 pmol/L. Further, the same system is demonstrated to sense a trace amount of Oversulfated Chondrootin Sulphate (OSCS) in heparin, which is a heparin adulterant, by utilizing the fact that OSCS serves as an inhibitor for heparinase activity, which leads to reverse modulation in the photo-physical features of the monomer/aggregate equilibrium of the TMPyP4+-heparin-heparinase system. The sensing mechanism has been thoroughly demonstrated by ground-state absorption, steady-state emission, and time-resolved emission measurements. The selectivity of the sensor was tested using lysozyme, α-amylase, pepsin, trypsin, lipase, and glucose oxidase in the heparinase selectivity study and the method is also validated using another method reported in the literature. The study provides a new approach for the development of optical methods for the detection of heparinase and oversulfated chondroitin sulfate, which is currently limited.

Analysis of Heparin Samples by Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy in the Solid State.

Heparin is a polydisperse, heterogeneous polysaccharide of the glycosaminoglycan (GAG) class that has found widespread clinical use as a potent anticoagulant and is classified as an essential medicine by the World Health Organization. The importance of rigorous monitoring and quality control of pharmaceutical heparin was highlighted in 2008, when the existing regulatory procedures failed to identify a life-threatening adulteration of pharmaceutical heparin with oversulfated chondroitin sulfate (OSCS). The subsequent contamination crisis resulted in the exploration of alternative approaches for which the use of multidimensional nuclear magnetic resonance (NMR) spectroscopy techniques and multivariate analysis emerged as the gold standard. This procedure is, however, technically demanding and requires access to expensive equipment. An alternative approach, utilizing attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) combined with multivariate analysis, has been developed. The method described enables the differentiation of diverse GAG samples, the classification of samples of distinct species provenance, and the detection of both established heparin contaminants and alien polysaccharides. This methodology has sensitivity comparable to that of NMR and can facilitate the rapid, cost-effective monitoring and analysis of pharmaceutical heparin. It is therefore suitable for future deployment throughout the supply chain.

Recent developments in sensing of oversulfated chondroitin sulfate in heparin. A review.

Oversulfated chondroitin sulfate (OSCS), a non-natural sulfated glycosaminoglycan, recognizes as a significant containment in the pharmaceutical heparin, and it could trigger adverse reactions. Chromatography-, electrophoresis-, electrochemistry-, and spectroscopy-related techniques are currently available for accurate and precise analysis of a trace amount of OSCS in heparin. Recently, emerging studies focus on developing colorimetric and fluorescent probes to monitor OSCS containments in heparin. Therefore, this current review aims to describe the sensing principle and procedure of the reported probes that are sensitive and selective toward OSCS in heparin without the interferences of other sulfated glycosaminoglycans. The reported OSCS-specific probes are comprehensively discussed according to the recognition elements of OSCS, including coralyne, AG73 peptides, positively charged tetraphenylethene derivatives, polythiophene polymer, and poly-L-lysine, protamine, superpositively charged green fluorescent proteins, and poly (diallyldimethylammonium chloride). The sensing of OSCS in heparin is generally achieved using, (i) the specific affinity of the recognition element with OSCS and heparin, (ii) heparinase-mediated hydrolysis of heparin, and (iii) OSCS-induced inhibition of heparinase activity. Additionally, coralyne-based DNA probes can detect OSCS in heparin in the presence of Ca2+ ions without the assistance of heparinase. This review will pave the way to design another sensing probe towards other sulfated contaminants, like dermatan sulfate.

(Plenary) Electrochemical/Optical Sensors in Medicine: Meeting Needs for the 21st Century

