Heparin Fragments Research Articles

Anticoagulation reversal (vitamin K, prothrombin complex concentrates, idarucizumab, andexanet-α, protamine).

Bleeding events are common in patients prescribed anticoagulants and can have devastating consequences. Several specific and nonspecific agents have been developed to reverse the effects of anticoagulant drugs or toxins. Vitamin K, as the oldest of these antidotes, specifically counteracts the effects of pharmaceuticals and rodenticides designed to deplete stores of vitamin K-dependent factors. In cases of life-threatening bleeding, the addition of prothrombin complex concentrates (PCCs) allows for the immediate replacement of coagulation factors. While the use of PCCs has been extended to the non-specific reversal of the effects of newer direct oral anticoagulants, the specific agents idarucizumab, targeting dabigatran and andexanet-α, binding factor Xa inhibitors, have recently been developed and are being preferentially recommended by most guidelines. However, despite having rapid effects on correcting coagulopathy, there is to date a lack of robust evidence establishing the clear superiority of direct oral anticoagulant-specific reversal agents over PCCs in terms of haemostatic efficacy, safety or mortality. For andexanet-α, a potential signal of increased thromboembolic risks, comparatively high costs and low availability might also limit its use, even though emerging evidence appears to bolster its role in intracranial haemorrhage. Protamine is the specific agent for the reversal of unfractionated heparin anticoagulation used mainly in cardiovascular surgery. It is much less effective for low molecular weight heparin fragments and is usually reserved for cases with life-threatening bleeding.

A Unique Enoxaparin Derived from Bovine Intestinal Heparin: A Single Purification Step of the Starting Material Assures a Bovine Enoxaparin Like the Standard from Porcine Origin.

Low-molecular-weight heparin represent a significant advancement in anticoagulant therapy with enoxaparin being a prominent example obtained exclusively through the fragmentation of porcine intestinal heparin. However, escalating demand and limited resources have raised concerns about enoxaparin supplementation. The current challenge involves exploring alternative heparin sources for large-scale enoxaparin production with bovine intestinal heparin emerging as a promising option. Our study demonstrates that enoxaparin derived from the available bovine heparin preparation differs significantly from the reference compound. Yet, the implementation of a straightforward purification step yields a preparation termed "high-anticoagulant bovine heparin". Fragmentation of this purified product through β-elimination produces enoxaparin akin to the standard from a porcine origin. To ensure physicochemical similarity, we employed various spectroscopic, enzymatic, and chromatographic tests to compare the new bovine-derived enoxaparin with the original porcine compound. Biological activity was confirmed through in vitro coagulation assays and assessments using an animal model of venous thrombosis. Our study affirms that the β-elimination reaction cleaves the bovine heparin chain without preferential breaks in regions with different sulfation patterns. Additionally, we scrutinized decasaccharides purified from enoxaparin preparations, providing a comprehensive demonstration of the similarity between products obtained from porcine and bovine heparin. In summary, our findings indicate that an enoxaparin equivalent to the original porcine-derived product can be derived from bovine heparin, given that the starting material undergoes a simple purification step.

Structural insights into the formation of repulsive netrin guidance complexes.

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding-deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex.

Abstract P2136: TpTe Interval Behavior And Mortality In Wistar Rats Submitted To Cardiac Ischemia And Reperfusion Treated With The Heparin Fragment Trisulfated Disaccharide

