Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin (original) (raw)
Related papers
Analytical Biochemistry, 2002
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.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2009
Background: Although protamine is effective as an antidote of heparin, there is a need to replace protamine due to its side effects. HIP peptide has been reported to neutralize the anticoagulant activity of heparin. The interaction of HIP analog peptides with heparin and heparin-derived oligosaccharides is investigated in this paper. Methods: Seven analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (HIP) were synthesized, and their interaction with heparin was characterized by heparin affinity chromatography, isothermal titration calorimetry, and NMR. Results: NMR results indicate the imidazolium groups of the His side chains of histidine-containing Hip analog peptide interact site-specifically with heparin at pH 5.5. Heparin has identical affinities for HIP analog peptides of opposite chirality. Analysis by counterion condensation theory indicates the peptide AC-SRPKAKAKAKAKDQTK-NH 2 makes on average ∼ 3 ionic interactions with heparin that result in displacement of ∼ 2 Na + ions, and ionic interactions account for ∼ 46% of the binding free energy at a Na + concentration of 0.15 M. Conclusions: The affinity of heparin for the peptides is strongly dependent on the nature of the cationic side chains and pH. The thermodynamic parameters measured for the interaction of HIP peptide analogs with heparin are strongly dependent on the peptide sequence and pH. General significance: The information obtained in this research will be of use in the design of new agents for neutralization of the anticoagulant activity of heparin. The site-specific binding of protonated histidine side chains to heparin provides a molecular-level explanation for the pH-dependent binding of β-amyloid peptides by heparin and heparan sulfate proteoglycan and may have implications for amyloid formation.
Biochemical and Biophysical Research Communications, 2006
The biological roles of heparin (HP) and heparan sulfate (HS) are mediated mainly through their interaction with proteins. In the present work, we provide a rapid method for screening HP/HS-protein interactions providing structural data on the key sulfo groups that participate in the binding. A library of polysaccharides structurally related to HP was prepared by immobilizing the biotinylated Nsulfated K5 polysaccharide (N-sulfoheparosan) on sensor chips followed by selective modification of this polysaccharide with enzymes that participate in HP/HS biosynthesis. The polysaccharides synthesized on the surface of the sensor chips differ in the number and position of sulfo groups present both on uronic acid and glucosamine residues. Surface plasmon resonance was used to measure the interaction of each member of this polysaccharide library with antithrombin III (ATIII), to afford structural information on sulfo groups required for this HP/HS-protein interaction. This method is viewed as widely applicable for the study of the structure-activity relationship (SAR) of HP/HS-protein interactions.
The Journal of biological chemistry, 1988
To study the structural requirements in heparin for interaction with heparin cofactor II (HC II) we have analyzed the properties of oligosaccharide fractions obtained after digestion of heparin by heparinase and gel filtration. No activation of HC II was detected in the presence of di-, tetra-, hexa-, octa-, deca-, or do-decasaccharides. The hexasaccharide pool was fractionated by ion-exchange chromatography, and the structure of the major species, obtained in a homogeneous state, was investigated by NMR. All the resonances were unambiguously assigned using correlation by homonuclear and heteronuclear scalar coupling. The six monosaccharide residues of this hexasaccharide were thus easily identified. The sequence was established through two-dimensional nuclear Overhauser effect experiments. The results indicate that this product is a hexasaccharide recently described by Linhardt et al. (Linhardt, R. J., Rice, K. G., Merchant, Z. M., Kim, Y. S., and Lohse, D. L. (1986) J. Biol. Chem....
Heparin interaction with protein-adsorbed surfaces
Journal of Colloid and Interface Science, 1986
Albumin and fibrinogen show no binding affinity to varied molecular weights ofhepafin at physiological pH. Human plasma fibronectin was shown to bind heparins in both the solution and adsorbed states. Fibronectin was shown to have six active binding sites for heparins which may be stericaUy blocked in some adsorbed states. 125I-Fibronectin monolayer concentrations were shown to be significantly different on polyvinyl chloride surfaces when compared to hydrophilic/hydrophobic silica, Biomer, Silastic, and polystyrene surfaces. Preadsorbing fibronectin to various substrates and then allowing heparins to interact with the protein monolayer made it possible to bind up to 0.2 t~g/cm 2 heparin in a plasma environment. This fibronectin-heparin complex was at least 85% stable in plasma and buffer solutions for up to 8 h time. The complex was observed to prolong blood clotting times two to three times over that of controls as assayed by Activated Partial Thromboplastin Times. All of the bound heparin was observed to be active by its ability to bind Factor X, in plasma. Monolayers of blood proteins adsorbed from human serum were not observed to be active in binding heparins. The fibronectin-heparin conjugate showed low activation of blood components compared to protein monolayers preadsorbed from human sera as assayed by Activated Partial Thromboplastin Time.