Over the past 40 years, miniaturized potentiometric and amperometric sensors for ions (K+, Ca++, Na+, Mg++, Cl-, H+), gases (O2 and CO2), and nutrients/metabolites (glucose, lactate, creatinine, urea) have revolutionized the practice of critical care medicine by providing tools to measure an array of these physiologically important species, simultaneously, in small volumes of undiluted whole blood. Indeed, all modern point-of-care whole blood analyzers used in hospitals worldwide now employ electrochemical sensor arrays as either single-use or multi-use devices for near-patient testing, especially in operating rooms, emergency rooms, intensive care units, etc. Some of the same or similar chemistries have also been adapted to create either single-use or continuous monitoring optical sensors (fluorescence, etc.). Further, nearly all blood glucometers as well as newer implantable subcutaneous glucose sensors now use electrochemical measurement principles to provide accurate glucose concentrations for millions of diabetic patients each and every day. A brief overview of these existing electrochemical/optical sensor technologies that have already had such a great impact in medicine will be provided during the introductory portion of this lecture.At the same time, there remain a number of unmet needs in medicine where electrochemical/optical chemical sensing devices could still play important analytical roles. Therefore, in the major portion of this presentation, the following research projects ongoing in our laboratories at the University of Michigan will be highlighted: 1) recent efforts to utilize electrochemical sensors for measurement of polyionic drugs and associated contaminants (including the anticoagulant heparin, low-molecular weight heparin, and inflammatory over-sulfated chondroitin sulfate (OSCS) contaminants in biomedical heparin preparations);1-2 2) research aimed at utilizing the same chemistry employed to make ion and polyion selective electrochemical sensors to create optical sensing films as well as optical sensing microfluidic devices that are able to quantitate species in volumes of << 1 µL;3 3) research related to the development of implantable electrochemical sensors for ions, gases, glucose, lactate, etc. that emit low levels of nitric oxide (NO) (a potent anti-platelet and antimicrobial agent) and that can potentially be used to continuously monitor critical care species intravenously in ICU patients with improved accuracy (see Fig. 1);4 and 4) efforts to use improved electrochemical/amperometric gas phase sensors for detecting nitric oxide (NO) in exhaled nasal/oral breath and also the use of NO and nitrogen dioxide (NO2) sensors to monitor and feedback control the levels of therapeutic gas phase NO being generated by novel photochemical and electrochemical gas phase generators.5-6 These gas phase NO generators can potentially replace use of very high cost compressed gas cylinders containing NO to provide controlled levels of NO gas on-demand for inhalation therapy of patients with pulmonary hypertension (including infants) and for use in the sweep gas of oxygenators to prevent clotting and Systemic Inflammatory Response Syndrome (SIRS) in patients undergoing open heart surgery.Literature Cited: L. Wang, S. Buchanan and M. E. Meyerhoff, “Rapid Detection of High Charge Density Polyanion Contaminants in Biomedical Heparin Preparations Using Potentiometric Polyanion Sensors,” Anal. Chem., 80, 9845-9847 (2008).K. Gemene and M. E. Meyerhoff, “Reversible Detection of Heparin and Other Polyanions by Pulsed Chronopotentiometric Polymer Membrane Electrode,” Anal. Chem., 82, 1612-1615 (2010).X. Wang, M. Sun, S. A. Ferguson, J. D. Hoff, Y. Qin, R. C. Bailey, and M. E. Meyerhoff, “Ionophore-Based Biphasic Chemical Sensing in Droplet Microfluidics,” Angew. Chem. Int. Ed., 58, 1-6 (2019).M. Frost and M. E. Meyerhoff, “Real-Time Monitoring of Critical Care Analytes in the Bloodstream with Chemical Sensors: Progress and Challenges,” Ann. Rev. Anal. Chem., 8, 171-192 (2015).J. Zajda, N. J. Schmidt, Z. Zheng, X. Wang and M. E. Meyerhoff, “Evaluation of Amperometric Platinized-Nafion-Based Gas Phase Sensor for Determining Nitric Oxide (NO) Levels in Exhaled Human Nasal Breath,” Electroanalysis 30, 1602-1607 (2018).Y. Qin, J. Zajda, H. Ren, J. Toomasian, T. Major, A. Rojas-Pena, B. Carr, T. Johnson , J. Haft, R. H. Bartlett, A. Hunt, N. Lehnert, and M. E. Meyerhoff, “Low Cost Portable Gas Phase Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Within the Sweep Gas/Cardiotomy Suction Air During Cardiopulmonary Bypass Surgery,” Mol. Pharmaceutics, 14, 3762-3771 (2017). Figure 1. Comparison of average error of PO2 levels using amperometric IV-oxygen sensing catheters with (n=4) and without (n=4) NO release chemistry placed into the arteries of pigs for 20 h. % deviation calculated vs. measurements made on discrete whole blood samples (heparinized syringe) on Radiometer blood-gas/electrolyte analyzer. Figure 1

Techniques for Detection of Clinical Used Heparins.

Heparins and sulfated polysaccharides have been recognized as effective clinical anticoagulants for several decades. Heparins exhibit heterogeneity depending on the sources. Meanwhile, the adverse effect in the clinical uses and the adulteration of oversulfated chondroitin sulfate (OSCS) in heparins develop additional attention to analyze the purity of heparins. This review starts with the description of the classification, anticoagulant mechanism, clinical application of heparins and focuses on the existing methods of heparin analysis and detection including traditional detection methods, as well as new methods using fluorescence or gold nanomaterials as probes. The in-depth understanding of these techniques for the analysis of heparins will lay a foundation for the further development of novel methods for the detection of heparins.