Introduction: The Tp-Te ekg interval reflects ventricular transmural dispersion, being used for prediction of ventricular arrhythmia and sudden death. The heparin fragment called Trisulfated Disaccharide (TD), which has no effect on blood coagulation and acts on the sodium-calcium exchanger accelerating the output of calcium from the cell, decreases cytoplasmic calcium concentrations in situations of calcium overload due to ischemia and reperfusion (I-R). Objective: To evaluate the behavior of the Tp-Te interval and mortality due to cardiac arest in Wistar rats submitted to surgicaly induced I-R under treatment with TD at reperfusion. Materials and Method: Wistar rats (n=8) with average weight of 300g were anesthetized and under assisted breathing were electrocardiographically monitored and submitted to surgical cardiac ischemia by ligation of the anterior descending artery for 10 min, with reperfusion thereafter. The Tp-Te interval was analyzed before ischemia, at 10 min after ischemia induction and at 10 min after reperfusion, The incidence of arrhythmias was determined throughout the recording period. In the study group intravenous (0.2 mg/kg) TD was injected concomitantly with the reperfusion. In the control group (n=8), the same procedure was performed, without the TD infusion. Results: During ischemia there was a similar alteration of the Tp-Te interval in both groups when compared to the pre-ischemic phase, with an increase of about 90% in magnitude, as well as the presence of ventricular arrhythmias, without animal mortality. During reperfusion Tp-Te interval returned to normal in the treated group and there was no mortality. In the control group, the magnitude of the Tp-Te increase was maintained. The presence of ventricular arrhythmias was similar, but total atrioventricular block and/or sustained arrhythmia occurred in 50% of the animals, followed by animals death. Conclusion: Treatment with TD at the time of reperfusion normalized the Tp-Te interval 10 min after TD infusion as well as reduced mortality in wistar rats submitted to I-R, and may be useful in the protection of organs submitted to the same I-R conditions (transplants, surgical and hemodynamic procedures), where calcium overload is determinant in morbidity and mortality.

Abstract P1095: Analysis Of Pro-arrhythmic Effects Of The Trisulfated Disaccharide Heparin Fragment, A Potential Antiarrhythmic Drug.

Background: The heparin fragment called trisulfated disaccharide (TD) acts on the sodium-calcium exchanger by removing excess calcium from cytoplasm with antiarrhythmic action demonstrated in animal models in the reversal of supraventricular and ventricular arrhythmia. However, it is known that drugs with antiarrhythmic potential can induce arrhythmic phenomena, limiting and interrupting the study of new potentially antiarrhythmic drugs. Objective: To evaluate the arrhythmogenic potential of TD in relation to the stimulation of hERG channels in human cardiomyocytes. Material and Methods: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in culture were plated onto a fibronectin coated 48 well microelectrode array plate at a density of 25,000 cells per well in iCell® maintenance medium. The plate was incubated for 7 days until a stable beating phenotype was achieved, and the cells were ready for drug treatment. They were dosed with 0.2% DMSO vehicle, E-4031 (0.05μM), or cisapride (0.05μM) as controls (arrhythmogenic drugs). They were also dosed with TD at four concentrations in triplicate. The activity of the cells was recorded prior to dosing and at 1 hour using the Axion Biosystems Maestro MEA system. Beat period (s), fast Na+ slope (V/s), fast Na+ amplitude (mV) and field potential duration (FPD) were evaluated. Results: E-4031 treatment resulted in a significant increase in beat period length (decrease in beat rate) and a decrease in the Na slope and amplitude. There was a very large increase in the FPD. Cisapride showed a very similar response. Both compounds caused an increase in the ratio of the FPD to the beat period length. When the cardiomyocytes were incubated with the different doses of TD, there was a slight increase in the beat period length (decrease in the beat rate), which was observed regarding all the doses. There was an increase in the FPD, which is consistent with the change in beat rates suggesting that the change is purely related to beating and not indicative of any hERG effect or other responses. The ratio of the FPD to the beat period length was the same. There was a slight decrease in the sodium slope and amplitude, but they were not significant. Therefore, the only significant change observed was the beat rate. Conclusion: TD when exposed to human cardiomyocytes did not present an arrhythmogenic potential in relation to the hERG channels, and this is a desirable behavior for an antiarrhythmic drug.

Heparanase Is a Putative Mediator of Endothelial Glycocalyx Damage in COVID-19 - A Proof-of-Concept Study.

Coronavirus disease 2019 (COVID-19) is a systemic disease associated with injury (thinning) of the endothelial glycocalyx (eGC), a protective layer on the vascular endothelium. The aim of this translational study was to investigate the role of the eGC-degrading enzyme heparanase (HPSE), which is known to play a central role in the destruction of the eGC in bacterial sepsis. Excess activity of HPSE in plasma from COVID-19 patients correlated with several markers of eGC damage and perfused boundary region (PBR, an inverse estimate of glycocalyx dimensions of vessels with a diameter 4-25 µm). In a series of translational experiments, we demonstrate that the changes in eGC thickness of cultured cells exposed to COVID-19 serum correlated closely with HPSE activity in concordant plasma samples (R = 0.82, P = 0.003). Inhibition of HPSE by a nonanticoagulant heparin fragment prevented eGC injury in response to COVID-19 serum, as shown by atomic force microscopy and immunofluorescence imaging. Our results suggest that the protective effect of heparin in COVID-19 may be due to an eGC-protective off-target effect.

