Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.     

A comparative study on the heparin‐binding proteomes of Toxoplasma gondii and Plasmodium falciparum

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin‐binding proteome of T. gondii. The parasite‐derived components were affinity‐purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin‐binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion‐related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry‐derived proteins were prominently identified. The profiling of the heparin‐binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin‐mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.     

Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

Heparin and heparin-derived components are widely applied anticoagulant drugs used for amongst other applications medical treatment of deep vein thrombosis and pulmonary embolism. Depolymerisation of native heparin results in complex mixtures of sulfated linear oligosaccharides that are usually not well characterised. In order to further characterise such mixtures, two on-line ion-pairing reverse-phased chromatography electrospray ionisation (ESI) mass spectrometry methods have been developed. One of the systems allows the determination of more than 200 components in a medium molecular weight heparin preparation, whereas the other system can be used to separate isomeric heparin oligosaccharides after previous separation according to size. This latter system allows semi-preparative isolation of isomeric heparin oligosaccharides. The experimental setup includes on-line cation exchange in order to prevent the ion-pairing reagent from entering the mass spectrometer.     

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.     

Use of insoluble heparin for isolation of DNA polymerase enzymes from murine myeloma

Heparin was found to be a potent inhibitor of a DNA polymerase present in the murine myeloma tumor MGPC-21. By increasing the KCl concentration of the reaction mixture, the inhibition of this enzyme could be completely reversed, suggesting that insoluble heparin might be a useful tool in the isolation of DNA polymerases. When heparin covalently bound to Sepharose was used, some of the DNA polymerases present in MOPC-21 myeloma tumors were separated and partially purified.     

Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans

Vaccinia virus complement control protein (VCP) inhibits both pathways of complement activation through binding the third and fourth components. A homolog of mammalian regulators of complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. This, the structure of any intact regulator of complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of complement.     

Heparin binding domain of human antithrombin III inferred from the sequential reduction of its three disulfide linkages. An efficient method for structural analysis of partially reduced proteins

Human antithrombin III (AT-III) was partially reduced under mild conditions in the absence or presence of low molecular weight heparin. Quantitation of reduced disulfide bonds was facilitated by the application of a water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid (S-DABIA). The study shows that the three disulfide linkages of AT-III can be sequentially reduced, with Cys8-Cys128 being the most sensitive, followed by Cys21-Cys95, while Cys247-Cys430 is the most resistant to the mild reduction conditions. The rate of reduction of Cys8-Cys128 and Cys21-Cys95 was significantly decreased in the presence of heparin. The reduction of Cys8-Cys128 was also found to correlate quantitatively with the loss of heparin-accelerated antithrombin activity, heparin binding affinity, and heparin-induced fluorescence enhancement. These results suggest that Cys8-Cys128 is required for the integrity of the heparin binding domain of AT-III and support previous findings that lysyl residues surrounding Cys128 (Lys107, Lys114, Lys125, and Lys136) constitute an important part of the heparin binding site in AT-III.     

Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focusing.

The mechanism of complexation of pI range 3.5--5 Ampholine to heparin in isoelectric focusing has been explored by the dye-binding technique at different pH values in solution. There is no significant interaction between heparin and Ampholine at pH 6.7. Weak, or selective, binding occurs at pH 5.1, and very strong interaction at pH 3.5. In the latter system, the Ampholine components appear to behave as polycations due to their ordered sequence of positive charges, each two methylene groups apart, which favors a strong binding to polyanions. In addition, there appear to be variable stoichiometries for the strong binding between heparin and Ampholine, depending on their relative amounts. It is proposed that at a low ratio of heparin to Ampholine (Ampholine excess), aggregation is perpendicular to the heparin chain, with the end ammonium charge of each Ampholine molecule neutralizing one negative charge along the heparin molecule; at higher ratios (heparin excess), the bound Ampholine segment is aligned parallel to the heparin molecule, so that on the average one Ampholine component neutralizes approx. three negative charges. The banding of heparin in isoelectric focusing in the pH range 3.0--4.5 can be explained by aggregation of the various components on heparin in amounts dependent upon the net charge on the Ampholine species at the given pH, and upon the changing stoichiometries as a function of the variation in ratio of heparin to Ampholine along the pH gradient. Binding of Ampholine to polygalacturonate was also demonstrated in excess Ampholine in a pH range dependent on the degree of protonation of the carboxyl groups of this acidic polysaccharide as well as on the net positive charge of the Ampholine. The aggregation seen at pH 4.2--4.5 led to the prediction and subsequent demonstration that polygalacturonate would also exhibit binding upon isoelectric focusing. This supports the hypothesis that aggregation of Ampholine on polyanions having sufficient charge density is a general phenomenon which can lead to spurious banding of certain polymers at appropriate pH ranges in isoelectric focusing. On the basis of their behavior in isoelectric focusing at pH 3.0--4.5, strength of aggregation of the polyanions studied appears to be heparin A = heparin B greather than polyglutamate greater than carboxyl-reduced heparin B greater than polygalacturonic acid.     

