Effects of glycosylation on heparin binding and antithrombin activation by heparin

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

Highly active heparin species with multiple binding sites for antithrombin

Porcine heparin has been fractionated by Sephadex G-100 gel filtration and affinity chromatography into mucopolysaccharide species with approximate molecular sizes of 20,000 daltons, and 7000 daltons, respectively. The larger component has a specific anticoagulant activity of 738 USP units/mg and contains two binding regions for antithrombin. The smaller component has a specific anticoagulant activity of 363 USP units/mg and possesses only a single interaction site for the inhibitor. These results provide the first demonstration that heparin molecules may bear multiple binding sites for antithrombin.     

Phosphate promotes glycation of antithrombin III which interferes with heparin binding

Nonenzymatic glycation of antithrombin III has been reported to cause the reduction of heparin-catalyzed thrombin-inhibiting activity in diabetes. The effect of in vitro nonenzymatic glycation of pure antithrombin III on heparin binding and heparin-potentiated activity under a variety of buffers and pH values was studied to further clarify the physiological significance of this reaction. The extent of glycation, measured by the fructosamine assay and [14C]glucose binding, was enhanced by the presence of phosphate ion (pH 7.45, 8.5 and 9.5) and increased linearly with increasing phosphate ion concentration from 0.01 to 0.2 M phosphate. Conversely, the heparin-catalyzed antithrombin activity decreased from 93.1% of controls for 0.01 M phosphate to 73.5% for 0.2 M phosphate as the extent of glycation increased. The increase in intrinsic fluorescence induced by binding of heparin to antithrombin III was also moderated by glycation of antithrombin III in a dose-dependent manner with a negative correlation coefficient of -0.94. Direct measurement of the heparin binding by affinity chromatography showed a decrease in the heparin-binding fraction which correlated with the degree of glycation and the decrease in heparin-catalyzed activity. These studies suggest that nonenzymatic glycation may be responsible for the reduction in antithrombin III activity observed in some diabetics.     

Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

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.     

Sequence variation in heparin octasaccharides with high affinity for antithrombin III

We have isolated from nitrous acid cleavage products of heparin two major octasaccharide fragments which bind with high affinity to human antithrombin. Octasaccharide S, with the predominant structure iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid-----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is sensitive to cleavage by Flavobacterium heparinase as well as platelet heparitinase and binds to antithrombin with a dissociation constant of (5-15) X 10(-8) M. Octasaccharide R, with the predominant structure iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is resistant to degradation by both enzymes and binds antithrombin with a dissociation constant of (4-18) X 10(-7) M. The occurrence of a 15-17% replacement of N-sulfated glucosamine 3,6-di-O-sulfate with N-sulfated glucosamine 3-O-sulfate and a 10-12% replacement of iduronic acid with glucuronic acid in both octasaccharides indicates that these substitutions have little or no effect on the binding of the oligosaccharides to the protease inhibitor. When bound to antithrombin, both octasaccharides produce a 40% enhancement in the intrinsic fluorescence of the protease inhibitor and a rate of human factor Xa inhibition of 5 X 10(5) M-1 s-1 as monitored by stopped-flow fluorometry. This suggests that the conformation of antithrombin in the region of the factor Xa binding site is similar when the protease inhibitor is complexed with either octasaccharide.     

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.     

Heterogeneity of rat skin heparin chains with high affinity for antithrombin.

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].     

Thermodynamic analysis of heparin binding to human antithrombin.

The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.     

Role of heparin in the interaction of serine proteinases with antithrombin III.

To characterize the mode of action of heparin, the kinetics of inhibition of thrombin, factor Xa, and plasmin by antithrombin III was studied without and in the presence of heparin. Following the concentration dependence of inactivation a linear dependence was found between the apparent first-order inactivation rate constant and the anti-thrombin III concentration. This behaviour is typical of enzyme-activator interaction. Values of kinetic constants of the inactivation reaction could be determined. Thus, heparin acts obviously as an activator of the enzymes and enhances their affinity for antithrombin III.     

Fractionation of low molecular weight heparin species and their interaction with antithrombin.

