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PubMed1995

The effect of sex and age on antithrombin biosynthesis in the rat.

Antithrombin is the primary inhibitor of Factor Xa and thrombin. Numerous reports have indicated that age and sex can influence antithrombin levels, but details of the regulation of antithrombin biosynthesis are not known. Thus, a characterization of antithrombin mRNA in eight tissues of young and old male and female rats was carried out. Liver produced the most mRNA, and hence contributes the majority of the plasma antithrombin, followed by the kidneys, with no age or sex related differences in mRNA levels being observed. Elevated amounts of mRNA were detected in aortas of old male rats compared to young ones. No antithrombin mRNA was detected in brain, lung, heart or skeletal muscle, and spleen showed low but variable levels. Plasma antithrombin protein was elevated in old female rats compared to young female or old male rats. These results show that the rat provides a potentially useful system to study the in vivo regulation of antithrombin biosynthesis.

Age Factors↗

PubMed1994

Antithrombin and its inherited deficiencies.

Human antithrombin is the major inhibitor of the coagulation serine proteases accounting for approximately 80% of the thrombin inhibitory activity of plasma. It is a member of the serpin family of serine protease inhibitors and in common with some other members of this family it undergoes a dramatic increase in its inhibitory activity in the presence of heparin and other sulphated glycosaminoglycans. Two functional domains in antithrombin are recognised, the reactive site domain which interacts with the active site serine residue of the protease and the heparin binding domain. The gene for antithrombin has been cloned and its entire nucleotide sequence determined. A deficiency or functional abnormality of antithrombin may result in an increased risk of thromboembolic disease. Such deficiencies are estimated to affect as many as 1:300 of the general population and 3 to 5% of patients with thrombotic disease. On the basis of functional and immunological antithrombin assays, antithrombin deficiency may be subdivided into Types I and II. Type I disease is due to a wide variety of heterogeneous DNA mutations whilst in Type II disease missense mutations leading to single amino acid substitutions have been identified in all cases. Clinically, Type I antithrombin deficiency is associated with recurrent thromboembolic disease whereas in Type II deficiency the risk of thrombosis is closely related to the position of the mutation within the protein. Thus, heterozygotes with mutations within the heparin binding domain of antithrombin have a relatively low risk of thrombosis compared to those with mutations at or close to the reactive site of the molecule.

Amino Acid Sequence↗

PubMed2000

Inhibition of antithrombin by protein SV-IV normalizes the coagulation of hemophilic blood.

The aim of the study was to evaluate the effect of the protein Seminal Vesicle Protein No. 4 (SV-IV), a potent inhibitor of antithrombin III (antithrombin), on the coagulation of blood obtained from patients affected by hemophilia A. In the coagulating blood of these patients, the antithrombin/thrombin ratio was found to be markedly higher (about 44) than in normal individuals (about 4. 4). This high ratio was related to the low efficiency of thrombin-generating reactions induced by the factor VIII deficiency and to the high levels of free (not bound to serine proteases) antithrombin present in the hemophilic serum (antithrombin concentration was the same in normal and hemophilic plasma). The elevated concentration of free antithrombin in hemophiliacs was primarily a consequence of a reduced consumption caused by the scarce availability in the hemophilic serum of factors Xa and IIa, which are serine proteases possessing strong binding affinity for antithrombin. Addition of SV-IV to coagulating hemophilic blood reduced markedly the serum antithrombin and thrombin-antithrombin complexes, normalizing, as a consequence, the clotting time and other coagulation parameters. Similar results were obtained by using appropriate concentration of factor VIII.

Antithrombin III↗

PubMed1994

Endogenous antithrombin associated with microvascular endothelium. Quantitative analysis in perfused rat hearts.

A recirculating Langendorff heart preparation is used to characterize the endogenous antithrombin associated reversibly with murine vascular endothelium. Rat hearts are perfused clear of blood and then recirculated with a physiological salt solution. Addition of heparin educes antithrombin activity continuously into the perfusate during 6 min of recirculation. This process contrasts with a more rapid equilibration of the system as assessed by displacement of [125I]thrombin with hirudin or with a heparin-antithrombin mixture. Perfusion of washed hearts with [125I]factor Xa, which evidences no significant binding to the coronary endothelium, identifies a minor fraction of the endogenous antithrombin that reacts immediately with factor Xa, i.e., at a rate indicative of heparin enhancement. This rapid-reacting antithrombin is not reproducibly detected with [125I]thrombin, which binds preferentially to thrombomodulin in this system. The amount of antithrombin reacting rapidly with factor Xa is too low to detect as a burst of antithrombin activity eluted into the perfusate when the hearts are perfused with heparin. It is concluded that the murine myocardial microvasculature harbors at least two pools of antithrombin, the minor of which is in an activated configuration characteristic of association with heparin. The major pool is in a more slowly accessible compartment or configuration.

