Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin.
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ABSTRACT:
SUBMITTER: Horner AA
PROVIDER: S-EPMC1148975 | biostudies-other | 1988 Apr
REPOSITORIES: biostudies-other
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Project description:Approximately half of all rat skin heparin proteoglycans have polysaccharide chains that have no sites with high binding affinity for antithrombin. The rest have chains with high-affinity antithrombin-binding-site densities ranging from zero to five sites per chain, with a high degree of variation. Proteoglycans vary in size because of diversity in the number of chains per molecule; the relationship between proteoglycan size and high-affinity antithrombin-binding-site density has not been studied previously. Polydisperse heparin proteoglycans from rat skin, labelled biosynthetically with 35S, were fractionated by gel filtration on Bio-Gel A-150m and arbitrarily divided into five fractions of decreasing average molecular size. Fractionation of these products on antithrombin-agarose showed that the proportion of proteoglycans with high affinity for antithrombin decreased from 39% to 25% as molecular size decreased. However, as the molecular size of high-affinity proteoglycans decreased, the proportion of their chains that had high affinity increased from 29% to 59%. Therefore molecular size is a significant factor in determining the proportion of high-affinity chains in heparin proteoglycans. A model of heparin biosynthesis is proposed in which areas of specific enzyme activity that control the synthesis of the antithrombin-binding-site sequence are sparsely and nonrandomly distributed on mast-cell Golgi membranes. It is postulated that the likelihood of a developing proteoglycan encountering one of these hypothetical areas is molecular-size-dependent.
| S-EPMC1133365 | biostudies-other
Project description:Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin.
| S-EPMC4913942 | biostudies-literature
Project description:Subfractions of 35S-labelled rat skin heparin proteoglycans with various degrees of high affinity for antithrombin were obtained by gradient elution from a column of antithrombin-agarose. Heparin chains released from the proteoglycan preparations by beta-elimination with alkali were re-fractionated on the same column. Proportions of chains with high affinity for antithrombin (HA-chains) ranged from 17% to 76%. These separations also revealed three overlapping subfractions of HA-chains. Their proportions varied in a manner consistent with a stepwise increase in the degree of affinity of HA-chains for antithrombin, this presumably being due to the biosynthesis of increasing numbers of antithrombin-binding sites per chain. The anticoagulant activity, with respect to thrombin neutralization, ranged from 32 units/mg to 287 units/mg. It is suggested that HA-chains may have from one to five or six antithrombin-binding sites. Thus the asymmetric distribution of these sites in rat skin heparin proteoglycans is much more marked than was realized from the earlier work of Horner & Young [(1982) J. Biol. Chem. 257, 8749-8754].
| S-EPMC1148051 | biostudies-other
Project description:Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin's anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described.
| S-EPMC5553205 | biostudies-literature
Project description:Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions. SUMMARY:Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-?B or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.
| S-EPMC5809256 | biostudies-literature
Project description:Rat skin heparin proteoglycan labelled biosynthetically with 35S was fractionated on a column of antithrombin-Sepharose into fractions with varying degrees of affinity for antithrombin. These were treated with NaOH to release heparin chains (Mr 60,000-100,000), by beta-elimination or incubated with serum to produce fragments of the same order of size as commercial heparin (Mr 5000-30,000), by endoglycosidase cleavage. Chains and fragments were then fractionated on antithrombin-Sepharose. The various fractions were deaminated with HNO2 at pH 1.5 followed by reduction with NaB3H4. Approx 90% of the incorporated 3H was associated with disaccharides. These were fractionated by high-performance ion-exchange chromatography. A unique minor component corresponding to the sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate) in the polysaccharide was found only in fractions with high affinity for antithrombin. The glucosamine residue linked to C-4 of this glucuronosyl unit was predominantly (or exclusively) N-sulphated rather than N-acetylated, pointing to a structural difference between the antithrombin-binding region of rat heparin and that of pig mucosal heparin. Calculations based on the distribution of the glucosaminyl 3-O-sulphate group showed that approximately two-thirds of the total antithrombin-binding regions present in the unfractionated material were accommodated by only 20% of the proteoglycan molecules, and by 10% of the polysaccharide chains. While most of the proteoglycan molecules thus lacked such regions (and hence affinity for antithrombin) a minor proportion of the polysaccharide chains contained on the average three binding regions per molecule. These findings support by direct chemical analysis an earlier proposal, based on anticoagulant activities of similar rat skin heparin fractions, that the distribution of antithrombin-binding sites in intact heparin proteoglycans is markedly non-random.
| S-EPMC1147467 | biostudies-other
Project description:[35S]Heparin proteoglycans were isolated from the skins and peritoneal mast cells of male rats aged 2 to 22 months. Their [35S]heparin chains were separated on antithrombin-agarose into fractions with high and low affinities for antithrombin. In skin, the proportion of 35S-labelled high-affinity heparin chains declined from 23% at 2 months to 8% at 12 months and did not change significantly between 12 and 22 months. In peritoneal mast cells, the proportion of 35S-labelled high-affinity heparin chains increased from 14% at 2 months to 21% at 4 months and then did not vary significantly until 15 months of age. By 21 months a consistent and significant decline to 8% occurred. The structures of high-affinity heparin proteoglycans did not change with age. Their decreased proportions, without change in their structure, may indicate that they are produced by a unique subset of mast cells, the proportion of which declines with age. [35S]heparan sulphate chains were isolated from skins and brains of rats in the same age range and fractionated on antithrombin-agarose. There were no significant variations in the proportions of 35S-labelled high-affinity heparan sulphate chains in skin (10%) or brain (24%) between 4 and 22 months of age.
| S-EPMC1136251 | biostudies-other
Project description:The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.
| S-EPMC1609903 | biostudies-literature
Project description:Heterogeneity in the composition and in the polydispersity of heparin has motivated the development of homogeneous heparin mimics, and peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in selected applications. Here, we report the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for vascular endothelial growth factor isoform 165 (VEGF165); these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF165 was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in selected cases, isothermal titration calorimetry (ITC). The shortest peptide, SP(a), showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SP(a) and PF4(ZIP) was indicated via SPR (K(D)=5.27 microM) and confirmed via ITC (K(D)=8.09 microM). The binding by SP(a) of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications.
| S-EPMC3100587 | biostudies-literature
Project description:The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A(NAc,6S)-GlcUA-A(NS,3,6S)-I(2S)-A(NS,6S)). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.
| S-EPMC3764794 | biostudies-literature