Preparations of low molecular weight porcine heparin with an average specific anticoagulant activity of 94 units/mg were fractionated into "active" and "relatively inactive" forms of the mucopolysaccharide of approximately 6000 daltons each. The active fraction was further subdivided into various species with descending but significant affinities for the protease inhibitor as well as decreasing but substantial anticoagulatn potencies. "Highly active" heparin (approximately 8% of the low molecular weight pool) possesses a specific anticoagulant activity of 350 +/- 10 units/mg. The relatively inactive fraction (67% of the low molecular weight pool) exhibits a specific anticoagulant activity of 4 +/- 1 units/mg. The binding of highly active heparin to antithrombin is accurately described by a single-site binding model with a KHep-ATDISS of approximately 1 X 10(-7) M. Variations in this binding parameter secondary to changes in environmental variables indicate that charge-charge interactions as well as an increase in entropy are critical to the formation of the highly active heparin-antithrombin complex. The interaction of relatively inactive heparin with the protease inhibitor is characterized by an apparent KHep-ATDISS of 1 X 10(-4) M. In large measure, this is due to small amounts of residual active mucopolysaccharide (0.5%). The ability of the highly active heparin to accelerate the thrombin-antithrombin interaction was also examined. We were able to demonstrate that the mucopolysaccharide acts as a catalyst in this process and is able to initiate multiple rounds of enzyme-inhibitor complex formation. The rate of enzyme neutralization is increased to a maximum of 2300-fold as the concentration of heparin is raised until the inhibitor is saturated with mucopolysaccharide. Further increases in heparin concentration result in a reduction in the speed of enzyme neutralization. This appears to be due to the formation of thrombin-heparin complexes. A mathematical model is given which provides a relationship between the initial velocity of enzyme neutralization and reactant concentrations.     

QCM-FIA with PGMA coating for dynamic interaction study of heparin and antithrombin III

In this work, we describe a method of constructing a film of linear poly(glycidyl methacrylate) (PGMA) polymer onto the surface of quartz crystal microbalance (QCM) electrode as a coating material that allows easy coupling of heparin molecules onto the electrode and facilitates the determination of the interaction between heparin and antithrombin III (AT III). The PGMA film was characterized with atomic force microscopy (AFM) and infra-red spectroscopy. The coupling of heparin was accomplished in one step solution reaction. A home-made quartz crystal microbalance-flow injection analysis (QCM-FIA) system with data analysis software developed in our laboratory was used to determine the interaction. The interactions between immobilized heparin and AT III were studied with various concentrations under various conditions. The obtained constants are kass=(1.49+/-0.12)x10(3)mol-1ls-1, kdiss=(3.94+/-0.63)x10(-2)s-1, KA=(3.82+/-0.33)x10(4)mol-1l.     

Contribution of monosaccharide residues in heparin binding to antithrombin III

The importance of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide iduronic acid(1)----N-acetylglucosamine 6-O-sulfate(2)----glucuronic acid(3)----N-sulfated glucosamine 3,6-di-O-sulfate(4)----iduronic acid 2-O-sulfate(5)----N-sulfated glucosamine 6-O-sulfate(6)----iduronic acid 2-O-sulfate(7)----anhydromannitol 6-O-sulfate(8) was determined by comparing with synthetic tetra- and penta-saccharides its ability to bind human antithrombin. The octasaccharide had an affinity for antithrombin of 1 X 10(-8) M (10.2 kcal/mol) measured by intrinsic fluorescence enhancement at 6 degrees C. The synthetic pentasaccharide, consisting of residues 2-6, had an affinity of 3 X 10(-8) M (9.6 kcal/mol). The same pentasaccharide, except lacking the 3-O-sulfate on residue 4, had an affinity of 5 X 10(-4) M (4.5 kcal/mol) measured by equilibrium dialysis. The tetrasaccharide, consisting of residues 2-5, bound antithrombin with an affinity of 5 X 10(-6) M (6.8 kcal/mol). The tetrasaccharide, consisting of residues 3-6, had an affinity of 5 X 10(-5) M (5.5 kcal/mol). Since the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 4-5 kcal/mol or a 40-50% loss in binding energy of the pentasaccharide, these two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.     

Purification and properties of guinea pig antithrombin III

Guinea pig antithrombin III has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography. The final product was homogeneous as judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of antithrombin III was determined by progressive inactivation of thrombin coagulant and amidolytic activity. Heparin cofactor activity was demonstrated by immediate inactivation of thrombin by antithrombin III in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin III occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities of heparin.