Regulatory role of heparin compounds with low molecular ligands of blood in plasma and thromocyte hemostasis

Aanalysis of the literature and our own research on the physiological effects of complex compounds of heparin with low molecular ligands (amino acids, regulatory peptides) is presented. It is proved that anticoagulative effects in blood flow were conditioned by the interaction of heparin with glioproline, immunopeptides, and other low molecular substances with formation of complex compounds. The presence of structural regions of binding of heparin and other components is established. It is indicated that in the blood of animals heparin complexes with low molecular ligands possess protective anticoagulative and antithrombotic effects. We made an attempt to reveal the possible mechanism of anticoagulative-fibrinolytic and antithrombotic action of complex compounds of heparin in the organism.     

FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho

Fibroblast growth factor-21 (FGF-21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-21-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor. Cells lacking betaKlotho did not respond to FGF-21; the introduction of betaKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells. Interestingly, FGF-21-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-21, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-21 receptor complex, mediating FGF-21 cellular specificity and physiological effects.     

Heparin: role in protein purification and substitution with animal-component free material

Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin’s activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.

     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition

Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.     

Heterogeneity of heparin: characterization of one hundred components with different anticoagulant activities by a combination of electrophoretic and affinity chromatography methods

A comparative study of heparin fractions obtained by affinity chromatography, electrofocusing, selective barium precipitation, polyacrylamide and agarose gel electrophoresis is reported. It is concluded that commercial heparin preparations are heterogeneous, containing at least 120 components which differ in molecular weight, in degree of affinity for antithrombin, and in their distribution in monomeric and dimeric forms. High anticoagulant activity for some heparin fractions was obtained by most of the methods used.     

Profiling heparin-chemokine interactions using synthetic tools.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.     

Analysis of the subproteomes of proteinases and heparin‐binding toxins of eight Bothrops venoms

Viperid snakes show the most complex snake‐venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2‐DE, and their subproteomes of proteinases were explored by 2‐D immunostaining and 2‐D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti‐coagulant and anti‐inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high‐affinity for heparin as composed of mainly serine proteinases and C‐type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin‐binding toxins. Only the non‐bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin‐bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.     

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.     

Mode of interaction between platelet factor 4 and heparin

Platelet factor 4 (PF4) is a platelet-derived protein capableof binding to, and thus neutralizing, the biological activitiesof heparin and heparan sulphate. The mode of binding of PF4to heparin was investigated in a comparative study also involvingantithrombin (AT; previously shown to selectively bind a specificoligosaccharide sequence) and fibronectin (FN; non-specificelectrostatic interaction). Heparin-derived saccharides wereincubated with each of the three proteins, followed by separationof free and protein-bound carbohydrate on a nitrocellulose filter.The interaction systems involved either (i) competition forthe protein ligand between ³H-labelled heparin and unlabelled,size-fractionated heparin oligosaccharides (isolated after deaminativecleavage with HNO₂) or (ii) direct binding of ³H-labelled oligosaccharides.Species smaller than octasaccharides were unable to bind AT,whereas binding to FN and PF4 increased continuously throughoutthe series, with increasing size of the oligosaccharides. Furtherseparation by anion-exchange chromatography showed that thePF4-binding and FN-binding octasaccharides represented essentiallyall components present in the initial octasaccharide fraction,the proportion of binding species increasing with charge (hencewith the degree of sulphation). The AT-binding octasaccharides,on the other hand, selectively represented only a few of thetotal octasaccharide components, without any correlation tooverall charge. These results indicate that the binding of PF4to heparin occurs by relatively nonspecific electrostatic interactions.The methodology delineated here may be generally useful in assessingspecificity in glycosaminoglycan—protein interactions. antithrombin fibronectin heparin platelet factor 4     

Characterization of the interaction between tumor necrosis factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin in extracellular matrix microenvironments

TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and site-directed mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-alpha-inhibitor (IalphaI). Experiments using the purified components of IalphaI indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6.IalphaI complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.     

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.