Activity Of Heparin Research Articles

Structural and activity variability of fractions with different charge density and chain length from pharmaceutical heparins.

Heparin is a structurally complex polysaccharide used as a clinical anticoagulant. It is comprised of a heterogeneous mixture of polysaccharide chains having a variety of sequences and lengths. The production methods and regulatory controls of pharmaceutical heparins have changed over the years. This study assesses the structural and activity uniformity of the polysaccharide chains comprising two contemporary heparin products. The heparin fractions with different sizes and charges were separated with size exclusion and ion exchange chromatography. The fractions were analyzed for their molecular weight properties, di- and tetrasaccharide compositions, and anti-factor IIa and anti-factor-Xa activities. The distribution of these properties through chains of different lengths and ones with different charge density were compared. The results demonstrate that with the increase in heparin purity, activity and molecular weight required by the current pharmacopeia, the uniformity of pharmaceutical heparin products have increased.

Blood collection vials and clinically used intravenous fluids contain significant amounts of nitrite

The biology of the inorganic anion nitrite is linked to nitric oxide (NO) as nitrite can be reduced to NO and mediate its biological activities. Thus, studies of nitrite biology require sensitive and selective chemical assays. The acetic and ascorbic acids method is selective for nitrite and measures it in biological matrices. However, one of the pitfalls of nitrite measurements is its ubiquitous presence in sample collection tubes. Here, we showed high levels of nitrite in collection tubes containing EDTA, sodium citrate or sodium heparin and smaller amounts in tubes containing lithium heparin or serum clot activator. We also showed the presence of nitrite in colloid and crystalloid solutions frequently administered to patients and found variable levels of nitrite in 5% albumin, 0.9% sodium chloride, lactated ringer's, and dextrose-plus-sodium chloride solutions. These levels of nitrite varied across lots and manufacturers of the same type of fluid. Because these fluids are administered intravenously to patients (including those in shock), sometimes in large volumes (liters), it is possible that infusions of these nitrite-containing fluids may have clinical implications. A protocol for blood collection free of nitrite contamination was developed and used to examine nitrite levels in whole blood, red blood cells, plasma and urine from normal volunteers. Nitrite measurements were reproducible, had minimal variability, and did not indicate sex-differences. These findings validated a method and protocol for selective nitrite assay in biological fluids free of nitrite contamination which can be applied for study of diseases where dysfunctional NO signaling has been implicated.

Non-Anticoagulant Heparins Are Hepcidin Antagonists for the Treatment of Anemia.

The peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs), which are heparin binding proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases.

Novel method for measurement of heparin anticoagulant activity using SPR

A novel method has been developed for the easy measurement of heparin's anticoagulant activity using surface plasmon resonance. The anticoagulant activity of target heparin was evaluated by measuring the competitive antithrombin III binding of analyte heparin in the solution phase and USP heparin immobilized on chip surface. Heparins, obtained from different animal sources, and low molecular weight heparins were analyzed. The results were reproducible and correlated well with the results of chromogenic assays (correlation coefficient r = 0.98 for anti-Xa and r = 0.94 for anti-IIa). This protocol provides many advantages, significantly minimizing time, cost and the complications of chromogenic assay methods.

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA

AbstractAnticoagulant therapy–associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

Comparison of Enoxaparin Sodium and Standard Heparin for Hemodialysis Anticoagulation

Background: Low-molecular-weight heparin (LMWH) has been suggested as an effective and safe anticoagulant for hemodialysis. The aim of our study was to investigate the effects of LMWH including minor bleeding, clotting formation in the extracorporeal dialysis circuit and their effect on the lipid profile in comparison with heparin. Methods: This randomized, crossover study with parallel design was conducted in 45 patients who required maintenance hemodialysis due to end-stage renal failure. Four patients with known bleeding disorders, receiving anticoagulant drugs and receiving drugs which could affect heparin activity were excluded. All patients were randomly assigned to receive either enoxaparin sodium (0.7 mg/kg) or standard heparin for a duration of 12 weeks, after which patients were crossed over to another therapy for a further 12 weeks. Enoxaparin sodium was administered 5 minutes before dialysis, injected into the arterial line pre- dialyser and heparin was administered 50 U/kg intravenously into the pre- dialyser arterial line followed by a maintenance dose of 1000 U per hour. Results: The mean age of the study population was of 65.18 (SD = 12.15) years. From these patients 22 (53%) were male and 19 (47%) were female. At the end of the first study phase, minor bleeding in patients receiving enoxaparin with dose of 0.25 mg/kg was significantly decreased in comparison to the patients receiving heparin (P: 0.03), although vascular compression time did not significantly differ between the heparin and enoxaparin groups. At the end of the second study phase, the enoxaparin group showed a significant increase in minor bleeding in comparison with the heparin group (P: 0. 04). In the enoxaparin arm, recurrent blood oozing from puncture sites led to the idea to reduce the dose of enoxaparin. After enoxaparin dose reduction, the frequency of minor bleeding decreased to 10% (from 19% to 10%) (P: 0.01). Vascular compression time was not statistically different in heparin and enoxaparin at the end of study (P: 0.1). There were no significant changes in serum lipids with either anticoagulant neither at the end of the 12th week nor at the end of the 24th week. Conclusions: This study suggests that a single-dose of enoxaparin is an effective and convenient alternative to standard heparin. The recommended dose of this study in Iranian patients is 0.25 mg/kg.

