Heparin Molecules Research Articles

Critical Influence of Cosolutes and Surfaces on the Assembly of Serpin-Derived Amyloid Fibrils

Many proteins and peptides self-associate into highly ordered and structurally similar amyloid cross-β aggregates. This fibrillation is critically dependent on properties of the protein and the surrounding environment that alter kinetic and thermodynamic equilibria. Here, we report on dominating surface and solution effects on the fibrillogenic behavior and amyloid assembly of the C-36 peptide, a circulating bioactive peptide from the α1-antitrypsin serine protease inhibitor. C-36 converts from an unstructured peptide to mature amyloid twisted-ribbon fibrils over a few hours when incubated on polystyrene plates under physiological conditions through a pathway dominated by surface-enhanced nucleation. In contrast, in plates with nonbinding surfaces, slow bulk nucleation takes precedence over surface catalysis and leads to fibrillar polymorphism. Fibrillation is strongly ion-sensitive, underlining the interplay between hydrophilic and hydrophobic forces in molecular self-assembly. The addition of exogenous surfaces in the form of silica glass beads and polyanionic heparin molecules potently seeds the amyloid conversion process. In particular, heparin acts as an interacting template that rapidly forces β-sheet aggregation of C-36 to distinct amyloid species within minutes and leads to a more homogeneous fibril population according to solid-state NMR analysis. Heparin’s template effect highlights its role in amyloid seeding and homogeneous self-assembly, which applies both in vitro and in vivo, where glycosaminoglycans are strongly associated with amyloid deposits. Our study illustrates the versatile thermodynamic landscape of amyloid formation and highlights how different experimental conditions direct C-36 into distinct macromolecular structures.

CTAB-Capped Gold Nanoparticles as a New Probe for Spectrophotometric Determination of Heparin

A simple, fast, and sensitive spectrophotometric method is reported for the determination of heparin concentration in human plasma samples. The assay is based on enhancing the extinction intensity of CTAB-capped gold nanoparticles in the presence of heparin molecules. The strong electrostatic interaction of the positively-charged gold nanoparticles with the negatively charged heparin molecules induces a marked increase in size of gold nanoparticles, which results in a dramatic rise in the extinction spectrum of gold nanoparticles. Since the enhanced extinction intensity at 528 nm is proportional to the concentration of heparin, quantitative determination of heparin is possible. Under optimum conditions, the enhanced extinction intensity is linearly correlated with the concentration of heparin in the range 0.18–11.98 U/mL with limit of detection 0.08 U/mL.

End-point immobilization of heparin on plasma-treated surface of electrospun polycarbonate-urethane vascular graft.

Vascular occlusion remains the leading cause of death all over the world, despite advances made in balloon angioplasty and conventional surgical intervention. Currently, autografts are the gold-standard grafts used to treat vascular occlusive disease. However, many patients with vascular occlusive disease do not have autologous vascular graft available. Therefore, there is a widely recognized need for a readily available, functional, small-diameter vascular graft (inner diameter of <6mm). This work addresses this critical need by developing a method of antithrombogenic modification of synthetic grafts.

A century of heparin: past, present and future

Heparin was discovered around 1922 by Howell (Baltimore) and was further developed by the teams of Best (Toronto) and Jorpes (Stockholm). Kakkar (London) propagated its routine use for the prevention of postoperative thrombosis from 1971 onwards. The discovery of low molecular weight heparins (1976, Johnson, London) and their development in the subsequent years led to the present arsenal of clinically useful drugs. In 1976, three teams independently found that a specific structure in heparin binds tightly to antithrombin. This enabled the teams of Lindahl (Stockholm) and Casu (Milan) to determine the pentasaccharide structure responsible for this binding and Petitou, from the Choay team (Paris), to synthesize it (1983). It was found (Olson and others) that heparin facilitates the interaction between antithrombin and a clotting enzyme by allosteric changes in the antithrombin (important for factor Xa) and by facilitating the approach of the enzyme to antithrombin via its "sliding" along the heparin molecule (important for thrombin). Antithrombin action therefore requires a minimum length of seven sugar units next to the pentasaccharide whereas anti-factor Xa action does not. The effect of heparin is almost entirely due to anti-thrombin action (B≐guin), so anti-factor Xa activity does not reflect the concentration of anticoagulant heparin. The anticoagulant effect is poorly reflected by the activated partial thromboplastin time. Because present clinical use is based on the latter tests, it is not generally known that the individual response to heparin shows an extremely wide variation. Individualization of heparin dosage is likely to improve clinical results.