Plasma Protein Binding Properties to Immobilized Heparin and Heparin?Albumin Conjugate
Artificial Organs, 2007
Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regarding hemocompatibility of foreign materials in contact with blood. Prothrombin, thrombin, antithrombin III (AT3), and fibrinogen were selected for analysis in an experimental model. Biomolecular interaction analysis employing surface plasmon resonance was utilized to record and analyze their binding properties in real time. Biotinylated heparin, heparin-albumin conjugate, and albumin, respectively, were immobilized onto streptavidin-coated sensors as ligands. Prothrombin did not bind to any of the ligand surfaces and no specific binding of any of the plasma proteins to albumin was observed. Binding kinetics of thrombin to heparin and to heparin-albumin conjugate were calculated using two different methods. For heparin, identical KD (equilibrium dissociation constant) values of 61 ¥ 10 -9 M were obtained with both methods. For the conjugate, only slightly different KD values of 111 ¥ 10 -9 and 104 ¥ 10 -9 M, respectively, were calculated. The affinity of thrombin toward the heparin-coated surface proved to be higher than its affinity toward the heparin conjugate. The binding pattern of AT3 to both heparin and heparinalbumin conjugate, although specific, was biphasic, possibly due to a conformational change during the binding process. Steady-state kinetic analysis revealed a KD value of 281 Ϯ 24 ¥ 10 -9 M for the heparin surface. For the conjugate surface, a KD of 53 Ϯ 5 ¥ 10 -9 M was calculated, indicating a higher affinity toward heparin-albumin conjugate. A high-affinity binding of fibrinogen to high-density surfaces of both heparin and the conjugate was observed. However, as binding to low-density surfaces was considerably reduced, specificity remained uncertain.
Analytical Biochemistry, 2013
Glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction of heparin currently used as an anticoagulant and antithrombotic drug, are arousing increasing interest in anticancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs heparins has not been studied yet in detail. In this work, ion pair reversed-phase highperformance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs heparins usually prepared from porcine mucosal heparin. The method was also found to be very effective in analyzing gs derivatives obtained from heparins of different animal and tissue origins. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs heparins contain mainly tetra-and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues was shown to be a marker of the gs-modified AT binding site within heparin chains.
The interaction of heparin with human plasmin
International Journal of Biochemistry, 1983
The interaction of heparin with human plasmin was investigated measuring plasmin activity and enzyme inactivation in the presence of heparin. Hydrolysis of synthetic substrates (H-oVal -Leu-Lys-pNA, H-D-Val-Phe-Lys-pNA and H-o-Pro-Phe-Lys-pNA) by plasmin was enhanced by heparin through an increase in k,,, values. 2. This effect was the consequence of a change of V,,, since K, values were not altered in the presence of heparin. The polysaccharide also enhanced the rate of enzyme inactivation using TLCK as an active site blocking reagent. 3. Furthermore, heparin increased the heat sensitivity of plasmin, when synthetic substrate H-D-Val-Leu-Lys-pNA was used but it did not affect enzyme activity towards N-benzoyl-L-arginine-ethylester substrate. 4. The data show that microenvironmental conformation around the active center of plasmin is influenced by heparin.
Interaction of Heparins with Fibroblast Growth Factors: Conformational Aspects
Current Pharmaceutical Design, 2007
Heparin and heparin-like oligo-and polysaccharides bind to fibroblast growth factors (FGFs) and modulate their ability to form active ternary complexes with FGF receptors (FGFRs). Considerable efforts have been made in recent years to identify the minimal heparin and heparan sulfate (HS) sequences that bind and activate individual FGFs. Heparin sequences involved in interaction with FGFs invariably contain at least one residue of 2-O-sulfated iduronic acid (IdoA2S), which adopts either the 1 C4 chair conformation or the equienergetic skew-boat 2 S0. In solution and in the absence of a binding protein, both these conformations are present in a dynamic equilibrium. In oligosaccharide-protein co-crystals, the protein selects those conformers that provide optimal contacts. The crystalline structure of a heparin hexasaccharide/FGF complex exhibits one of the two IdoA2S residues in the active site of the growth factor in 1 C4 conformation and the other (outside the active site) in 2 S0 conformation. NMR studies suggest that active conformations of heparin/HS oligosaccharides in solution could be distinct from those adopted in crystals. Heparin tetrasaccharides in the presence of FGF1 and FGF2 have both their IdoA2S residues prevalently in the 1 C4 form. Current NMR and molecular modelling studies are being extended to longer heparin oligosaccharides as well as to heparins with "glycol-split" residues along their chains.
Biochemistry, 2012
Heparin and related heparan sulfate interact with a number of cytokines and growth factors thereby playing an essential role for many physiological and pathophysiological processes by involving both signal transduction and the regulation of the tissue distribution of cytokines/growth factors. Follistatin (FS) is an autocrine protein with a heparin-binding motif that serves to regulate the cell proliferative activity of the paracrine hormone, and member of the TGF-β family, activin A (ActA). Follistatin is currently under investigation as an antagonist of another TGF-β family member myostatin (Mstn) to promote muscle growth in diseases associated with muscle atrophy. In the present study, we employ surface plasmon resonance (SPR) spectroscopy to dissect the binding interactions between the heparin polysaccharide and both free follistatin (FS288), and its complexes (FS288-ActA, FS288-Mstn). FS288 complexes show much higher heparin binding affinity than FS288 alone. SPR solution competition studies using heparin oligosaccharides showed that the binding of FS288 and its complex to heparin is chain length dependent. Full chain heparin or large oligosaccharides, having 18 to 20 sugar residues show the highest binding activity for FS288 and FS288-ActA, whereas smaller heparin molecules could interact with the FS288-Mstn complex. These interactions were also analyzed in normal physiological buffers and at different salt concentrations and pH values. Unbound follistatin was much more sensitive to all salt concentrations above 150 mM. Heparin binding of the FS288-ActA complex was disrupted at 500 mM salt, whereas it was actually increased for the FS288-Mstn complex. At acidic pH values, heparin binding to FS288 and FS288-ActA binding was enhanced. While slightly acidic pH values (pH 6.2 and 5.2) enhanced FS288-Mstn binding to heparin, at pH 4 heparin-binding was inhibited. Overall these studies demonstrate that specific ligand binding to FS288 differentially regulates its affinity and behavior for heparin molecules.