Interaction Of Heparin Research Articles

Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa.

The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.

Low density lipoprotein receptor-related protein (LRP) is a heparin-dependent adhesion receptor for connective tissue growth factor (CTGF) in rat activated hepatic stellate cells

Connective tissue growth factor (CTGF) is a cysteine-rich, extracellular matrix-associated heparin-binding protein implicated in a variety of fibrotic disorders. CTGF is initially synthesized as a mosaic protein containing four discrete structural modules (CTGF 1–4) but this is susceptible to proteolytic cleavage yielding isoforms comprising modules 3 and 4 (CTGF 3–4) or module 4 alone (CTGF 4). In this study, we show that cultured rat hepatic stellate cells (HSCs) produce CTGF 1–4 and CTGF 3–4 following treatment with transforming growth factor-β and that CTGF is a cell adhesion factor for activated HSCs. Low density lipoprotein receptor-associated protein (LRP) is a receptor for CTGF 1–4 or CTGF 3–4, but not CTGF 4, whereas cell surface heparan sulfate proteoglycans (HSPGs) are binding sites for all CTGF isoforms. Prior occupancy of LRP with other LRP ligands, receptor associated protein, anti-LRP, or a thrombospondin type I peptide (TEWSACSKTCG) resulted in a 50% decrease in the adhesion of activated HSCs to CTGF 1–4 or CTGF 3–4 whereas there was no effect on CTGF 4-mediated adhesion. Co-incubation of CTGF with heparin or perturbation of cell surface HSPGs with heparinase or sodium chlorate completely blocked adhesion of activated HSCs to all CTGF isoforms. Freshly isolated HSCs demonstrated only weak binding to CTGF but strong binding to fibronectin. Thus HSC adhesion is at least partially promoted by CTGF through its binding to LRP, a process that is heparin-dependent. CTGF–LRP interactions are likely mediated by module 3 and CTGF–heparin interactions occur principally in module 4, although additional motifs may account for the heparin-dependency of LRP binding. These data show that LRP and HSPGs are utilized by HSCs for binding to CTGF and suggest that these cell surface molecules may be involved in mediating CTGF activity or adhesive signaling during the activation process.

Interaction of fibrin(ogen) with heparin: further characterization and localization of the heparin-binding site.

The beta chain 15-42 sequence of the fibrin(ogen) E region was implicated in heparin binding [Odrljin et al. (1996) Blood 88, 2050-2061]; whether heparin binds to other fibrin(ogen) regions remains to be clarified. To address this question, we studied the interaction of heparin with fibrinogen, fibrin, and their major fragments D(1), D-D, E(1), E(3), and alphaC, which together cover the entire structure of the molecule, by ligand blotting, surface plasmon resonance, and fluorescence. All three techniques revealed that at physiological ionic conditions only fibrin(ogen) and the E(1) fragment bind heparin, indicating that the only physiologically relevant heparin-binding site of fibrin(ogen) is located in its E region. To test whether the beta15-42 sequence is sufficient to form this site or some additional sequences are also involved, we tested the interaction of heparin with a number of beta15-42-containing fragments. The synthetic beta15-42 peptide bound heparin weakly (K(d) = 44.5 microM) while the recombinant beta15-57 and beta15-64 fragments exhibited almost 7-fold higher affinity (K(d) = 6.4 and 7.1 microM, respectively), indicating that the beta43-57 region is also important for heparin binding. At the same time the recombinant dimeric disulfide-linked (beta15-66)(2) fragment which mimics the dimeric arrangement of the beta chains in fibrin bound heparin with high affinity (K(d) = 66 nM), almost 100-fold higher than that for the monomeric fragments. This affinity was similar to those determined for fibrin and the E(1) fragment (K(d) = 72 and 70 nM, respectively) suggesting that (beta15-66)(2) mimics well the heparin-binding properties of the latter two. Altogether, these results indicate that the only heparin-binding site in fibrin(ogen) is formed by NH(2)-terminal portions of the beta chains, including residues beta15-57, and that dimerization is essential for high-affinity binding.