Preparation and Application of Nanosensor in Safeguarding Heparin Supply Chain

Heparin has been in clinical use as an anticoagulant for the last eight decades and used worldwide in more than 100 million medical procedures every year. This lifesaving drug is predominantly obtained from ~700 million pig intestines or bovine organs through millions of small and medium-sized slaughterhouses. However, the preparations from animal sources have raised many safety concerns, including the contamination of heparin with potential pathogens, proteins, and other impurities. In fact, contaminated heparin preparations caused 149 deaths in several countries, including the United States, Germany, and Japan in 2008, highlighting the need for implementing sensitive and simple analytical techniques to monitor and safeguard the heparin supply chain. The contaminant responsible for the adverse effects in 2008 was identified as oversulfated chondroitin sulfate (OSCS). We have developed a very sensitive, facile method of detecting OSCS in heparin lots using a nanosensor, a gold nanoparticle-heparin dye conjugate. The sensor is an excellent substrate for heparitinase enzyme, which cleaves the heparin polymer into smaller disaccharide fragments, and therefore facilitates recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, because OSCS inhibits the enzyme. The newly designed nanosensor can detect as low as 1 × 10-9% (w/w) OSCS, making it the most sensitive tool available to date for the detection of trace amounts of OSCS in pharmaceutical heparins. In this report, we describe a simple methodology for the preparation of nanosensor and its application in the detection of OSCS contaminants.

(Plenary) Electrochemical/Optical Sensors in Medicine: Meeting Needs for the 21st Century

Over the past 40 years, miniaturized potentiometric and amperometric sensors for ions (K+, Ca++, Na+, Mg++, Cl-, H+), gases (O2 and CO2), and nutrients/metabolites (glucose, lactate, creatinine, urea) have revolutionized the practice of critical care medicine by providing tools to measure an array of these physiologically important species, simultaneously, in small volumes of undiluted whole blood. Indeed, all modern point-of-care whole blood analyzers used in hospitals worldwide now employ electrochemical sensor arrays as either single-use or multi-use devices for near-patient testing, especially in operating rooms, emergency rooms, intensive care units, etc. Some of the same or similar chemistries have also been adapted to create either single-use or continuous monitoring optical sensors (fluorescence, etc.). Further, nearly all blood glucometers as well as newer implantable subcutaneous glucose sensors now use electrochemical measurement principles to provide accurate glucose concentrations for millions of diabetic patients each and every day. A brief overview of these existing electrochemical/optical sensor technologies that have already had such a great impact in medicine will be provided during the introductory portion of this lecture.At the same time, there remain a number of unmet needs in medicine where electrochemical/optical chemical sensing devices could still play important analytical roles. Therefore, in the major portion of this presentation, the following research projects ongoing in our laboratories at the University of Michigan will be highlighted: 1) recent efforts to utilize electrochemical sensors for measurement of polyionic drugs and associated contaminants (including the anticoagulant heparin, low-molecular weight heparin, and inflammatory over-sulfated chondroitin sulfate (OSCS) contaminants in biomedical heparin preparations);1-2 2) research aimed at utilizing the same chemistry employed to make ion and polyion selective electrochemical sensors to create optical sensing films as well as optical sensing microfluidic devices that are able to quantitate species in volumes of << 1 µL;3 3) research related to the development of implantable electrochemical sensors for ions, gases, glucose, lactate, etc. that emit low levels of nitric oxide (NO) (a potent anti-platelet and antimicrobial agent) and that can potentially be used to continuously monitor critical care species intravenously in ICU patients with improved accuracy (see Fig. 1);4 and 4) efforts to use improved electrochemical/amperometric gas phase sensors for detecting nitric oxide (NO) in exhaled nasal/oral breath and also the use of NO and nitrogen dioxide (NO2) sensors to monitor and feedback control the levels of therapeutic gas phase NO being generated by novel photochemical and electrochemical gas phase generators.5-6 These gas phase NO generators can potentially replace use of very high cost compressed gas cylinders containing NO to provide controlled levels of NO gas on-demand for inhalation therapy of patients with pulmonary hypertension (including infants) and for use in the sweep gas of oxygenators to prevent clotting and Systemic Inflammatory Response Syndrome (SIRS) in patients undergoing open heart surgery.Literature Cited: 1. L. Wang, S. Buchanan and M. E. Meyerhoff, “Rapid Detection of High Charge Density Polyanion Contaminants in Biomedical Heparin Preparations Using Potentiometric Polyanion Sensors,” Anal. Chem., 80, 9845-9847 (2008).2. K. Gemene and M. E. Meyerhoff, “Reversible Detection of Heparin and Other Polyanions by Pulsed Chronopotentiometric Polymer Membrane Electrode,” Anal. Chem., 82, 1612-1615 (2010).3. X. Wang, M. Sun, S. A. Ferguson, J. D. Hoff, Y. Qin, R. C. Bailey, and M. E. Meyerhoff, “Ionophore-Based Biphasic Chemical Sensing in Droplet Microfluidics,” Angew. Chem. Int. Ed., 58, 1-6 (2019).4. M. Frost and M. E. Meyerhoff, “Real-Time Monitoring of Critical Care Analytes in the Bloodstream with Chemical Sensors: Progress and Challenges,” Ann. Rev. Anal. Chem., 8, 171-192 (2015).5. J. Zajda, N. J. Schmidt, Z. Zheng, X. Wang and M. E. Meyerhoff, “Evaluation of Amperometric Platinized-Nafion-Based Gas Phase Sensor for Determining Nitric Oxide (NO) Levels in Exhaled Human Nasal Breath,” Electroanalysis 30, 1602-1607 (2018).6. Y. Qin, J. Zajda, H. Ren, J. Toomasian, T. Major, A. Rojas-Pena, B. Carr, T. Johnson , J. Haft, R. H. Bartlett, A. Hunt, N. Lehnert, and M. E. Meyerhoff, “Low Cost Portable Gas Phase Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Within the Sweep Gas/Cardiotomy Suction Air During Cardiopulmonary Bypass Surgery,” Mol. Pharmaceutics, 14, 3762-3771 (2017). Figure 1