Investigations of the effectiveness of heparin variants as inhibitors of histones

BackgroundExtracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models. ObjectivesTo compare the effectiveness of unfractionated heparin (UFH), low‐molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones MethodsBinding affinities between heparin variants and histone subunits were determined by Bio‐layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP‐1 cells with histones ± heparin variants. ResultsUnfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (Kd <1 pM–66.7 nM) whereas fondaparinux exhibited a low affinity (Kd of 3.06 µM–81.1 mM). UFH, LMWH, and Vasoflux neutralize histone‐mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone‐mediated impairment of APC generation in a dose‐dependent manner. ConclusionsThe ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin‐binding pentasaccharide. In contrast, the ability of heparin to neutralize histone‐mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones.

Chemoenzymatic Synthesis of D‐Glucaro‐δ‐lactam Containing Oligosaccharides as Putative Heparanase Inhibitors

AbstractHeparanase is an endo‐β‐glycosidase that cleaves heparan sulfate chains at the level of the glycosidic bond of selected glucuronic acid residues. Heparin fragments, that can compete with heparan sulfate for binding to heparanase, have been reported as weak heparanase inhibitors. To reinforce their inhibitory activity, a known β‐glucuronidase inhibitor has been introduced at their reducing end to directly interfere with the active site of the enzyme. We report here the synthesis of such oligosaccharides, displaying D‐glucaro‐δ‐lactam at their reducing end. Thanks to the trichloroacetimidate glycosylation method, we could devise experimental conditions allowing O‐glycosylation of the lactam ring, and we thus chemically prepared trisaccharides that were further elongated and sulfated using enzymatic methods. Heparanase inhibitory activity was not significantly improved by the presence of the D‐glucaro‐δ‐lactam ring.

Blockage of undesirable endocytosis of recombinant human growth/differentiation factor-5 in Chinese hamster ovary cell cultures requires heparin analogs with specific chain lengths.

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis lowers the yield of recombinant human bone morphogenetic proteins (rhBMPs), such as rhBMP-2 and rhBMP-4, from Chinese hamster ovary (CHO) cell cultures. Exogenous recombinant human growth/differentiation factor-5 (rhGDF-5), a member of the BMP family, bound to cell surface HSPGs and was actively internalized into CHO cells. Knockdown of heparan sulfate (HS) synthesis enzymes in CHO cells revealed that the chain length and N-sulfation of HS affected the binding of rhGDF-5 to HSPGs and subsequent rhGDF-5 internalization. To increase product yield by minimizing rhGDF-5 internalization in recombinant CHO (rCHO) cell cultures, heparin, and dextran sulfate (DS) of various polysaccharide chain lengths, which are structural analogs of HS, were examined for blockage of rhGDF-5 internalization. Heparin fragments of four monosaccharides (MW of 1.2kDa) and DS (MW of 15kDa) did not inhibit rhGDF-5 internalization whereas unfractionated heparin and DS of 200kDa could significantly inhibit it. Compared to the control cultures, supplementation with unfractionated heparin or DS of 200kDa at 1g L-1 resulted in more than a 10-fold increase in the maximum rhGDF-5 concentration. Taken together, the supplementation of structural HS analogs improved rhGDF-5 production in rCHO cell cultures by inhibiting rhGDF-5 internalization.

CORONAVIRUS-2019 PANDEMIC, CURRENT METHODS OF DIAGNOSIS, TREATMENTS AND ENGINEERING OF THE SAFE AND EFFECTIVE MODERN VACCINES - AN OVERVIEW

Following the Coronavirus-2019/SARS-COV-2 attack on the front-line of epithelial cells of alveoli, the neurons in the olfactory bulb, intestinal lining and endothelial lining of blood vessels after inhalation, all organs supported by the circulatory system are affected simultaneously. Dr. Avindra Nath at NINDS identified the swelling of olfactory bulb by MRI in the acute phase resulting in multi-system organ failure. We have access to a few drugs for viral elimination like specific monoclonal antibody made by Regeneron and Lilly, anti-inflammatory drug, corticosteroids, Dexamethasone and anti-viral drug Remdesivir. The blood-thinners, heparin, low molecular weight heparin fragments and anti-platelet medication saved many lives from micro- and macro-clots and pulmonary embolism. Best approach is to avoid viral exposure before the arrival of safe and effective new generations of vaccines. In this brief review, an attempt has been made to explain the mechanism of viral entry via the ACE2 receptor and the modes of multiple interventions for both diagnosis and therapy and in rare occasions, a lung transplantation. My lifetime investigations on the diagnosis of heart muscle damage, and the mechanism of arterial thrombosis and drug development in cardiovascular diseases assisted me in identifying one of the major aspects of the blood clots, thrombi and emboli in the COVID-19 patients.