Ionic modulation of the effects of heparin and 6-aminohexanoic acid on plasminogen activation by streptokinase: The role of ionic strength, divalent cations and chloride

Studies were conducted on the effect of heparin or 6-aminohexanoic acid (6-AH) on the activation of glutamic plasminogen (Glu-Plg) by streptokinase in the presence of different concentrations of buffer, NaCl and divalent cations. Heparin and 6-AH inhibited streptokinase-mediated activation of Glu-Plg using 10 mM Tris-HCl buffer pH 7.4. This inhibition was partially reversed by the addition of 0.2-1.0 mM of Mg ions. Increasing the ionic strength of Tris-HCl buffer from 10 to 50 mM or addition of 50-150 mM of NaCl to 50 mM Tris-HCl pH 7.4 inhibited the activation of Glu-Plg by streptokinase while decreasing the % inhibition by heparin over the control samples. Double reciprocal plot of the activation of Glu-Plg by streptokinase using 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl showed that the addition of heparin lowered Vmax by 50% without affecting Km. To determine whether the inhibitory effect of heparin was specifically directed towards Glu-Plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of heparin over the controls were plotted against the inverse of the volume fraction of Glu-Plg or streptokinase after serial dilutions. The results indicated that the dilutions of streptokinase but not of Glu-Plg influenced the ratios, suggesting an interaction of heparin with streptokinase. Addition of 6-AH reversed the inhibitory effect of NaCl on the activation of Glu-Plg by streptokinase and the results of the near UV CD spectra of Glu-Plg showed that addition of 6-AH enhanced the spectra in this region with an increase in the ellipticity which was not affected by addition of NaCl.

Two Different Heparin-binding Domains in the Triple-helical Domain of ColQ, the Collagen Tail Subunit of Synaptic Acetylcholinesterase

ColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparin-binding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina.

Capillary electrophoresis: a tool for studying interactions of glycans/proteoglycans with growth factors

Heparan sulfate (HS) and heparin bind to various growth factors and modulate their activities. Interactions of heparin and HS with members of the fibroblast growth factor (FGF) family are prerequisites for binding of FGFs to their high affinity cell receptors. The sulfation patterns of distinct oligosaccharide domains within heparin and HS chains determine their high affinity binding with basic FGF (bFGF). In order to study the structural basis of interactions of HS with bFGF, we developed a capillary electrophoresis (CE) method in order to monitor the ability of HS-derived oligosaccharides to bind this growth factor. HS was degraded to Δ-di- and Δ-oligosaccharides with digestion with heparitinase and the obtained Δ-saccharides were analyzed by capillary zone electrophoresis (CZE), using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Under these conditions all differently sulfated HS Δ-disaccharides and the various Δ-oligosaccharide groups were resolved. Following incubation of the digest with bFGF and re-electrophoresis of the mixture, the bFGF interacting oligosaccharide groups were easily detected and identified. In view of the obtained results, CE is a multipotent analytical tool for determining disaccharide composition in HS, separating the various oligosaccharide groups produced by the action of heparitinase and identifying those interacting with bFGF.

A kinetics and modeling study of RANTES(9-68) binding to heparin reveals a mechanism of cooperative oligomerization.

Heparan sulfate (HS) and heparin bind to virtually all chemokines and have been shown to play critical roles in the regulation of their activities. However, both binding mechanisms and structural features involved in chemokine-HS interactions remain poorly defined. In the study presented here, we analyzed the binding of heparin to RANTES(9-68), a N-terminally truncated form of the CC-chemokine RANTES. Using biochemical and surface plasmon resonance (BIAcore system) approaches, we showed that the RANTES(9-68)-heparin interaction was characterized by a complex binding model that involved dimerization of the chemokine through a mechanism of positive cooperativity. Since RANTES(9-68) remains monomeric in solution, we concluded that heparin induced chemokine dimerization. The structure of a complex involving a RANTES dimer and a heparin heptadecasaccharide was proposed by molecular modeling. This model was used to design a dimer of "head to head" coupled octasaccharides that would fit the internal symmetry of the chemokine dimer. This engineered oligosaccharide bound RANTES(9-68) much better than a natural heparin fragment of the same length, further supporting the interaction process and the proposed structural model. Altogether, the data reported here provide a basis for understanding the mechanisms by which HS modulates RANTES functions.

Protein–heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization

Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein–heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.

Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III

Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). The dissociation constant (K(d)), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. The conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K(d) values were 17 nM and 100 nM respectively. The CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K(d) values. The synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-d-glucosamine; G, beta-d-glucuronic acid; A*, N,3,6-O-sulphated alpha-d-glucosamine; I, 2-O-sulphated alpha-l-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500 Da heparin was displaced by protamine. In conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.

Heparin-protein interactions.

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins.

Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

It has been shown previously that the long chain fragments of heparin bind to the beta-strand cationic belt of the three-finger cobra cardiotoxin (or cytotoxin, CTX) and hence enhance its penetration into phospholipid monolayer under physiological ionic conditions. By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown by (1)H NMR study that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the beta-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX toward LPC. The Fourier-transform infrared spectra and NMR nuclear Overhauser effect of Hep-6.CTX and CTX.LPC complex in aqueous buffer also supplemented the aforementioned observation. Thus, the detected conformational change may presumably be the result of structural coupling between the connecting loops and its beta-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane-bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.