Quantitative Analysis of Impurities in Unfractionated Heparin of Bovine Origin.

The USP heparin sodium monograph lists impurities with specifications developed for porcine derived products. Most of these impurities are of biological origin and are present in porcine intestinal mucosa, the tissue source used in the production of porcine heparin. One of the specified impurities, oversulfated chondroitin sulfate (OSCS), has been introduced in the monograph to detect intended adulteration of heparin products with this impurity. The evaluation of bovine intestinal heparin as an alternative source of pharmaceutical heparin included an evaluation of bovine heparin with the current USP heparin sodium monograph methods. This evaluation included a comparison of impurity quantities observed in multiple bovine intestinal heparin samples against the specifications found in the USP heparin sodium monograph. The impurities investigated in this study were protein, galactosamine, nucleotidic impurities, and OSCS. Bovine intestinal heparin met the requirements in the tests for protein, galactosamine, and nucleotidic impurities. A potential issue was observed with the strong anion exchange high performance liquid chromatography (SAX-HPLC) used to analyze for the presence of OSCS. While the OSCS was well-resolved from the bovine heparin peak, the resolution of dermatan sulfate from heparin did not consistently meet system suitability requirements in the current USP Heparin sodium monograph. The overall levels of impurities observed in bovine intestinal mucosal heparin were comparable to those observed in porcine intestinal mucosal heparin. Bovine intestinal mucosal heparin can be produced with acceptable impurity levels that align with these important quality attributes found in porcine heparin.

Mass spectrometric evidence for the mechanism of free-radical depolymerization of various types of glycosaminoglycans

Glycosaminoglycans (GAGs) are large, complex carbohydrate molecules that interact with a wide range of proteins involved in physiological and pathological processes. Several naturally derived GAGs have emerged as potentially useful therapeutics in clinical applications. Natural polysaccharides, however, generally have high molecular weights with a degree of polydispersity, making it difficult to investigate their structural properties. In this study, we establish a free-radical–mediated micro-reaction system and use hydrophilic interaction chromatography (HILIC)–Fourier transform mass spectrometry (FTMS) to profile the degraded products of various types of GAGs, heparin, chondroitin sulfate A, NS-heparosan, and oversulfated chondroitin sulfate (OSCS), to reveal the free-radical degradation mechanism of GAGs. The results show that the bulk fragments of GAGs generated by free-radical degradation can maintain their basic structural units and sulfate substituents. In addition, an abundance of oligomers modified with oxidation at their reducing ends or by dehydration also appeared. We discovered that these modifications were related in terms of the degree of sulfation and the α- or β-linkage of HexNY (Y = SO3− or Ac), and especially that the different linkage of the disaccharide unit is the main factor in modification. In addition, the method based on micro-free-radical reaction and HILIC-FTMS is both effective and sensitive, thus suggesting its broad practical value for the structural characterization and in the biological structure-function studies of GAGs.