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12)

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (KD = 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.

Evaluation of Unfractionated Heparin Dosing for Thromboprophylaxis using Anti-Xa Levels in Obese Critically Ill Patients: A Retrospective Study

Purpose: This study aims to evaluate whether current dosing recommendations of UFH for the prevention of thromboembolism is appropriate in obese critically ill patients utilizing Anti-Xa levels. Materials and methods: A pilot, observational and retrospective chart review of critically ill patients was evaluated. Patients aged ≥ 18 years were included if they received subcutaneous UFH for thromboprophylaxis and had a recorded Anti-Xa level taken four to five h after the administration of UFH. Identified patients were categorized into two groups: BMI<30 kg/m2 or BMI ≥ 30 kg/m2. Anti-Xa heparin activity levels were then compared. Statistical analysis was performed using Student’s t-test. Results: A total of 130 adult ICU patients were included in this study. For all critically ill patients receiving 5,000 units of subcutaneous heparin every 8 h, there was no statistically significant difference in Anti-Xa levels between the groups (p=0.287). There was no incidence of DVT/ PE reported from either group. Conclusion: This study suggests that there is no significant difference in Anti- Xa levels between obese critically ill patients and non-obese patients receiving traditional UFH dosing for thromboprophylaxis.

Comparative Studies on the Anticoagulant Actions of Recombinant Thrombomodulin and Heparin and Their Neutralization By FEIBA As Measured By Thromboelastography

INTRODUCTIONThromboelastographic (TEG) analysis represents a global approach to monitor the clotability of the native whole blood and is sensitive to the cellular and plasma components of the blood which affect the rate or structure of a thrombus or its breakdown. This method can be used for the evaluation of the point of care hemostatic status of whole blood. rTM (recombinant Thrombomodulin) is currently developed for various clinical indications and exerts multiple actions on blood and its components to reduce thrombogenesis without increasing a bleeding risk. This is in contrast to Heparins which mediate their actions accompanied by some bleeding risk. FEIBA (Factor VII Inhibitor Bypass Activity) contains non activated Factors II, IX and X and activated factor VII. It has been approved by the FDA for use in Hemophilia A and B patients with inhibitors for routine prophylaxis and to prevent or reduce the frequency of bleeding episodes. The purpose of this study is to compare the anticoagulant effects related to bleeding risk of rTM and Unfractionated Heparin (UH) at various levels and their neutralization by FEIBA. MATERIALS AND METHODSCitrated Whole Blood samples were supplemented with rTM or UH in a concentration range of 0-10ug/ml. (n=10). TEG analysis was performed on a TEG 5000 system in which the clotting was initiated by recalcification of the whole blood and set parameters as R time, K Time, MA and Angle were measured. The relative neutralization profiles of the UH and rTM by FEIBA at 1 and 0.1 U/ml were also investigated. All results were analyzed in terms of Mean Values ± Standard Deviation. RESULTSIn comparison to UH, rTM exhibited much weaker anticoagulant effects which were evident in all parametric evaluations.At 1ug/ml rTM did not produce any anticoagulant effects as evident by TEG profile. At higher levels greater than 2.5ug/ml, rTM produced a concentration dependent anticoagulant effect which altered the TEG profile of the whole blood. In contrast, UH produced relatively stronger anticoagulant effects and at a concentration greater than 2.5ug/ml, totally inhibited the clot formation in the TEG analysis. The anticoagulant effects of a 1ug/ml rTM as compared to 1ug/ml UH are tabulated in the following table.At higher concentrations, UH produced strong anticoagulation of whole blood whereas rTM at a concentration of 10ug/ml produced weaker effects. FEIBA at a concentration of 1U/ml completely neutralized the anticoagulant effect of rTM at 10ug/ml and UH at 0.5ug/ml. Furthermore, FEIBA at a concentration of 0.1U/ml neutralized the anticoagulant effects of rTM completely at a concentration of 10ug/ml. At this level, it only partially neutralized the anticoagulation effects of Heparin at 0.5ug/ml. The neutralization of anticoagulation effects of rTM at 10ug/ml and UH at 0.5ug/ml by FEIBA at 0.1U/ml are tabulated in the following table. CONCLUSIONThese studies suggest that rTM is a relatively weaker anticoagulant in comparison to UH. Furthermore, in the given indications, the expected concentration of rTM is in the range of 0.5-1.5ug/ml. At these concentrations, this agent is not expected to produce any anticoagulant effects. In contrast UH, at therapeutic concentrations of 1-2ug/ml produces, strong anticoagulant effect. While these TEG studies indicate that rTM is an antithrombotic agent mediating its effects via multiple mechanisms whereas heparins primarily produce anticoagulant effects. Moreover, the supratheraputic levels of thrombomodulin and heparin are neutralizable by FEIBA. [Display omitted] [Display omitted] DisclosuresTsuruta:Asahi Kasei Pharma America: Employment.

