Role of heparin in the realization of the hypoglycemic action of insulin

The presence of reactive heparin in blood circulation is necessary for realization of the hypoglycemic action of insulin. This is confirmed by the fact that after heparin binding by protamin sulfate both endogenous and exogenous insulin do not exhibit any hypoglycemic activity. In animals with different basal concentration of heparin in the blood, the blockade of insulin action is attained by application of different doses of protamin sulfate, respectively. Based on the data obtained one can determine an approximate blood heparin concentration necessary for realization of the hypoglycemic action of insulin.     

Immunoelectrophoretic quantitation of antithrombin III. Electroimmunodiffusion in agarose gels containing heparin.

Antithrombin III (AT-III), being an alpha2-globulin, will have an electrophoretic mobility in the presence of heparin like prealbumin in agarose gels. This phenomenon was utilized to quantitate AT-III from serum and plasma by electroimmunodiffusion (EID) for 90 min agarose gels containing 75 USP units of heparin/ml gel. The method permits a rapid quantitation of AT-III from serum, citrated plasma and EDTA plasma, and a positive correlation was observed between these values and those obtained by single radial immunodiffusion (SRI). This is in contrast to quantitation of AT-III by EID in gels containing no heparin where the values for plasma showed poor correlation with those obtained by SRI.     

Substrate interactions with the alpha-subunit of the Escherichia coli tryptophan synthase. A kinetic study of the wild-type alpha-subunit.

Small-angle X-ray scattering has been used to determine the size and shape of human antithrombin III and its complex with heparin. The scattering data obtained show that antithrombin III behaves like an ellipsoid with semi-axes of 1.9, 3.7, and 5.2 nm. The antithrombin III-heparin complex produces a scattering curve very similar to that of pure antithrombin III, indicating that there is no major change in size and shape upon binding of heparin. The nature of the heparin binding site is discussed.     

Improved cleavage of bovine ICSI ova cultured in heparin-containing medium

Although heparin plays an important role in bovine sperm capacitation, there is no direct evidence for a role in embryonic development. The present study was designed to examine the effect of heparin on early development of bovine zygotes obtained by intracytoplasmic sperm injection (ICSI). Spermatozoa were treated with or without heparin, and the ICSI ova were cultured in a chemically defined medium + BSA, with or without heparin. Treatment of spermatozoa with heparin before ICSI or heparin in post-ICSI culture medium for 18 or 24h had a beneficial effect on pronuclear formation, cleavage rate (63% versus 76-83%), and number of cells in blastocysts (68 cells versus 82-109 cells; P < 0.05).     

Effect of heparin on the extent of inhibition of thrombin by heparin cofactor.

A heparin preparation obtained by gel chromatography is compared to unfractionated heparin with respect to the effects of heparin on the reaction between thrombin and heparin cofactor. Whereas both preparations enhance the rate of inhibition of thrombin by heparin cofactor, the extent of inhibition is decreased by the unfractionated, but not by the fractionated heparin. The decreased extent of inhibition is accounted for by residua of unreacted and undegraded heparin cofactor and thrombin, as demonstrated by gel electrophoresis in dodecyl sulfate. However both heparin preparations enhance the rate of degradation by thrombin of the thrombin-heparin cofactor complex.     

The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine.

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469--3473]. This activity is attributed to an enzyme provisionally named 'macromolecular heparin depolymerase'. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.     

Effects of glycosylation on heparin binding and antithrombin activation by heparin

Antithrombin (AT), a serine protease inhibitor, circulates in blood in two major isoforms, α and β, which differ in their amount of glycosylation and affinity for heparin. After binding to this glycosaminoglycan, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. In this process, β‐AT presents the higher affinity for heparin, being suggested as the major AT glycoform inhibitor in vivo. However, either the molecular basis demonstrating the differences in heparin binding to both AT isoforms or the mechanism of its conformational activation are not fully understood. Thus, the present work evaluated the effects of glycosylation and heparin binding on AT structure, function, and dynamics. Based on the obtained data, besides the native and activated forms of AT, an intermediate state, previously proposed to exist between such conformations, was also spontaneously observed in solution. Additionally, Asn135‐linked oligosaccharide caused a bending in AT‐bounded heparin, moving such polysaccharide away from helix D, which supports its reduced affinity for α‐AT. The obtained data supported the proposal of an atomic‐level, solvent and amino acid residues accounting, putative model for the transmission of the conformational signal from heparin binding exosite to β‐sheet A and the reactive center loop, also supporting the identification of differences in such transmission between the serpin glycoforms involving helix D, where the Asn135‐linked oligosaccharide stands. Such intramolecular rearrangements, together with heparin dynamics over AT surface, may support an atomic‐level explanation for the Asn135‐linked glycan influence over heparin binding and AT activation. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme-template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme-template complex to the same extent as by an RNA-synthesizing enzyme-template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.     

Changes in numbers and heparin content of peritoneal fluid mast cells of growing rats measured by flow cytofluorometry

A cytofluorometric method, based on berberine staining of mast cell heparin, was used for flow cytofluorometric counting and heparin quantitation of mast cells in crude peritoneal suspensions of growing rats. The automatic flow cytofluorometric counting of mast cells correlated well with hemocytometer cell counts. The mean mast cell heparin content obtained by flow cytofluorometry showed good agreement with such obtained by cytofluorometry of microscopically identified mast cells. The number of peritoneal mast cells and the mean mast cell heparin content was found to increase as the animals grew older. The results of the microscope fluorometric measurements suggested that the heparin content was normally distributed within mast cell populations of both young and old rats. However, the heparin distributions obtained by flow cytofluorometry were often positively skewed but did not fulfill the condition of the log-normal distribution.     