Heparin's solution structure determined by small-angle neutron scattering.

Heparin is a linear, anionic polysaccharide that is widely used as a clinical anticoagulant. Despite its discovery 100 years ago in 1916, the solution structure of heparin remains unknown. The solution shape of heparin has not previously been examined in water under a range of concentrations, and here is done so in D2 O solution using small-angle neutron scattering (SANS). Solutions of 10kDa heparin-in the millimolar concentration range-were probed with SANS. Our results show that when sodium concentrations are equivalent to the polyelectrolyte's charge or up to a few hundred millimoles higher, the molecular structure of heparin is compact and the shape could be well modeled by a cylinder with a length three to four times its diameter. In the presence of molar concentrations of sodium, the molecule becomes extended to nearly its full length estimated from reported X-ray measurements on stretched fibers. This stretched form is not found in the presence of molar concentrations of potassium ions. In this high-potassium environment, the heparin molecules have the same shape as when its charges were mostly protonated at pD ≈ 0.5, that is, they are compact and approximately half the length of the extended molecules.

Development and characterization of an innovative heparin coating to stabilize and protect liposomes against adverse immune reactions

Liposomes have been recognized as excellent drug delivery systems, but when they come in direct contact with different blood components they may trigger an immediate activation of the innate immune system. The aim of the present study was to produce long-circulating, blood-compatible liposomes by developing a construct of liposomes covered by a novel unique heparin complex (CHC; 70 heparin molecules per complex) to avoid recognition by the innate immune system. Unilamellar, cationic liposomes were produced by hand extrusion through a 100-nm polycarbonate membrane. Coating of liposomes with the macromolecular CHC was accomplished by electrostatic interactions. Dynamic light scattering as well as QCM-D measurements were used to verify the electrostatic deposition of the negatively charged CHC to cationic liposomes. The CHC-coated liposomes did not aggregate when in contact with lepirudin anti-coagulated plasma. Unlike previous attempts to coat liposomes with heparin, this technique produced freely moveable heparin strands sticking out from the liposome surface, which exposed AT binding sites reflecting the anticoagulant potentials of the liposomes. In experiments using lepirudin-anticoagulated plasma, CHC-coated liposomes, in contrast to non-coated control liposomes, did not activate the complement system, as evidenced by low C3a and sC5b-9 generation and reduced leakage from the liposomes. In conclusion, we show that liposomes can be successfully coated with the biopolymer CHC, resulting in biocompatible and stable liposomes that have significant application potential.

Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells

Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93%. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation.

Application Of Phenol/Amine Copolymerized Film Modified Magnesium Alloys: Anticorrosion And Surface Biofunctionalization

Magnesium metal as degradable metallic material is one of the most researched areas, but its rapid degradation rate restricts its development. The current anticorrosion surface modification methods require expensive equipment and complicated operation processes and cannot continue to introduce biofunction on modified surface. In this study, the GAHD conversion coatings were fabricated on the surface of magnesium alloys (MZM) by incubating in the mixture solution of gallic acid (GA) and hexamethylenediamine (HD) to decrease the corrosion rate and provide primary amines (-NH2), carboxyl (-COOH), and quinone groups, which is supposed to introduce biomolecules on MZM. Chemical structures of the MZM-GAHD and MZM-HEP-GAHD were explored by analyzing the results of FTIR and XPS comprehensively. Furthermore, it was proved that the heparin (HEP) molecules were successfully immobilized on MZM-GAHD surface through carbodiimide method. The evaluation of platelet adhesion and clotting time test showed that MZM-HEP-GAHD had higher anticoagulation than MZM-GAHD. Through electrochemical detection (polarization curves and electrochemical impedance spectroscopy Nyquist spectrum) and immersion test (Mg(2+) concentration and weight loss), it was proved that compared to MZM, both the MZM-GAHD and MZM-HEP-GAHD significantly improved the corrosion resistance. Finally, in vivo experimentation indicated that mass loss had no significant difference between MZM-1:1, MZM-HEP-1:1, and MZM. However, the trend still suggested that MZM-1:1 and MZM-HEP-1:1 possessed corrosion resistance property.