Aliphatic Polyethers with Sulfate, Carboxylate, and Hydroxyl Side Groups-Do They Show Anticoagulant Properties?

There is increasing interest in the synthesis of low molecular weight heparin and heparan sulfate mimetic polymers because of their various potential biomedical applications. The functional activity of heparin and heparan sulfate is believed to arise from the presence of a number of functional groups, such as hydroxyl, carboxylate and sulfate groups. The design and synthesis of novel heparin-mimetic polymers with a particular functionality poses a formidable challenge and requires carefully control of the selective conversion of functional groups on the polymer chain. Here, this study describes a simple and efficient synthetic protocol for the preparation of heparin-mimetic linear polyglycidol copolymers based on the selective conversion of primary hydroxyl groups to carboxylic acids under ruthenium-catalyzed selective dehydrogenation in basic aqueous solution. To achieve the anticoagulant activity of these polymers, primary hydroxyl groups are selectively converted to sulfate groups. The anticoagulant activity of the heparin mimics is studied by rotational thromboelastometry using EXTEM and INTEM assays. The environmentally benign process described herein provides an attractive route for the synthesis of heparin-mimetic polymers with tailored functions such as anticoagulant activity.

The role of heparins and nano-heparins as therapeutic tool in breast cancer.

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.

Controlled Radical Polymerization as an Enabling Approach for the Next Generation of Protein-Polymer Conjugates.

Protein-polymer conjugates are unique constructs that combine the chemical properties of a synthetic polymer chain with the biological properties of a biomacromolecule. This often leads to improved stabilities, solubilities, and in vivo half-lives of the resulting conjugates, and expands the range of applications for the proteins. However, early chemical methods for protein-polymer conjugation often required multiple polymer modifications, which were tedious and low yielding. To solve these issues, work in our laboratory has focused on the development of controlled radical polymerization (CRP) techniques to improve synthesis of protein-polymer conjugates. Initial efforts focused on the one-step syntheses of protein-reactive polymers through the use of functionalized initiators and chain transfer agents. A variety of functional groups such as maleimide and pyridyl disulfide could be installed with high end-group retention, which could then react with protein functional groups through mild and biocompatible chemistries. While this grafting to method represented a significant advance in conjugation technique, purification and steric hindrance between large biomacromolecules and polymer chains often led to low conjugation yields. Therefore, a grafting from approach was developed, wherein a polymer chain is grown from an initiating site on a functionalized protein. These conjugates have demonstrated improved homogeneity, characterization, and easier purification, while maintaining protein activity. Much of this early work utilizing CRP techniques focused on polymers made up of biocompatible but nonfunctional monomer units, often containing oligoethylene glycol meth(acrylate) or N-isopropylacrylamide. These branched polymers have significant advantages compared to the historically used linear poly(ethylene glycols) including decreased viscosities and thermally responsive behavior, respectively. Recently, we were motivated to use CRP techniques to develop polymers with rationally designed and functional biological properties for conjugate preparation. Specifically, two families of saccharide-inspired polymers were developed for stabilization and activation of therapeutic biomolecules. A series of polymers with trehalose side-chains and vinyl backbones were prepared and used to stabilize proteins against heat and lyophilization stress as both conjugates and additives. These materials, which combine properties of osmolytes with nonionic surfactants, have significant potential for in vivo therapeutic use. Additionally, polymers that mimic the structure of the naturally occurring polysaccharide heparin were prepared. These polymers contained negatively charged sulfonate groups and imparted stabilization to a heparin-binding growth factor after conjugation. A screen of other sulfonated polymers led to the development of a polymer with improved heparin mimesis, enhancing both stability and activity of the protein to which it was attached. Chemical improvements over the past decade have enabled the preparation of a diverse set of protein-polymer conjugates by controlled polymerization techniques. Now, the field should thoroughly explore and expand both the range of polymer structures and also the applications available to protein-polymer conjugates. As we move beyond medicine toward broader applications, increased collaboration and interdisciplinary work will result in the further development of this exciting field.