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants.

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Galactosaminoglycans: Medical Applications and Drawbacks.

Galactosaminoglycans (GalAGs) are sulfated glycans composed of alternating N-acetylgalactosamine and uronic acid units. Uronic acid epimerization, sulfation patterns and fucosylation are modifications observed on these molecules. GalAGs have been extensively studied and exploited because of their multiple biomedical functions. Chondroitin sulfates (CSs), the main representative family of GalAGs, have been used in alternative therapy of joint pain/inflammation and osteoarthritis. The relatively novel fucosylated chondroitin sulfate (FCS), commonly found in sea cucumbers, has been screened in multiple systems in addition to its widely studied anticoagulant action. Biomedical properties of GalAGs are directly dependent on the sugar composition, presence or lack of fucose branches, as well as sulfation patterns. Although research interest in GalAGs has increased considerably over the three last decades, perhaps motivated by the parallel progress of glycomics, serious questions concerning the effectiveness and potential side effects of GalAGs have recently been raised. Doubts have centered particularly on the beneficial functions of CS-based therapeutic supplements and the potential harmful effects of FCS as similarly observed for oversulfated chondroitin sulfate, as a contaminant of heparin. Unexpected components were also detected in CS-based pharmaceutical preparations. This review therefore aims to offer a discussion on (1) the current and potential therapeutic applications of GalAGs, including those of unique features extracted from marine sources, and (2) the potential drawbacks of this class of molecules when applied to medicine.

The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

Dual Role of a Fluorescent Peptidyl Probe Based on Self-Assembly for the Detection of Heparin and for the Inhibition of the Heparin-Digestive Enzyme Reaction.

The detection of fluorescent probes for biomolecules and control of the function of a complex through a recognition process have not been investigated intensively. A fluorescent peptidyl probe (1) based on the self-assembly stimulated by heparin was synthesized. The fluorescent probe with an aggregation-induced emission fluorophore formed a self-assembling complex with heparin, resulting in a sensitive and selective turn-on response to heparin compared to its biological competitors. The detection limits for heparin were measured to be 138.0 pM (R2 = 0.976) in aqueous solution and 2.6 nM (R2 = 0.996) in aqueous solution containing human serum. Nanosized aggregates formed through the self-assembly of the complex showed potent resistance against the heparin-digestive enzyme. The dual role of the probe for the detection of heparin and the inhibition of heparinase-mediated digestion through the recognition process was used for the real-time monitoring of the enzyme activity of heparinase for the digestion of heparin. Furthermore, the dual role of the probe was applied for the detection of the oversulfated chondroitin sulfate contaminant in heparin.

Sulfated Hyaluronan Alters Endothelial Cell Activation in Vitro by Controlling the Biological Activity of the Angiogenic Factors Vascular Endothelial Growth Factor-A and Tissue Inhibitor of Metalloproteinase-3

Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer, or atherosclerosis are often associated with abnormal angiogenesis, which requires innovative biomaterial-based treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and oversulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis, and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.

Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe

The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R2=0.99) in aqueous solutions, 2.98nM (R2=0.98) in 1% serum samples, and 3.43nM (R2=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.

Modulating luminescence of Tb3+ with biomolecules for sensing heparin and its contaminant OSCS

The detection of heparin (Hep) and its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance in clinics but remains challenging. Here, we report a sensitive and selective time-resolved luminescence (TRL) biosensing system for Hep by modulating the photoluminescence of Tb3+ with guanine-rich ssDNA and Hep-specific AG73 peptide (RKRLQVQLSIRT). With the developed system, Hep including both unfractionated Hep (UFH) and the low molecular weight Hep (LMWH) has been successfully detected with a satisfactory detection limit. Owing to the highly specific interaction between Hep and AG73 peptide, major interfering substances in Hep detection, such as Hep analogs of chondrotin sulfate (Chs) and hyaluronic acid (HA), did not interfere with Hep detection. The established TRL sensing system was then successfully used for monitoring Hep metabolism in living rats by microdialysis. Moreover, the proposed TRL sensing system was further applied to analyze OSCS contaminant in Hep with heparinases treatment by exploring the inhibition effects of OSCS on the activity of heparinases. As low as 0.002% of OSCS in Hep was identified.

