Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.     

Anticoagulantly active heparin-like molecules from vascular tissue

Mucopolysaccharides were isolated from calf cerebral microvasculature and calf aorta. The only complex carbohydrates that exhibited anticoagulant activity were heparin-like components. The biologic potencies of calf cerebral and aortic heparin-like species were 2.92 units/mg of anti-factor Xa activity and 2.85 units/mg of anti-factor IIa activity, as well as 0.56 unit/mg of anti-factor Xa activity and 0.19 unit/mg of anti-factor IIa activity, respectively. Additional experiments revealed that the anticoagulantly active aortic components were significantly present only within the intima. The above populations of heparin-like species were affinity fractionated with antithrombin. The highly active component obtained from calf cerebral microvasculature exhibited an anti-factor Xa activity of 40.7 units/mg as well as an anti-factor IIa activity of 36.8 units/mg, constituted about 4.2% of the initial mass of the starting material, and represented about 75% of the biologic potency of the starting material. The highly active component derived from calf aorta exhibited an anti-factor Xa activity of 55.4 units/mg as well as an anti-factor IIa activity of 11.3 units/mg, constituted about 0.3% of the initial mass of the starting material, and represented about 60% of the biologic potency of the starting material. The highly active cerebral microvascular species possessed a molecular weight and charge density similar to that of heparan sulfate whereas the highly active aortic species displayed a molecular weight and charge density equivalent to that of a hexadecasaccharide fragment of heparin.     

Structure and activity of a unique heparin-derived hexasaccharide

A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.     

Isolation and characterization of raw heparin from dromedary intestine: evaluation of a new source of pharmaceutical heparin

Heparin, a heterogeneous anionic polysaccharide, is the glycosaminoglycan (GAG) used clinically an anticoagulant. This anticoagulant activity is primarily derived from its binding to the serine protease inhibitor antithrombin III, a potent inhibitor of thrombin (factor IIa) and factor Xa. Heparin is a complex natural product and its in vitro synthesis is not yet possible due to the difficulty of organizing the many biosynthetic enzymes required for its synthesis. The principle natural sources for heparin include porcine intestine and bovine lung. These two sources pose concerns for religious and health reasons, respectively. To circumvent these concerns, GAG from the intestinal tissue of one humped camel was isolated. Chemical characterization of this newly isolated GAG and spectroscopic analysis by 1D and 2D 1H-NMR were undertaken. Unsaturated disaccharide compositional analysis was performed on the enzymatically depolymerized GAG and the molecular weight of the isolated GAG was determined by gradient polyacrylamide gel electrophoresis. Anticoagulant activity of the newly isolated GAG was tested by using an anti-factor Xa assay. The results of these studies suggest that the GAG from one humped camel intestine is a mixture of heparin and heparan sulfate and represents an alternative source of heparin.     

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

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.     

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (GlcpA) and idopyranosyluronic acid (IdopA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (GlcpNAc) residue was lost on workup. The GlcpA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6-O-sulfo glycopyranosylamine (GlcpNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in GlcpNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained GlcpNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.     

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.     

The relative molecular mass dependence of the anti-factor Xa properties of heparin.

The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III.     

Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa.

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.     

Antithrombin in vertebrate species: conservation of the heparin-dependent anticoagulant mechanism

Heparin is thought to regulate the rate of mammalian blood clotting by enhancing the activity of antithrombin, an inhibitor of coagulation enzymes. The present study establishes that this same inhibitor is present in the blood plasma of each of the terrestrial vertebrate groups including mammals, birds, reptiles, and amphibians. In each case, an inhibitor with remarkably similar properties to human antithrombin was isolated by affinity chromatography on immobilized porcine heparin. The purified vertebrate inhibitors all show the following physical and functional homologies to human antithrombin: (i) heparin-enhanced inhibition of both bovine thrombin and human Factor Xa, (ii) molecular masses of approximately 60,000, and (iii) heparin-induced increases in ultraviolet fluorescence. Also, the heparin-binding interaction of vertebrate antithrombins is highly selective with each demonstrating the same rigid specificity for heparin species fractionated on the basis of their affinity for human antithrombin. This common ability of vertebrate antithrombins to discriminate among heparins is accomplished by a nearly unvarying equilibrium binding constant for the high-affinity heparin species. Thus, the present results suggest that the anticoagulant relationship of heparin and antithrombin was established at an early point in the evolution of the coagulation system and has been highly conserved since that time.     

