Dependence of antithrombin III and thrombin binding stoichiometries and catalytic activity on the molecular weight of affinity-purified heparin

Heparin was fractionated by affinity chromatography on immobilized antithrombin III followed by gel filtration on Sephadex G-100. Eighteen fractions were obtained ranging in molecular weight from 9,700 to 34,300 as determined by sedimentation equilibrium. The binding stoichiometries of antithrombin III and thrombin interactions with the heparin of these fractions were measured, using changes in intrinsic and extrinsic fluorescence. Catalytic activity also was measured for each of the heparin fractions. As the molecular weight of heparin varied from about 10,000 to 30,000, the average number of antithrombin and thrombin sites/heparin molecule varied from 1.0 to 2.1 and 2.4 to 6.8. In addition, the molar specific activity increased 5.7-fold, an increase which correlated directly with the product of the number of antithrombin III and thrombin molecules bound. Thus as the number of bound molecules increased with increased molecular weight, the rate of reaction/bound antithrombin III increased in proportion to the number of bound thrombin molecules and vice versa. This can be explained by assuming that heparin functions as a template for both proteins, that all bound thrombin and antithrombin III molecules are accessible to each other, and that the rate at which a bound molecule reacts is proportional to the number of molecules of its interacting counterpart bound. These observations and conclusions are similar to those of Hoylaerts et al. (Hoylaerts, M., Owen, W. G., and Collen, D. (1984) J. Biol. Chem. 259, 5670-5677), who demonstrated that the rate at which single molecules of antithrombin III, covalently attached to heparin, react increases as the thrombin binding capacity (chain length) of heparin increases.     

Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor Xa

Oligosaccharides (10-20 monosaccharide units) with high affinity for antithrombin, as well as larger high-affinity heparin fractions (having relative molecular masses between 6,000 and 21,500), all markedly accelerated the inhibition of Factor Xa by antithrombin. Moreover, all high-affinity oligosaccharides and heparins enhanced, to a similar extent, the amount of free proteolytically modified antithrombin cleaved at the reactive bond by Factor Xa. In contrast, a minimum high-affinity heparin size of approximately 18 monosaccharide units was required to significantly accelerate the inactivation of thrombin by antithrombin and to enhance the production of modified antithrombin by this enzyme. All high-affinity fractions studied had similar affinities for antithrombin, as determined by fluorescence titrations. In competition experiments, binary complexes of antithrombin with octadecasaccharide or larger high-affinity heparins, but not with smaller oligosaccharides, displaced inactivated 125I-thrombin from matrix-linked low-affinity heparin. Moreover, similar binary complexes with 3H-labeled octadecasaccharide or larger chains, but not with smaller oligosaccharides, were capable of binding to matrix-linked inactivated thrombin. These results indicate that simultaneous binding of antithrombin and thrombin to high-affinity heparin is a prerequisite to the acceleration of the antithrombin-thrombin reaction and that the minimum heparin sequence capable of binding both proteins comprises approximately 18 monosaccharide units. Similar complex formation apparently is not required for the acceleration of the antithrombin-Factor Xa reaction.     

[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages.]

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.     

Kinetic analysis of various heparin fractions and heparin substitutes in the thrombin inhibition reaction

Kinetic characteristics of several heparin preparations and substitute heparins were determined to help understand the bases for activity differences. Several materials were highly active in factor Xa inhibition and the reaction rate at constant factor Xa concentration appeared to be predicted by the extent of intrinsic antithrombin III fluorescence change induced by the polysaccharide. Heparin fractions of different molecular weight and affinity for antithrombin III showed similar kinetic parameters in catalysis of the thrombin-antithrombin III reaction when these parameters were expressed on the basis of antithrombin III-binding heparin. The latter was determined by stoichiometric titration of the antithrombin III fluorescence change by the heparin preparation. However, the various heparin fractions showed very different specific activities per mg of total polysaccharide. This indicated that functional heparin molecules had similar kinetic properties regardless of size or antithrombin III-binding affinity and is possible because the Km for antithrombin III is determined by diffusion rather than by binding affinity. Substitute heparins and depolymerized heparin were poor catalysts for thrombin inhibition, due at least partially to their affinity for thrombin. This latter binary interaction inhibits thrombin reaction in the heparin-catalyzed reaction.     