Investigation of the heparin–thrombin interaction by dynamic force spectroscopy

BackgroundThe interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. MethodsIn this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin–thrombin interaction from an energy viewpoint at the molecular scale. ResultsWell-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. ConclusionsAn increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. General significanceThese results contribute to understanding the role of specific and nonspecific forces that drive heparin–thrombin interactions under applied force or flow conditions.

Inhibition of urinary macromolecule heparin on aggregation of nano-COM and nano-COD crystals.

Purpose: This research aims to study the influences of heparin (HP) on the aggregation of nano calcium oxalate monohydrate (COM) and nano calcium oxalate dihydrate (COD) with mean diameter of about 50 nm. Method: The influences of different concentrations of HP on the mean diameter and Zeta potential of nano COM and nano COD were investigated using a nanoparticle size Zeta potential analyzer. Results: HP could be adsorbed on the surface of nano COM and nano COD crystals, leading to an increase in the absolute value of Zeta potential on the crystals and an increase in the electrostatic repulsion force between crystals. Consequently, the aggregation of the crystals is reduced and the stability of the system is improved. The strong adsorption ability of HP was closely related to the -OSO3− and -COO− groups contained in the HP molecules. X-ray photoelectron spectroscopy confirmed the coordination of HP with Ca2+ ions of COM and COD crystals. Conclusion: HP could inhibit the aggregation of nano COM and nano COD crystals and increase their stability in aqueous solution, which is conducive in inhibiting the formation of calcium oxalate stones.

The Effects of Metal Ions on Heparin/Heparin Sulfate-Protein Interactions.

Heparin/heparin sulfate (HS) interacts with a number of proteins thereby playing an essential role in the regulation of many physiological processes. The understanding of heparin/HS-protein interactions at the molecular level is of fundamental importance to biology and will aid in the development of highly specific glycan-based therapeutic agents. The heparin-binding proteins (HBPs) interact with sulfated domains of heparin/HS chains primarily through ionic attraction between negatively charged groups in HS/heparin chains and basic amino acid residues within the protein. Reports in literature have been shown that heparin molecules have a high affinity for a wide range of metal ions. In the present study, we used surface plasmon resonance (SPR) to study the effects of metal ions (under physiological and non-physiological concentrations) on heparin/HS-protein interactions. The results showed that under non-physiological of metal ion concentration, different metal ions showed different effects on heparin binding to fibroblast growth factor-1 (FGF1) and interleakin-7 (IL7). While the effects of individual metal ion at physiological concentrations had little impact on protein binding, the mixed metal ions reduced the FGF1/heparin or IL7/heparin binding affinity, changing its binding profile.

Facile conjugation of heparin onto titanium surfaces via dopamine inspired coatings for improving blood compatibility

Immobilization of heparin on biomaterials surface has been proven to be an effective strategy for preventing thrombus formation. However, the procedures of most immobilization methods (physical adsorption, covalent linkage, electrostatic interaction) are complicated and time-consuming. In the present study, heparin with various concentrations immobilized on a titanium (Ti) substrate via polydopamine layer for improving blood compatibility was investigated. Water contact angle measurement showed that the immobilization of heparin resulted in an increase of the hydrophilicity. X-ray photoelectron spectroscopy (XPS) and Toluidine Blue O (TBO) tests displayed that the heparin molecules were successfully immobilized on Ti surface. The evaluations of blood compatibility (hemolysis rate, APTT, platelet adhesion and activation, fibrinogen conformational change) showed that the immobilization of heparin decreased hemolysis rate, prolonged blood coagulation time, reduced platelets adhesion and activation, and induced less fibrinogen conformational change. Moreover, a significant inhibition of blood coagulation and platelet adhesion was obtained when the heparin concentration was higher than 5 mg/mL, indicating that only with a certain surface densities could heparin perform its anticoagulant properties well. The results suggest that the immobilization of heparin via polydopamine layer can confer excellent antithrombotic properties, and the heparin immobilization method via polydopamine layer provides an alternative approach for other biomolecules immobilization on biomaterials surface. Thus it is envisaged that this method will be potentially useful for the surface modification of blood-contacting biomaterials.