Resonance Rayleigh scattering spectra for studying the interaction of heparin with some basic phenothiazine dyes and their analytical applications

In near neutral medium, binding of heparin with some basic phenothiazine dyes such as methylene blue (MB), Azure B or toluidine blue (TB) can result in a significant enhancement of resonance Rayleigh scattering (RRS) and appearance of a new RRS spectrum. Their maximum scattered wavelengths appear at 346, 338 and 329nm for MB, Azure B and TB, respectively. The optimum conditions of these reactions, the influencing factors and the relationship between the RRS intensity and the concentration of heparin have been investigated. A new method for the determination of trace amounts of heparin based on the RRS method has been developed. The methods exhibit high sensitivities, the detection limits for heparin are 9.0ngml−1 for MB, 14.5ngml−1 for Azure B and 26.0ngml−1 for TB. Among them the MB is the most sensitive and has better selectivity, so it was applied to the determination of heparin in heparin sodium injection samples with satisfactory results. The mechanisms of the effect are discussed.

Interaction of oligopeptides with heparin

This heparin affinity chromatography study revealed some very interesting aspects with respect to oligopeptide retention. The first and most surprising effect observed was that oligopeptides with no aromatic groups were not retained on the column while those with aromatic groups were retained. The aromatic interaction was determined to be related to a charge transfer -like interaction as electron donating groups on the aromatic moiety increased retention on the column and electron withdrawing groups on the aromatic moiety deceased retention time. Column retention was dependent on oligopeptide size; the tripeptides were not retained while tetrapeptides and the larger hexapeptides were effectively retained if they contained some aromatic functionality. Retention was pH dependent; low pH's of ∼2.5 were most effective while neutral pH was not effective in retaining the oligopeptides on the heparin affinity column. In addition to the aromatic interaction, retention was dependent on the hydrophobicity of the oligopeptides as well as their ability to hydrogen bond as determined by eluent solvent effects on the heparin affinity column. These results have led us design and carry out other experiments to determine more accurately the type and the degree of oligopeptide to heparin interaction.

Inhibition of antiphospholipid antibody (APA) activity with unfractionated (UF) and low molecular weight (LMW) heparin.

Objective: Numerous studies have compared heparin interactions with platelet-endothelial cells and plasma proteins to investigate possible mechanisms of action. Unfractionated (UF) heparin has been utilized as the treatment of choice in women with recurrent pregnancy loss (RPL) and antiphospholipid antibodies (APA). Recently, some investigators have advocated low molecular weight (LMW) heparin because it has been associated with a low incidence of osteoporosis, a low risk of bleeding and requires less frequent administration. Thus, we compared the competitive interactions of UF and LMW heparin on APA binding to phospholipids in the sera of patients with RPL and APA. Design: Patients with positive levels of IgG APA (≥30 phospholipid units) and ≥2 consecutive pregnancy losses met the criteria for the Antiphospholipid Antibody Syndrome (APS). First, the sera were assayed in an APA ELISA to determine the interactions of APA binding to phospholipids with UF heparin and LMW heparin in dose response manner. Next, the sera from all patients with positive levels of APA were fractionated by heparin affinity chromatography to compare the binding avidity between UF heparin and LMW heparin. Materials/Methods: The sera of 11 women with IgG antibodies to the phospholipids cardiolipin (CL) and phosphatidylserine (PS) were selected based on a positive test (≥30 phospholipid units) by a standard APA ELISA. The serum samples were reassayed with a modified ELISA utilizing increasing doses of UF heparin and LMW heparin (0, 16, 32, 64, 128, and 256 IU). The 50% inhibitory concentration of UF heparin and LMW heparin for each sample was determined. Affinity chromatography with UF and LMW heparin was performed on all patient sera and fractioned into two pools. The fractions containing the protein that passed through the column and the fractions that were absorbed to heparin then eluted, were pooled separately and reassayed for APA activity by a standard ELISA. Results: All patient sera containing IgG CL and PS antibodies demonstrated a dose response inhibition of binding to phospholipids with the addition of both UF or LMW heparin. APA binding activity in the ELISA was inhibited ≥80% in all patient samples with 50 to 500 IU of UF and LMW heparin. To decrease the mean IgG CL activity (63.4 GPL) by 50%, 93.4 IU ± 57.9 of UF heparin compared to 52.5 IU ± 28.2 of LMW heparin was required. Similarly, 104.5 IU ± 68.6 of UF heparin compared to 84.2 IU ± 50.0 of LMW heparin was required to inhibit the mean IgG PS activity (81.3 GPL) by 50%. Per IU of activity, LMW heparin was more effective in reducing the binding of APA in most of the individual samples. After absorption by affinity chromatography with UF or LMW heparin, 80% of the activity was bound to heparin while 20% of the APA activity passed through the column. The majority of the APA activity was recovered after elution from the column (absorbed to heparin). Conclusions: APA binds directly to heparin (UF and LMW) as demonstrated by affinity chromatography. Heparin (UF and LMW) directly inhibits APA binding to phospholipids in a dose response fashion as demonstrated by inhibition curve. These data suggest that on a per unit basis LMW heparin inhibits the binding of APA more effectively than UF heparin. Clinical studies are in progress to determine if these in vitro findings correlate with possible outcomes when treating women with APS.