Catheter Closure Through a Venous Approach of Patent Ductus Arteriosus in Small Pediatric Patients Using Combined Angiographic and Echocardiographic Guidance

The standard technique of catheter closure of patent ductus arteriosus (PDA) may be associated with arterial complications particularly in small pediatric patients. The aim of this study was to evaluate whether catheter closure of PDA in small children using an exclusive venous approach is a safe and effective alternative to closure with the standard technique. One hundred-twelve patients, aged 2 to 24months, were randomly assigned in a 1:1 ratio to catheter closure of PDA using the standard technique (group 1) and an exclusive venous approach (group 2), respectively. In group 2, the procedure was guided using hand injections of contrast media through the delivery sheath and 2-dimensional and color Doppler echocardiography. Group 1: the PDA diameter ranged from 2 to 5.5mm and the device diameter ranged from 4 to 8mm. The PDA occluders were permanently implanted in all patients. Five losses of the arterial pulses that were restored with intravenous infusion of heparin and recombinant tissue plasminogen activator (rtPA), and 4 groin hematomas were the main complications of the procedure. Group 2: the mean PDA diameter ranged from 2.5 to 6mm and the device diameter ranged from 3 to 8mm. The PDA occluders were permanently implanted in all but 2 patients. There were no complications. Complete echocardiographic closure of PDA at 1-month follow-up was observed in all 110 patients. Exclusive transvenous PDA occlusion is an effective and safe technique that prevents the arterial complications of the standard approach in small children.

Analytical comparison of a US generic enoxaparin with the originator product: The focus on comparative assessment of antithrombin-binding components

Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.

Neutralization of anticoagulant activity of heparin by N-[(2-hydroxy-3-trimethylammonium) propyl] chloride derivatives of chitosan

Alkylated derivatives of low molecular weight chitosan with different substitution degrees of 98, 40, and 9% (I, II, and III respectively) have been synthesized. The structure of the obtained derivatives was defined by spectral assays (IR-spectroscopy and proton magnetic resonance). Chitosan derivatives were characterized with positive zeta-potential (33–51 mV) and solubility from 2 to 100 mg/mL in pH 7.4 and 25°C. It was shown that, at a concentration of 0.0014–0.0029 mg/mL, derivative I, as well as protamine sulfate, could be used to neutralize the anticoagulant activity of unfractionated or low molecular weight heparin. At a concentration of 0.0029–0.58 mg/mL, derivative I enhanced platelet aggregation, which would be necessary when hemostatic compounds or materials were used. Derivatives II and III enhanced platelet aggregation to a lesser extent.

Heparin centenary – an ever-young life-saving drug

On the centenary of the discovery of heparin, the International Journal of Cardiology agreed to publish a collection of mini reviews that summarize the historical development of this ever-young life-saving drug. The present articles deal not only with the historical milestones, but also with current and future perspectives regarding the development of heparin in terms of its structure, as well as on-going biochemical, biological and clinical research. Attention is focused on recent applications of heparin derivatives to non-anticoagulant or antithrombotic therapies, providing particular emphasis on their inhibitory activities, including their potential as anti-cancer agents. In the Chapter, entitled ‘Recent innovations in the structural analysis of heparin’, some recent technological advances are described for the problem of monitoring the purity and reproducibility of pharmaceutical heparin. These now permit sensitive detection of non-heparin impurities, as well as the detection of heparin from different animal sources, to be made in pharmaceutical heparin samples. In ‘Past, present, and future perspectives of heparin in clinical settings and the role of impaired renal function’, the author traces the history of heparin and the development of low molecular weight heparin, highlighting the large number of clinical trials in which it has been involved, and reviewing its efficacy among patients with impaired renal function. In the final chapter, ‘Old and new applications of non-anticoagulant heparin’, the authors survey some of the many non-anticoagulant activities of heparin and its derivatives, including glycol-split heparin, which has demonstrated promising activities in a wide-range of situations.