Animals↗

PubMed1990

Evidence that arginine-129 and arginine-145 are located within the heparin binding site of human antithrombin III.

Arginyl residues of human antithrombin III have been implicated to involve in the heparin binding site [Jorgensen, A. M., Borders, C. L., & Fish, W. W. (1985) Biochem, J. 231, 59-63]. We have performed chemical modification of antithrombin with (p-hydroxyphenyl)glyoxal (HPG) in order to determine the locations of these arginine residues. Antithrombin was modified with 12 mM HPG in the absence and presence of heparin (2-fold by weight to antithrombin). In the absence of heparin, about 3-4 mol of arginines/mol of antithrombin were modified within 60 min, and the modification led to the loss of 95% of the inhibitor's heparin cofactor activity as well as heparin-induced fluorescence enhancement and 50% of its progressive inhibitory activity. In the presence of heparin, the extent of modification was diminished by 30% and modified antithrombin retained approximately 70% of its heparin cofactor activity. Peptide mapping and subsequent sequence analysis revealed that selective HPG modification occurred at Arg129 and Arg145 and that their modifications were protected upon binding of heparin to antithrombin. We conclude that Arg129 and Arg145 are situated within the heparin binding site of human antithrombin III.

Amino Acid Sequence↗

PubMed2003

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability.

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.

Amino Acid Substitution↗

PubMed1998

Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites.

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.

Allosteric Regulation↗

PubMed2002

Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators.

Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity ( approximately 10.7 microM) comparable to the reference trisaccharide DEF ( approximately 4.5 microM), it accelerated the inhibition of factor Xa only 10-fold as compared to the approximately 300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K(D) = 3.5 microM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M(-1) s(-1)). On the contrary, the K(D) and k(ACT) were found to be lower approximately 7.4- and approximately 2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion ( approximately 55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected approximately 4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.

Antithrombins↗

PubMed2004

Acute normovolemic hemodilution does not reduce antithrombin concentration for cardiac surgery.

BACKGROUND: Acute normovolemic hemodilution (ANH) is used to reduce allogeneic blood transfusion with cardiac surgery. This procedure involves pre-operatively removing and storing a volume of whole blood and replacing the volume with crystalloid. The stored blood is then available for transfusion, if required. Hemodilution associated with ANH may reduce the effectiveness of heparin anticoagulation due to dilution of antithrombin. The aim of this study was to determine if antithrombin concentrations are reduced in patients who undergo one unit of ANH during cardiac surgery. METHODS: Patients scheduled for cardiac surgery (n = 71) were grouped according to whether they did or did not undergo ANH pre-operatively. Antithrombin concentrations were measured before and after ANH. This study had 80% power to detect a difference in reduction of antithrombin concentration of 6% between groups following ANH with an alpha error of <0.05. The effect of one unit ANH was expected to cause a difference of 12% or greater. RESULTS: No significant difference in the concentration of antithrombin between ANH patients and those that did not have ANH, nor was there a difference in the decrease in antithrombin between groups. CONCLUSIONS: The results indicate that one unit of ANH does not significantly reduce the concentration of antithrombin prior to cardiac surgery. Thus patients who undergo one unit of ANH are not at increased risk due to dilution of antithrombin.

Aged↗

PubMed2002

Antithrombin III activity and platelet count are more likely to decrease in twin pregnancies than in singleton pregnancies.

BACKGROUND: It is not known whether antithrombin III activity and platelet count are more likely to decrease in women with twin pregnancies than singleton pregnancies. METHODS: We enrolled 56 consecutive women with twin pregnancies and 692 consecutive women with singleton pregnancies. Antithrombin III activity and platelet count were determined at 26-31 weeks and again at 32-40 weeks of gestation. Thrombin-antithrombin complex, and plasmin-alpha2 plasmin inhibitor complex were measured simultaneously in some women. RESULTS: In twin pregnancies, both antithrombin III activity (111 +/- 8.2%) and platelet count (244 +/- 60 x 109/l) at 28.6 +/- 1.0 weeks decreased over time to 91 +/- 12% and 205 +/- 59 x 109/l, respectively, at 35.2 +/- 1.2 weeks; these decreases were significantly greater than in singleton pregnancies. However, a small number of women with singleton pregnancies had a significant decrease in antithrombin III activity in the absence of preeclampsia. The serum thrombin-antithrombin complex was significantly higher in twin pregnancies than in singleton pregnancies (9.7 +/- 4.2 ng/ml vs. 6.7 +/- 4.4 ng/ml, respectively, p < 0.01), and this increase correlated significantly with the decrease in antithrombin III activity in twin pregnancies (r = -0.459, p < 0.001). CONCLUSIONS: Antithrombin III activity and platelet count show a greater decrease in twin than in singleton pregnancies, perhaps due to a greater increase in thrombin generation.