Heparin fragments induce cervical inflammation by recruiting immune cells through Toll-like receptor 4 in nonpregnant mice.

Inflammation is a hallmark in the human cervix remodelling. A possible candidate inducing the inflammatory driven ripening of the cervix is the matrix component heparan sulphate, which has been shown to be elevated in late pregnancy in the cervix and uterus. Heparin and a glycol-split low molecular weight heparin (gsHep) with low anticoagulant potency has been shown to enhance myometrial contraction and interleukin (IL)-8 production by cervical fibroblasts. The aim of this study was to investigate the mechanism by which heparin promotes cervical inflammation. Wild-type, Toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88) and Interferon regulatory factor 3 (IRF3)-deficient mice were treated by deposition of gsHep into the vaginas of nonpregnant mice. To identify which cells that responded to the heparin fragments, a rhodamine fluorescent construct of gsHep was used, which initially did bind to the epithelial cells and were at later time points located in the sub-mucosa. The heparin fragments induced a strong local inflammatory response in wild-type mice shown by a rapid infiltration of neutrophils and to a lesser extent macrophages into the epithelium and the underlying extracellular matrix of the cervix. Further, a marked migration into the cervical and vaginal lumen was seen by both neutrophils and macrophages. The induced mucosal inflammation was strongly reduced in TLR4- and IRF3-deficient mice. In conclusion, our findings suggest that a TLR4/IRF3-mediated innate immune response in the cervical mucosa is induced by gsHep. This low anticoagulant heparin version, a novel TLR4 agonist, could contribute to human cervical ripening during the initiation of labour.

Structural basis for the design of bisubstrate inhibitors of protein kinase CK2 provided by complex structures with the substrate-competitive inhibitor heparin.

The Ser/Thr kinase CK2, a member of the superfamily of eukaryotic protein kinases, has an acidophilic substrate profile with the substrate recognition sequence S/T-D/E-X-D/E, and it is inhibited by polyanionic substances like heparin. The latter, a highly sulphated glucosamino glycan composed mainly of repeating 2-O-sulpho-α-l-idopyranuronic acid/N,O6-disulpho-α-d-glucosamine disaccharide units, is the longest known substrate-competitive CK2 inhibitor. The structural basis of CK2's preference for anionic substrates and substrate-competitive inhibitors is only vaguely known which limits the value of the substrate-binding region for the structure-based development of CK2 bisubstrate inhibitors. Here, a tetragonal and a monoclinic co-crystal structure of CK2α, the catalytic subunit of CK2, with a decameric heparin fragment are described. In the tetragonal structure, the heparin molecule binds to the polybasic stretch at the beginning of CK2α's helix αC, whereas in the monoclinic structure it occupies the central substrate-recognition region around the P+1 loop. Together, the structures rationalize the inhibitory efficacy of heparin fragments as a function of chain length. The monoclinic CK2α/heparin structure, in which the heparin fragment is particularly well defined, is the first CK2 structure with an anionic inhibitor of considerable size at the central part of the substrate-recognition site. The bound heparin fragment is so close to the binding site of ATP-competitive inhibitors that it can guide the design of linkers and pave the way to efficient CK2 bisubstrate inhibitors in the future.

Heparin stimulates biofilm formation of Escherichia coli strain Nissle 1917.