Heparin binding site, conformational change, and activation of antithrombin.

Alignment of the heparin-activated serpins indicates the presence of two binding sites for heparin: a small high-affinity site on the D-helix corresponding in size to the minimal pentasaccharide heparin, and a longer contiguous low-affinity site extending to the reactive center pole of the molecule. Studies of the complexing of antithrombin and its variants with heparin fractions and with reactive center loop peptides including intermolecular loop-sheet polymers all support a 3-fold mechanism for the heparin activation of antithrombin. Binding to the pentasaccharide site induces a conformational change as measured by circular dichroism. Accompanying this, the reactive center becomes more accessible to proteolytic cleavage and there is a 100-fold increase in the kass for factor Xa but only a 10-fold increase for thrombin, to 6.4 x 10(4) M-1 s-1. To obtain a 100-fold increase in the kass for thrombin requires in addition a 4:1 molar ratio of disaccharide to neutralize the charge on the extended low-affinity site. Full activation requires longer heparin chains in order to stabilize the ternary complex between antithrombin and thrombin. Thus, addition of low-affinity but high molecular weight heparin in conjunction with pentasaccharide gives an overall kass of 2.7 x 10(6) M-1 s-1, close to that of maximal heparin activation.     

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.     

Molecular-size-dependent variations in the proportions of chains with high binding affinities for antithrombin in rat skin heparin proteoglycans.

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.     

Measurement of the affinities of heparins, naturally occurring glycosaminoglycans, and other sulfated polymers for antithrombin III and thrombin

Heparin, other glycosaminoglycans, and synthetic sulfated polymers have antithrombotic and anticoagulant activities, which may be mediated through a range of interactions with different proteins. A simple, quantitative method has been developed for assessing the affinity of interaction between sulfated polymers and proteins in the liquid phase. This has been used to compare the binding of a range of glycosaminoglycans and other sulfated polymers to antithrombin III and thrombin, a major inhibitor of and a central protease in the coagulation system, respectively. The results are consistent with the binding of naturally occurring glycosaminoglycans to antithrombin III solely through the well-defined antithrombin III-binding pentasaccharide found in heparin, the apparent affinity of a preparation depending upon its content of this pentasaccharide. Highly sulfated synthetic polymers will, however, bind antithrombin III by a second mechanism. The affinity of heparin for thrombin decreased with decreasing molecular weight. However, results obtained with heparan sulfate preparations did not indicate any clear relationship between either molecular weight or sulfate content and thrombin binding, but suggested that there may be an oligosaccharide sequence containing N-sulfate residues which confers high affinity for thrombin. In addition, some of the synthetic sulfated polymers bound thrombin with very high affinity.     

Heparin binding to antithrombin III: Variation in binding sites and affinity

Heparin fractions of different molecular weights and anticoagulant activities were prepared by chromatography on protamine-Sepharose, and the association constants and stoichiometry for binding to antithrombin III were determined by measurement of enhancement of tryptophan fluorescence. A 7,900 molecular weight heparin preparation bound to antithrombin III with a stoichiometry of close to 2:1, whereas 14,300 and 21,600 molecular weight fractions bound at approximately 1:1 with the protein. Apparent association constants were 0.66 × 10⁶ M^?1 for the low molecular weight preparation and 2.89 × 10⁶ M^?1 for the high molecular weight material. Maximal fluorescence enhancement was greater with the higher molecular weight heparin. These results suggest a model of heparin-antithrombin III binding in which two sites on antithrombin III can accommodate one large heparin molecule with high affinity or two smaller molecules with low affinity.     

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides: STRUCTURAL FEATURES AND BIOLOGICAL PROPERTIES

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.     

Mechanism of inhibition of mammalian DNA topoisomerase I by heparin.