Docking server for the identification of heparin binding sites on proteins.

Many proteins of widely differing functionality and structure are capable of binding heparin and heparan sulfate. Since crystallizing protein–heparin complexes for structure determination is generally difficult, computational docking can be a useful approach for understanding specific interactions. Previous studies used programs originally developed for docking small molecules to well-defined pockets, rather than for docking polysaccharides to highly charged shallow crevices that usually bind heparin. We have extended the program PIPER and the automated protein–protein docking server ClusPro to heparin docking. Using a molecular mechanics energy function for scoring and the fast Fourier transform correlation approach, the method generates and evaluates close to a billion poses of a heparin tetrasaccharide probe. The docked structures are clustered using pairwise root-mean-square deviations as the distance measure. It was shown that clustering of heparin molecules close to each other but having different orientations and selecting the clusters with the highest protein–ligand contacts reliably predicts the heparin binding site. In addition, the centers of the five most populated clusters include structures close to the native orientation of the heparin. These structures can provide starting points for further refinement by methods that account for flexibility such as molecular dynamics. The heparin docking method is available as an advanced option of the ClusPro server at http://cluspro.bu.edu/.

Importance of interdisciplinary collaboration regarding updated activated partial thromboplastin time reporting for heparin.

Importance of interdisciplinary collaboration regarding updated activated partial thromboplastin time reporting for heparin.

Modifying the vessel walls in porcine kidneys during machine perfusion

BackgroundEndothelial glycocalyx regulates the endothelial function and plays an active role in maintaining vascular homeostasis. During ischema and reperfusion, the glycocalyx is rapidly shed into the blood stream. A Corline heparin conjugate (CHC; Corline systems AB, Uppsala, Sweden) consists of 70 heparin molecules that have the capacity to adhere strongly to biological tissues expressing heparin affinity. We hypothesized that CHC could be used to restore disrupted glycocalyx in vivo in kidneys from brain-dead pigs. Materials and methodsBrain death was induced in male landrace pigs (n = 6) by inflating a balloon catheter in the epidural space until obtaining negative cerebral perfusion. The recovered kidneys (n = 5 + 5) were perfused by hypothermic machine perfusion using two Lifeport kidney transporters (Organ Recovery Systems, Chicago, IL). CHC (50 mg) (including 25 mg biotinylated CHC) or 50 mg unfractionated heparin (control) was added to the perfusion fluid in the respective machines. In one case, the kidneys were used only for dose escalation of CHC with the same procedure. ResultsCHC was detected by immunofluorescence and confocal microscopy in the inner surface of the vessel walls. The binding of CHC in the kidney was confirmed indirectly by consumption of CHC from the perfusion fluid. ConclusionsIn this first attempt, we show that CHC maybe used to coat the vessel walls of perfused kidneys during hypothermic machine perfusion, an approach that could become useful in restoring endothelial glycocalyx of kidneys recovered from deceased donors to protect vascular endothelium and possibly ameliorate ischemia and reperfusion injuries.

Pulse radiolysis and spectrophotometric studies on the binding of organic cations with heparin

Here we present the spectroscopic and pulse radiolysis studies of the interactions of heparin and some organic cations:methylene blue (MB), 1-methylnicotinamide (MNA+), and its dimer 1,3-bis(1-methylnicotinamide)propane (bis(MNA+)).The interaction between heparin and some cationic dyes (e.g. methylene blue) in aqueous solution leads to the changes in the absorption spectra of those dyes. This effect, called metachromasia, has been successfully used to study the interactions of a number of cationic dyes with heparin and other glycosaminoglycans.Using methylene blue, as a model compound, we have shown that the changes in the rate constant for the reaction of solvated electrons with cationic dye in the presence of heparin (pulse radiolysis) are well correlated with the accompanying changes in the absorption spectra (metachromasia). The same mathematical formalism was applied to absorption and rate constants changes. Using such formalism, we have determined the total number of binding sites per heparin molecule and the equilibrium constants for MNA+ and bis(MNA+).