Escherichia coli strain Nissle 1917 (EcN), a gut probiotic competing with pathogenic bacteria, has been used to attenuate various intestinal dysfunctions. Heparin is a sulfated glycosaminoglycan enriched in the human and animal intestinal mucosa, which has a close connection with bacterial biofilm formation. However, the characteristics of heparin affecting bacterial biofilm formation remain obscure. In this study, we investigated the influence of heparin and its derivatives on EcN biofilm formation. Here, we found that heparin stimulated EcN biofilm formation in a dose-dependent manner. With the addition of native heparin, the EcN biofilm formation increased 6.9- to 10.8-fold than that without heparin, and was 1.4-, 3.1-, 3.0-, and 3.8-fold higher than that of N-desulfated heparin (N-DS), 2-O-desulfated heparin (2-O-DS), 6-O-desulfated heparin (6-O-DS), and N-/2-O-/6-O-desulfated heparin (N-/2-O-/6-O-DS), respectively. Depolymerization of heparin produced chain-shortened heparin fragments with decreased molecular weight. The depolymerized heparins did not stimulate EcN biofilm formation. The OD570 value of EcN biofilm with the addition of chain-shortened heparin fragments was 8.7-fold lower than that of the native heparin. Furthermore, the biofilm formation of Salmonella enterica serovar Typhimurium was also investigated with the addition of heparin derivatives, and the results were consistent with that of EcN biofilm formation. We conclude that heparin stimulated EcN biofilm formation. Both the sulfation and chain-length of heparin contributed to the enhancement of EcN biofilm formation. This study increases the understanding of how heparin affects biofilm formation, indicating the potential role of heparin in promoting intestinal colonization of probiotics that antagonize pathogen infections.

Abstract 202: In Vitro Effect of Trisulfate Disaccharide on ICa (L-type), INa, IK, and the Na/Ca Exchange Current Recorded From Rat Ventricular Myocytes by Patch Clamp

Background: Ion channels are pharmacological targets for antiarrhythmic action, and drugs currently used for this purpose are generally not specific to a site of action and may act on several channels and even trigger proarrhythmic phenomena. Trisulfate disaccharide (TD) is an heparin fragment known to act on the sodium calcium exchanger (NCX), reducing intracellular calcium in overload situations and reversing arrhytmias, but its action on other ionic currents is unknown. Objective: To evaluate by patch clamp the action of TD at different concentrations in NCX and ionic currents in situations of intracellular calcium overload. Materials and Methods: Adult rat myocytes were obtained from a sample from ventricles. Currents were measured using the whole-cell variant of the patch clamp method. Creation of voltage clamp pulses and data acquisition was controlled by a computer with pClamp software. Peak inward current amplitude was measured for ion currents. For Na/Ca exchange current a ramp voltage protocol was employed. Three different concentrations of Cai (300nM, 400nM and 600nM) were used in separate experiments. One drug concentration was applied per cell (10, 30 and 100 micromolar each). The current sensitive to 5mM nickel was taken as the Na/Ca exchange current. The effects of TD on the INa, L-type Ca, and the potassium currents, transiente outward current (Ito), inwardly rectifying potassium current (IK1), and sustained current (Isus) recorded from adult rat ventricular myocytes were also examined in the same conditions. Results: TD concentration-dependently increased the inward Na/Ca exchange current in all intracellular calcium concentration. The effects of TD on the INa, L-type Ca, and the potassium currents, Ito, IK1 and Isus was associated with less than 30% mean reduction on any current at the highest concentration of TD tested (100 micromolar) and still below the positive block controls for different channels that is above 40% block. Conclusion: TD acts on NCX under different concentrations used, without affecting other ionic currents, suggesting specificity in the mechanism of action and possibly not exerting a pro-arrhythmic activity, this effect being desirable for its possible use in reversal of cardiac arrhythmias.

PC-FACS

PC-FACS

Single Molecule Identification and Quantification of Glycosaminoglycans Using Solid-State Nanopores.

Glycosaminoglycans (GAGs) are a class of polysaccharides with potent biological activities. Due to their complex and heterogeneous composition, varied charge, polydispersity, and presence of isobaric stereoisomers, the analysis of GAG samples poses considerable challenges to current analytical techniques. In the present study, we combined solid-state nanopores-a single molecule sensorwith a support vector machine (SVM)-a machine learning algorithm for the analysis of GAGs. Our results indicate that the nanopore/SVM technique could distinguish between monodisperse fragments of heparin and chondroitin sulfate with high accuracy (>90%), allowing as low as 0.8% (w/w) of chondroitin sulfate impurities in a heparin sample to be detected. In addition, the nanopore/SVM technique distinguished between unfractionated heparin (UFH) and enoxaparin (low molecular weight heparin) with an accuracy of ∼94% on average. With a reference sample for calibration, a nanopore could achieve nanomolar sensitivity and a5-Log dynamic range. We were able to quantify heparin with reasonable accuracy using multiple nanopores. Our studies demonstrate the potential of the nanopore/SVM technique to quantify and identify GAGs.