We have previously shown that heparin is a potent inhibitor of a mammalian DNA topoisomerase I. We have now investigated the mechanism of its inhibition. This was carried out first by scrutinizing the structural features of heparin molecules responsible for the inhibition. Commercial heparin preparation was fractionated by antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity fractions, of which only the high-affinity fraction of heparin is known to contain a specific oligosaccharide sequence responsible for the binding to antithrombin III. These fractions all exhibited essentially similar inhibitory activities. Furthermore, when chemically sulphated to an extent comparable with or higher than heparin, otherwise inactive glycosaminoglycans such as heparan sulphate, chondroitin 4-sulphate, dermatan sulphate and neutral polysaccharides such as dextran and amylose were converted into potent inhibitors. Sulphated dermatan sulphate, one of the model compounds, was further shown to bind competitively to the same sites on the enzyme as heparin. These observations strongly suggested that topoisomerase inhibition by heparin is attributable primarily, if not entirely, to the highly sulphated polyanionic nature of the molecules. In a second series of experiments we examined whether heparin inhibits only one or both of the topoisomerase reactions, i.e. nicking and re-joining. It was demonstrated that both reactions were inhibited by heparin, but the nicking reaction was more severely affected than was the re-joining reaction.     

Studies on the mechanism of the rate-enhancing effect of heparin on the thrombin-antithrombin III reaction.

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticogulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

A method for rapid quantitation and preparation of antithrombin III-high-affinity heparin fractions

An electrophoretic method for the quantitation and preparation of antithrombin III-high-affinity heparin using agarose beds is described. The method allows the determination of high-affinity heparin fractions in several samples in one single step. The incubation mixture containing heparin and antithrombin III is submitted to agarose gel electrophoresis in 0.06 m barbital buffer, pH 8.6. A sharp separation between free antithrombin III, the complex antithrombin III-heparin, and free heparin occurs under these conditions. Around 30% of heparin molecules present in commerical preparations bind to antithrombin. This bound heparin has an anticoagulant activity of 240 IU. Negligible binding of other sulfated mucopolysaccharides to antithrombin III was observed. The whole procedure takes less than 6 h and can also be used as a semipreparative method for high-affinity heparin.     

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.     

Kinetic analysis of the role of zinc in the interaction of domain 5 of high-molecular weight kininogen (HK) with heparin

Previous investigations have shown that HK and its light chain bind heparin, preventing the enhancement of antithrombin inhibition of thrombin and potentiating the inhibition of plasma kallikrein by antithrombin. We found that both HK and HKa bound heparin, but HK exhibited a greater affinity. We therefore localized the binding sites for heparin on HK. HK domains 5 and 6 of the light chain as well as domain 3 from the heavy chain, expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli, were tested for binding to immobilized heparin by surface plasmon resonance using a BiaCore 2000 instrument. GST-D5, but not GST-D3, GST-D6, or GST, bound to heparin when the recombinant domains were present at a concentration of 70 nM. To localize more precisely the amino acid sequences on D5, both of the subdomains, histidine-glycine-rich GST-(K420-D474) and histidine-glycine-lysine-rich GST-(H475-S626), were expressed and tested for binding to immobilized heparin. The K(d) was much lower for GST-(K420-D474) than for GST-(H475-S626) in the presence or absence of Zn(2+). GST-(K420-D474) was effective in decreasing the rate of inactivation of thrombin by antithrombin in the presence of heparin and Zn(2+), while GST-(H475-S626) had no effect. We conclude that the binding of heparin to HK is a complex function of Zn(2+) interacting with histidines in the sequence K420-D474 to create high-affinity binding sites. HK has the potential to be an important modulator of heparin therapy.     

Multiple complexes of thrombin and heparin

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.     

1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site

Heparin inhibits blood coagulation by binding to the protease inhibitor antithrombin, thus promoting inactivation of the protease "factors" of the coagulation cascade mechanism. The article provides an overview of the studies, by different research groups, that led to the structural elucidation of the antithrombin-binding region of heparin. These studies were triggered by the finding that only a fraction of heparin molecules were capable of binding with high affinity to antithrombin, and further that this fraction essentially accounted for the anticoagulant activity of the unfractionated material. Oligosaccharides obtained by selective, partial depolymerization of heparin were fractionated on immobilized antithrombin, and the smallest high-affinity molecules recovered were subjected to structural analysis, in conjunction with various modification steps. The results were interpreted in terms of a binding site for antithrombin constituted by a pentasaccharide sequence with an internal unique 3-O-sulfated glucosamine unit, in addition to sugar residues and sulfate groups present elsewhere also in the polysaccharide. The structure/function relations deduced were verified by chemical synthesis of several pentasaccharide variants with the predicted bioactivities.     

A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.     

Activation of heparin cofactor II by heparin oligosaccharides

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.