Biologically inspired membrane design with a heparin-like interface: prolonged blood coagulation, inhibited complement activation, and bio-artificial liver related cell proliferation.

In the present work, inspired by the chemical structure of heparin molecules, we designed a polyethersulfone (PES) membrane with a heparin-like surface for the first time by physically blending sulfonated polyethersulfone (SPES), carboxylic polyethersulfone (CPES), and PES at rational ratios. Evaporation and phase-inversion membranes of PES/CPES/SPES were prepared by evaporating the solvent in a vacuum oven, and by a liquid-liquid phase separation technique, respectively. Scanning electron microscopy (SEM) images revealed that the structures of the PES/CPES/SPES membranes were dependent on the proportions of the additives and no obvious phase separation was detected. The blood compatibility of the modified membrane surfaces was characterized in terms of bovine serum fibrinogen (BFG) adsorption, platelet adhesion, thrombin-antithrombin (TAT) generation, percentage of platelets positive for CD62p expression, clotting times (activated partial thromboplastin time (APTT) and prothrombin time (PT)), and complement activation on C3a and C5a levels. The results indicated that the blood compatibility of PES matrix was improved due to the biologically inspired membrane design with a heparin-like interface by introducing functional sulfonic acid and carboxylic acid groups. Furthermore, cell morphology observation and cell culture assays demonstrated that the modified membranes showed better performance in bio-artificial liver related cell proliferation than the pristine PES membrane. In general, the intriguing PES/CPES/SPES membranes, especially the phase-inversion one, showed improved blood and cell compatibility, which might have great potential application in the blood purification field.

Importance of IdoA and IdoA(2S) ring conformations in computational studies of glycosaminoglycan–protein interactions

Glycosaminoglycans (GAGs) interact with chemokines and growth factors in the extracellular matrix and, therefore, mediate cell communication processes. Heparin is one of the most studied GAGs, for which many experimental structures of its complexes with proteins are available. One of the monosaccharide components of heparin, sulfated iduronic acid (IdoA(2S)), is observed to adopt both 1C4 and 2S0 ring conformations. Despite the biological relevance of the sugar ring conformations for heparin–protein interactions, it is very challenging to take into account the conformational space of IdoA(2S) sugar ring for computational studies. Therefore, instead of systematically analyzing several ring conformations, which represents a combinatorial problem for a periodic heparin molecule, often only one ring conformation is taken into account. Here, we use docking and molecular dynamics (MD) to estimate how crucial this assumption could be for the conclusions being made in computational studies of heparin–protein interactions. We show that both docking solutions and free energy calculations from MD simulations are significantly affected by the conformations adopted by IdoA and IdoA(2S) rings. Therefore, in the application of computational approaches to heparin–protein systems the ring conformations should be treated properly to avoid misleading conclusions.

Severe Hypoglycemia Associated with Levofloxacin in a Healthy Older Woman

Severe Hypoglycemia Associated with Levofloxacin in a Healthy Older Woman

Anti-angiogenic activity of heparin functionalised cerium oxide nanoparticles

Cerium oxide nanoparticles (nanoceria) are widely reported to be non-cytotoxic and modulate intracellular reactive oxygen species (ROS). In this study, nanoceria (dxRD = 12 nm) were functionalised with either 130 or 880 molecules of unfractionated heparin using the organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria with a low level of heparin functionalisation were found to scavenge intracellular ROS to the same extent as unfunctionalised nanoceria and significantly more than cells exposed to medium only. In contrast, nanoceria with the highest level of heparin functionalisation were not as effective at scavenging intracellular ROS. Nanoceria were localised predominantly in the cytoplasm, while heparin-nanoceria were localised in both the cytoplasm and lysosomes. Together these data demonstrated that the level of nanoceria surface functionalisation with heparin determined the intracellular localisation and ROS scavenging ability of these particles. Additionally, heparin-nanoceria were effective in reducing endothelial cell proliferation indicating that they may find application in the control of angiogenesis in cancer in the future.