The Molecular weight of heparin fragments interferes with the protection of the hepatocyte subjected to injury by ischemia and reperfusion

Background: Calcium overload is responsible for hepatic cell death after ischemia and reperfusion (I/R). Sodium-calcium exchanger (NCX) decreases the cytoplasmic calcium by acceleration of calcium extrusion. Heparin fragments of different molecular weights interact with the NCX decreasing calcium. However, they have adverse effects upon the coagulation process, except Trisulfate Disaccharide(TD). Aims: To evaluate and compare the effects of heparin fragments with different molecular weights in the cellular protection in Wistar rats liver cells subjected to ischemia and reperfusion. Materials and methods: Male Wistar Rats were subjected to surgical ischemia of the median and lateral liver lobes for 60 minutes and reperfusion for 4 hours and divided in 4 Groups (n=6): Control: IV infusion of saline; Enox: Enoxaparin (4200 Da) IV infusion (2mg/kg); Fond: Fondaparinux (3000 Da) IV infusion (0.03 mg/Kg); TD: Trisulfate Disaccharide (585 Da) IV infusion (0.2 mg/kg). Saline or heparin fragments (0.4 ml) were injected into the penile vein, 10 minutes before reperfusion. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) were quantified after 4h of Reperfusion. Results: There were decreases in ALT and AST in Enox, Fond and TD when compared with the control group (p < 0.05). There was no difference between Enox and Fond groups, but TD group animals presented lower ALT levels compared to Enox and Fond groups. Conclusion: Decreased ALT and AST levels after administration of Enoxaparin, Fondaparinux and Trisulfate Disaccharide in the animal model of liver I/R suggests hepatocellular protection with a greater effectiveness of the smaller molecular weight fragment (TD).

Depolymerization of heparin by dielectric barrier discharge: Effect of operating modes and anticoagulant potential analysis of low-molecular-weight products

Low-molecular-weight heparin and ultra-low-molecular-weight heparin are widely used as anticoagulant and antithrombotic drugs. An atmospheric pressure dielectric-barrier discharge procedure was developed to depolymerize heparin to low- or ultra-low-molecular-weight fragments. Dielectric-barrier discharge treatment of heparin resulted in several well-separated and uniform fragments. The structural character of digested fragments below 3,000 Da was elucidated. Expected digestion products such as sulfated disaccharide, sulfated tetrasaccharide, and pentasaccharides with alternatingly-linked N-acetyl glucosamine and iduronic acid were detected. The anticoagulant potential of the digested heparin was tested. The results indicated that dielectric-barrier discharge treatment can produce various molecular weight (MW) heparin fragments and maintain anticoagulant potency.

Mo2031 - The Molecular Weight of Heparin Fragments Interferes with the Protection of the Hepatocyte Subjected to Injury by Ischemia and Reperfusion

Mo2031 - The Molecular Weight of Heparin Fragments Interferes with the Protection of the Hepatocyte Subjected to Injury by Ischemia and Reperfusion

Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate.

Cleavage of heparan sulfate proteoglycans (HSPGs) by the enzyme heparanase modulates tumour-related events including angiogenesis, cell invasion, and metastasis. Metalloshielding of heparan sulfate (HS) by positively charged polynuclear platinum complexes (PPCs) effectively inhibits physiologically critical HS functions. Studies using bacterial P. heparinus heparinase II showed that a library of Pt complexes varying in charge and nuclearity and the presence or absence of a dangling amine inhibits the cleavage activity of the enzyme on the synthetic pentasaccharide, Fondaparinux (FPX). Charge-dependent affinity of PPC for FPX was seen in competition assays with methylene blue and ethidium bromide. The dissociation constant (Kd ) of TriplatinNC for FPX was directly measured by isothermal titration calorimetry (ITC). The trend in DFT calculated interaction energies with heparin fragments is consistent with the spectroscopic studies. Competitive inhibition of TAMRA-R9 internalization in human carcinoma (HCT116) cells along with studies in HCT116, wildtype CHO and mutant CHO-pgsA745 (lacking HS/CS) cells confirm that HSPG-mediated interactions play an important role in the cellular accumulation of PPCs.