Control of the heparosan <Emphasis Type="Italic">N</Emphasis>-deacetylation leads to an improved bioengineered heparin

The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.     

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.     

Crystallization of macromolecular heparin

X-ray fibre-diffraction photographs were obtained from oriented films of the sodium salt of macromolecular heparin (molecular weight approx. 10(6)) prepared from rat skin. Two distinct molecular chain conformations corresponding to two different crystal lattices were observed as a function of relative humidity. The first conformation, obtained at 78% relative humidity, has a layer-line spacing of 1.73nm, which can be interpreted as an approximate twofold helix. On increasing the relative humidity to 84% a second phase with a layer-line repeat of 1.65nm is obtained with the reflexions indexing on a triclinic unit cell similar to that obtained previously (Nieduszynski & Atkins, 1973) for pig mucosal heparin.     

Extension and structural variability of the antithrombin-binding sequence in heparin

Oligosaccharides with different affinities for antithrombin were isolated following partial deaminative cleavage of pig mucosal heparin with nitrous acid. The smallest high-affinity component obtained was previously identified as an octasaccharide with the predominant structure: (Formula: see text). The interaction of this octasaccharide, and of deca- and dodecasaccharides containing the same octasaccharide sequence, with antithrombin was studied by spectroscopic techniques. The near-ultraviolet difference spectra, circular dichroism spectra, and fluorescence enhancements induced by adding these oligosaccharides to antithrombin differed only slightly from the corresponding parameters measured in the presence of undegraded high-affinity heparin. Moreover, the binding constants obtained for the oligosaccharides and for high-affinity heparin were similar (1.0-2.9 X 10(7) M-1 at I = 0.3). In contrast, two hexasaccharides corresponding to units 1-6 and 3-8, respectively, of the above sequence showed about a 1000-fold lower affinity for antithrombin, and also induced considerably different spectral perturbations in antithrombin. Since the 1-6 hexasaccharide contains a reducing-terminal anhydromannose residue instead of the N-sulfated glucosamine unit 6 of the intact sequence, these results strongly support our previous conclusion that the N-sulfate group at position 6 is essential to the interaction with antithrombin. The low affinity of the hexasaccharide 3-8 provides further evidence that a pentasaccharide sequence 2-6 constitutes the actual antithrombin-binding region in the heparin molecule. Structural analysis of the various oligosaccharides revealed natural variants with an N-sulfate group substituted for the N-acetyl group at position 2. The preponderance of N-acetyl over N-sulfate groups at this position may be rationalized in terms of the mechanism of heparin biosynthesis, assuming that the D-gluco configuration of unit 3 is an essential feature of the antithrombin-binding region.     

Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site

Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of approximately 2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.     

Isolation of nuclear envelopes with polyanions

Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei are established and a morphological and biochemical study of such isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4 degrees C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outher leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating the pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37 degrees C, in the presence of phosphate ions.     

An Inter-subunit Disulfide Bond Affects Affinity of Human Lung Extracellular Superoxide Dismutase to Heparin

Human extracellular superoxide dismutase (EC-SOD) was purified to homogeneity from lung tissue and the nature of the binding of heparin to EC-SOD was investigated. The enzyme was purified using three column chromatographic steps, and 127 μg of purified EC-SOD was obtained. A specific anti-human EC-SOD antibody was obtained by immunization with the purified enzyme. Western blot analysis of the heparin affinity chromatography product indicated that the presence of the inter-subunit disulfide bond affects the affinity of EC-SOD for heparin. The affinity of EC-SOD for heparin is a very important feature of the enzyme because it controls the distribution of the enzyme in tissues. The present study suggests that, not only the processing of the C-terminal region but inter-subunit disulfide bonds also play a role in determining the tissue distribution of EC-SOD. Moreover, the results obtained here also suggest that the redox state of the tissues might regulate the function of the EC-SOD.     

pH-sensitive genipin-cross-linked chitosan microspheres for heparin removal

Chitosan hydrogel microspheres were obtained by cross-linking chitosan in its inverse emulsion using genipin as cross-linker. The genipin-cross-linked chitosan microspheres (ChGp) swell significantly in water at pH values below 6.5 and shrink to a smaller extent at pH values above 6.5. ChGp microspheres bind heparin in water. The kinetics of heparin binding was found to be pH dependent and was faster and more efficient at a lower pH. That can be also controlled by the weight of ChGp microspheres used. Rate and efficiency of heparin adsorption at pH 7.4, which is typical of blood, could be increased by quaternization of ChGp microspheres using glycidyltrimethylammonium chloride (GTMAC). The polymeric material obtained thus can be potentially useful for heparin removal in biomedical applications.     

Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca2+ and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca2+ dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a Kd of approximately 21 nM for the interaction of annexin V with end-chain immobilized heparin and a Kd of approximately 49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The Kd of a heparin octasaccharide binding to annexin V was approximately 1 microM with a binding stoichiometry of 1:1.     

The determination of iduronic acid and glucuronic acid in heparins by a new technique: Re-evaluation of the variable hexuronic acid composition of mammalian heparins

A new technique for the quantitative determination of the uronic acid components of heparin is described. Heparin was deamination with organic nitrite and methanolyzed. The monomeric derivatives of uronic acids were quantitated by gas chromotography. Depolymerization to monomeric units appeared essentially complete as judged from the yield of uronic acid derivatives. By applying the method the relative contents of iduronic acid and glucuronic acid in a number of heparin samples were estimated. In all samples examined the content of iduronic acid was larger than that of glucuronic acid. A species specific difference in the iduronic acid to glucuronic acid ration of heparin was noted. Noticeable difference of this ratio was observed also between different organs of a species of animal and among heparin fractions obtained from an organ.