The Analysis of Heparin–Protein Interactions Using Evanescent Wave Biosensor with Regioselectively Desulfated Heparins as the Ligands

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin–protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.

Characterization of Slit Protein Interactions with Glypican-1

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.

Resonance Rayleigh scattering study of the interaction of heparin with some basic diphenyl naphthylmethane dyes.

In a near-neutral medium, a combination of heparin with some basic diphenyl naphthylmethane dyes such as victoria blue 4R (VB4R), victoria blue B (VBB), or night blue (NB) can result in a significant enhancement of resonance. Rayleigh scattering (RRS) and their maximum scattering wavelengths lambdamax appear at 523, 534, and 540 nm for VB4R, VBB, and NB, respectively. The characteristics of RRS spectra of the heparin-dye complexes, the influencing factors, and the optimum conditions of these reactions have been investigated. The RRS intensity is directly proportional to the concentration of heparin in the range of approximately 0 to 0.4 microg/mL for all systems. The methods exhibit high sensitivities, and the detection limits for heparin are 3.35 ng/mL for the VB4R system, 6.62 ng/ mL for the VBB system, and 6.29 ng/mL for the NB system. Because the VB4R system is the most sensitive, it was taken as an example to study the selectivity of the method. A new method for the determination of trace amounts of heparin based on RRS technique has been developed. Moreover, the enhancement reasons of RRS and the relationship between RRS spectral characteristics of the heparin-dye complex and its absorption spectra have been primarily discussed.

Certain High Molecular Weight Heparin Chains Have High Affinity for Vitronectin

Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the interaction of heparin, vitronectin, and the complement system. Previous studies demonstrated that heparin diminishes vitronectin inhibition of complement activity. Numerous studies have also demonstrated that heparin exerts a net inhibitory effect on complement. We used two dimensional affinity resolution electrophoresis (2DARE) to examine this apparent paradox. 2DARE allowed simultaneous determination of binding affinity of heparin for vitronectin as well as the Mr of the heparin species. In the 2DARE experiment, the interaction of heparin with vitronectin caused retardation of the movement of the heparin through the tube gel in the first dimension. The degree of the retardation of movement was used to calculate the approximate Kd of that interaction. The heparin from the tube gel was then subjected to a second dimension electrophoresis to determine the Mr of the heparin. 2DARE analysis of the interaction of heparin with vitronectin clearly demonstrated that a sub-population of heparin chains with Mr > 8000 bound vitronectin with high affinity whereas most high Mr chains and all lower Mr chains showed little to no affinity for vitronectin. Our findings are consistent with the hypothesis that a unique binding domain exists in certain heparin chains for vitronectin.

Identification and Characterization of a Conformational Heparin-binding Site Involving Two Fibronectin Type III Modules of Bovine Tenascin-X

Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.

Structure and biological interactions of heparin and heparan sulfate

Publisher Summary This chapter discusses the structural aspects and structure–activity relationships of heparin/heparan sulfate (HS). The molecular mechanism behind the anticoagulant/antithrombotic effects is also presented. Heparin has been in clinical use for several past years preventing and curing thromboembolic disease. HS is now recognized as a family of multiple, closely related yet distinct polysaccharide species. In fact, heparin may well be considered merely another member of this family. Owing to the structural diversity of the heparin/HS family, the polysaccharides are capable of binding a large number of different proteins, and thus may be conceived as important regulators of a variety of biological processes. Several biological effects exerted by exogenous heparin are now being considered in terms of reinforcing or competing with HS functions. Biological activities of heparin/HS other than anticoagulant/antithrombotic are also reviewed in the chapter. Treatment of heparin/HS with individual lyases generates a variety of oligosaccharides. Heparin preparations from pig intestinal mucosa incubated with heparin lyase I currently generate five major di- and oligo-saccharides, the most abundant one arising from sequences of the trisulfated disaccharide IdoA2SO 3 -GlcNSO 3 6SO 3 . Probably because the enzyme requires at least three consecutive units of this trisulfated disaccharide for effective action, about one-third of these sequences usually remain as tetrasaccharides.