Bioengineering of Human Heparin: A Next Generation Anti‐coagulant

Heparin used clinically is manufactured by extraction from porcine and bovine tissues. Recently, contaminating oversulfated chondroitin sulfates found in batches of porcine heparin led to the deaths of approximately 100 people in the US, which was caused by an uncontrolled anaphylactoid‐type response. This has led researchers to explore alternative methods of producing heparin including chemical synthesis, chemoenzymatic synthesis and metabolic engineering of cells to express high levels of the enzymes involved in heparin biosynthesis. However, these approaches are yet to produce heparins with anticoagulant activity similar to heparin used in the clinic. The aim of this research was to recombinantly express serglycin in human cells as an alternative approach to producing anticoagulant heparin.The serglycin recombinantly expressed in human embryonic kidney (HEK‐293) cells was decorated with heparin, heparan sulfate and chondroitin sulfate. The disaccharide composition of the recombinantly expressed heparin was analysed by fluorophore assisted carbohydrate electrophoresis and contained 15% tri‐sulfated disaccharides as well as 40% di‐sulfated, 25% monosulfated and 20% unsulfated disaccharides. The disaccharide composition of low molecular weight heparin was also analysed and contained a higher proportion of sulfated disaccharides than the recombinantly expressed heparin being 40% trisulfated, 45% disulfated, 10% monosulfated and 5% unsulfated disaccharides.The activated partial clotting time assay is used clinically to monitor heparin levels in patients after its administration and was used in this study to determine the anticoagulant activity of the recombinant human heparin and to compare this to the activity of low molecular weight porcine heparin. In this assay porcine heparin was approximately 20 times more potent in its anticoagulant activity than the recombinant human heparin on a weight basis.These data demonstrate for the first time the successful production of human heparin from cells transfected with the human serglycin gene. This is a significant step towards alternative sources of heparin that has distinct advantages for patient safety due to the use of production methods with a lower inherent risk of contamination. Human cell‐derived heparin will also provide an alternative source to animal‐derived heparin that will enable its use by patients who, due to religious beliefs and/or dietary choices, have so far been excluded from heparin treatment.Support or Funding InformationFunding provided by the Australian Research Council.

In vitro and in vivo characterization of a reversible synthetic heparin analog

BackgroundThe global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. Objective/methodsWe sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. ResultsThe anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. ConclusionsThese data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile.

Reversible Electrochemical Sensor for Detection of High-Charge Density Polyanion Contaminants in Heparin.

We present a simple, rapid, and inexpensive electrochemical sensor based on a reversible pulsed chronopotentiometric polyanion-selective membrane electrode for the detection and quantification of oversulfated chondroitin sulfate (OSCS) and other high charge-density polyanions that could potentially be used to adulterate heparin. The membrane is free of ion exchanger and is formulated with plasticized poly(vinyl chloride) (PVC) and an inert lipophilic salt, tridodecylmethylammonium-dinonylnaphthaline sulfonate (TDMA-DNNS). The neutral salt is used to reduce membrane resistance and to ensure reversibility of the sensor. More importantly, TDMA(+) is used as the recognition element for the polyanions. Here an anodic galvanostatic current pulse is applied across the membrane to cause the extraction of the polyanions from the sample into the membrane and potential is measured at the sample-membrane interface. The measured electromotive force (emf) is proportional to the concentration and the charge density of the polyanions. High charge-density polyanion contaminants and impurities in heparin can be detected using this method since the overall equilibrium potential response of polyions increases with increasing charge density of the polyions. Here, first the potential response of pure heparin is measured at a saturation concentration, the concentration beyond which further addition of heparin does not produce a change in potential response. Then the potential response of heparin tainted with different quantities of the high charge-density contaminant is measured at a fixed total polyion concentration (heparin concentration + contaminant concentration). The latter gives a greater negative potential response due to the presence of the high charge-density contaminant. The increase in the negative potential response can be used for detection and quantification of high charge-density contaminants in heparin. We demonstrate here that 0.3% (w/w) OSCS as well as 0.1% (w/w) dextran sulfate can be detected in heparin at 20-mg/mL total polyion concentration. It has also been shown that 1% (w/w) of dextran sulfate can readily be detected in heparin at only 2-mg/mL total polyanion concentration with a linear response (R(2) = 0.994).