Antithrombin binding of low molecular weight heparins and inhibition of factor Xa

Fluorescence and stopped flow methods were used to compare clinically used heparins with regard to their ability to bind to antithrombin and to accelerate the inactivation of factor Xa. Titration of antithrombin with both low molecular weight heparin (LMWH) (enoxaparin, fragmin and ardeparin) and unfractionated heparin (UFH) produced an equivalent fluorescence increase and indicates similar affinity of all heparin preparations to antithrombin. However, relative to UFH enoxaparin, the LMWH with the smallest average molecular mass, contained only 12% material with high affinity for antithrombin. The rate of factor Xa inhibition by antithrombin increased with the concentration of the examined heparins to the same limiting value, but the concentration required for maximal acceleration depended on the preparation. According to these data the high affinity fraction of the heparin preparations increased the intrinsic fluorescence and inhibitory activity equally without additional effects by variations in chain length and chemical composition. In contrast, in the presence of Ca UFH accelerated the inhibition of factor Xa by antithrombin 10-fold more efficiently than comparable concentrations of the high affinity fractions of enoxaparin and fragmin. The bell-shaped dependence of this accelerating effect suggests simultaneous binding of both proteins to heparin. In conclusion, under physiologic conditions the anti-factor Xa activity of heparin results from a composite effect of chain length and the content of material with high affinity to antithrombin. Thus, the reduced antithrombotic activity of LMWH relative to UFH results from a smaller content of high affinity material and the absence of a stimulating effect of calcium.     

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.     

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.     

Synthesis and detection of N-sulfonated oversulfated chondroitin sulfate in marketplace heparin

N-sulfonated oversulfated chondroitin sulfate (NS–OSCS), recently reported as a potential threat to the heparin supply, was prepared along with its intermediate derivatives. All compounds were spiked into marketplace heparin and subjected to United States Pharmacopeia (USP) identification assays for heparin (proton nuclear magnetic resonance [¹H NMR], chromatographic identity, % galactosamine [%GalN], anti-factor IIa potency, and anti-factor Xa/IIa ratio). The U.S. Food and Drug Administration (FDA) strong-anionic exchange high-performance liquid chromatography (SAX–HPLC) method resolved NS–OSCS from heparin and OSCS and had a limit of detection of 0.26% (w/w) NS–OSCS. The %GalN test was sensitive to the presence of NS–OSCS in heparin. Therefore, current USP heparin monograph tests (i.e., SAX–HPLC and %GalN) detect the presence of NS–OSCS in heparin.     

Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xa bridging complex. Demonstration by rapid kinetics studies

Heparin catalyzes the inhibition of factor Xa by antithrombin mainly through an allosteric activation of the serpin inhibitor, but an alternative heparin bridging mechanism has been suggested to enhance the catalysis in the presence of physiologic calcium levels due to calcium interactions with the Gla domain exposing a heparin binding exosite in factor Xa. To provide direct evidence for this bridging mechanism, we studied the heparin-catalyzed reaction of antithrombin with factor Xa, Gla-domainless factor Xa (GDFXa), and a heparin binding exosite mutant of GDFXa in the absence and presence of calcium using rapid kinetic methods. The pseudo-first-order rate constant for factor Xa inhibition by antithrombin complexed with a long-chain approximately 70-saccharide heparin showed a saturable dependence on inhibitor concentration in the presence but not in the absence of 2.5 mM Ca(2+), indicating the formation of an intermediate heparin-serpin-proteinase encounter complex with a dissociation constant of approximately 90 nM prior to formation of the stable serpin-proteinase complex with a rate constant of approximately 20 s(-1). Similar saturation kinetics were observed for the inhibition of GDFXa by the antithrombin-heparin complex, except that Ca(2+) was not required for the effect. By contrast, no Ca(2+)-dependent saturation of the inhibition rate constant was detectable over the same range of inhibitor concentrations for reactions of either a short-chain approximately 26-saccharide high-affinity heparin-antithrombin complex with factor Xa or the long-chain heparin-antithrombin complex with the heparin binding exosite mutant, GDFXa R240A. These findings suggest that binding of full-length heparin chains to an exosite of factor Xa in the presence of Ca(2+) produces a chain-length-dependent lowering of the dissociation constant for assembly of the intermediate heparin-antithrombin-factor Xa encounter complex, resulting in a several 100-fold rate enhancement by a heparin bridging mechanism.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Characterization of rat factors X and Xa: demonstration of factor Xa in rat plasma.

We have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl2 eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a Ki of 2.2 x 10(-11) M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6 x 10(4) M-1.min-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat alpha-1-antitrypsin and alpha-2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides.

The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin.

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.