Past, present, and future perspectives of heparins in clinical settings and the role of impaired renal function

Heparin, whose discovery goes back one hundred years, was first detected as a thromboplastin from liver tissue, and its anticoagulant action was only identified later. The procoagulant action of heparin, which was later characterized as an immunologic reaction by binding to platelet-factor IV, presenting as heparin-induced thrombocytopenia, remains as a side effect. For more than 60 years heparin has been the immediate anticoagulant of choice in many clinical indications. Further development of heparins resulted in the production of low-molecular weight heparins and Fondaparinux, which substituted heparin for many indications and has received many more new indications, including administration for non-anticoagulant purposes. This development is still ongoing and has resulted in more than 300 registered clinical trials at the end of 2015. All types of heparins are still investigated in patients with impairment of renal function to improve the safety of treatment. New therapeutic strategies for the prevention and treatment of thromboembolism, as well as of the non-anticoagulant actions of natural and modified types of heparins, are studied intensively. The clinical study designs include treatment with vitamin-K and non-vitamin K oral anticoagulants. Consequently, heparins, low-molecular weight heparins and Fondaparinux play an important role in the human health care system.

Structural characterization and anti-inflammatory activity of two novel polysaccharides from the sea squirt, Ascidiella aspersa

It is now recognized that certain polysaccharides can exhibit anti-inflammatory activity, including the glycosaminoglycan (GAG) heparin that is widely used as an anti-coagulant drug. However, it would be desirable to identify molecules that retain the anti-inflammatory actions of heparin, but that are devoid of significant anti-coagulant activity. In the present study we have identified a number of novel GAG and GAG-like polysaccharides (VRP327) from marine organisms, most of which were resistant to digestion by heparinase II and chondroitinase ABC. Fourier transform infra-red spectrum (FTIR) revealed species with variable degrees of sulphation and monosaccharide analysis revealed a range of sugar compounds, which in some cases included sugars not present in mammalian GAGs. 1H NMR spectra of these species are consistent with the structures of complex polysaccharides. From an initial screening cascade to remove compounds having significant anti-coagulant activity and no overt cytotoxicity, we identified a high molecular weight oversulphated dermatan sulphate (VRP327) isolated from the tunicate Ascidiella aspersa which was fully characterised by NMR spectroscopy. This material was depolymerised to produce well characterized low molecular weight fractions which were demonstrated to be non-toxic, with low levels of anti-coagulant activity, and to have demonstrable anti-inflammatory activity assessed in several in vitro and in vivo models. The identification of low molecular weight polysaccharides having significant anti-inflammatory activity without significant anti-coagulant activity may provide novel templates for the development of a novel class of anti-inflammatory drugs.

Specific inhibition of secreted NRG1 types I–II by heparin enhances Schwann Cell myelination

Primary cultures of mixed neuron and Schwann cells prepared from dorsal root ganglia (DRG) are extensively used as a model to study myelination. These dissociated DRG cultures have the particular advantage of bypassing the difficulty in purifying mouse Schwann cells, which is often required when using mutant mice. However, the drawback of this experimental system is that it yields low amounts of myelin. Here we report a simple and efficient method to enhance myelination in vitro. We show that the addition of heparin or low molecular weight heparin to mixed DRG cultures markedly increases Schwann cells myelination. The myelin promoting activity of heparin results from specific inhibition of the soluble immunoglobulin (Ig)-containing isoforms of neuregulin 1 (i.e., NRG1 types I and II) that negatively regulates myelination. Heparin supplement provides a robust and reproducible method to increase myelination in a simple and commonly used culture system. GLIA 2016;64:1227-1234.

Abstract 117: Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies

Objectives: Anastomotic neo-intimal hyperplasia (ANIH) remains a limiting factor in the long-term success of prosthetic vascular grafts. Understanding of the molecular mechanisms involved in the host response to prosthetic material has set the stage for using this material to deliver siRNA and other modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. To create practical and effective drug delivery from a prosthetic material, we have combined a platform of polyethylene terephthalate (PET) fabric with an applied cryogel carrying the biologic agents, resulting in a bioactive prosthetic graft system (BPGS). Methods: Hybrid grafts were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun, knitted, or woven PET. Arg-Gly-Asp peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Human aortic endothelial cell (HAoEC) adherence and proliferation were assessed with a resazurin-based assay, and confocal microscopy was used to visualize cell interaction with the graft material. Heparin activity of the material in buffer solution was measured using an anti-Xa assay. Results: SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. HAoEC incorporated, adhered, and proliferated over 7 days for all materials (Figure 1). Anti-Xa assay confirmed continued activity of heparin from all grafts over 7 days. Conclusion: We have created a BPGS with a biocompatible cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity. This system allows application of the gel and biologic agents to PET prior to implantation, and provides flexibility in combining bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving strategies.