Adult↗

PubMed1986

Location of antithrombin-binding regions in rat skin heparin proteoglycans.

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.

Animals↗

PubMed1988

Effect of oversulphated chondroitin and dermatan sulphate upon thrombin and factor Xa inactivation by antithrombin III or heparin cofactor II.

The kinetics of inhibition of human thrombin and Factor Xa by antithrombin III or heparin cofactor II were examined under pseudo-first-order conditions as a function of the concentration of naturally occurring oversulphated chondroitin and dermatan sulphates. The sulphated glycosaminoglycans (GAGs) studied were chondroitin sulphate D (CSD) (GlcA-2-SO4-GalNAc-6-SO4), chondroitin sulphate K (CSK) (GlcA-3-SO4-GalNAc-4-SO4), chondroitin sulphate H (CSH) (IdA-GalNAc-4,6-diSO4) and polysulphated dermatan sulphate (DPS) (IdA-2-SO4 or -3-SO4-GalNAc-4,6-diSO4). The data for the antithrombin III inhibition of thrombin showed a low degree of maximal potentiation of this interaction (congruent to 10-fold), which would appear to be characteristic of GAGs devoid of the high-affinity antithrombin III binding site. In contrast there was a greater potentiation of the inhibition of thrombin by heparin cofactor II with DPS showing an activity comparable to heparin in this interaction at a concentration two orders of magnitude lower than dermatan sulphate. DPS potentiated antithrombin III-Factor Xa interaction by 1200-fold, similar to that shown by high-affinity heparin of 6 kDa. The antithrombin III-Factor Xa interaction was potentiated by all other GAGs studied to a degree similar to that of heparin pentasaccharide with high affinity for antithrombin III. The findings suggest more stringent structural requirements for GAG stimulation of antithrombin-thrombin interaction than for antithrombin-Factor Xa or heparin cofactor-thrombin interaction, which may also be of significance in physiological control of haemostasis.

Antithrombin III↗

PubMed1988

The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type.

The effect of various well-characterized heparin preparations on the inactivation of human Factor XIa by human antithrombin III was studied. The heparin preparations used were unfractionated heparin and four heparin fractions obtained after anion-exchange chromatography. Inactivation of Factor XIa was monitored with S2366 as chromogenic substrate and followed pseudo-first-order reaction kinetics under all reaction conditions tested. Enhancement of the rate of inhibition of Factor XIa in the presence of unfractionated heparin correlated to the binding of antithrombin III to heparin. From the kinetic data a binding constant of 0.1 microM was inferred. The maximum rate enhancement, achieved at saturating heparin concentrations, was 30-fold. The rate enhancement achieved in the presence of each of the heparin fractions could also be correlated to the binding of antithrombin III to the heparin. The binding constant inferred from the kinetic data varied from 0.10 to 0.28 microM and the number of binding sites for antithrombin III varied from 0.06 to 0.74 site per heparin molecule. The maximum rate enhancements, achieved at saturating heparin concentrations, were strongly dependent on the type of heparin used and varied from 7-fold for fraction A to 41-fold for fraction D. Therefore, although the stimulation of Factor XIa inactivation by antithrombin III could be quantitatively correlated to the binding of antithrombin III to heparin, the heparin-catalysed inhibition of Factor XIa is dependent not only upon the degree of binding of antithrombin III to heparin but also upon the type of heparin to which antithrombin III is bound.

Antithrombin III↗

PubMed1991

Heterogeneous distribution of antithrombin-binding sites in rat brain heparan sulphate proteoglycans.

Heparan sulphates with high binding affinity for antithrombin (HA-HS), labelled in vivo with [35S]sulphate, were extracted from rat brains and purified by chromatography on DEAE-cellulose and on antithrombin-agarose. HA-HS proteoglycans (HA-HSPG) were then separated from HA-HS chains on Sepharose CL-6B. The total HA-HSPG product was rechromatographed on antithrombin-agarose. Six HA-HSPG subfractions with differing degrees of affinity for antithrombin were recovered and treated with NaOH to release their chains. Rechromatography of these six 35S-labelled HS chain preparations on antithrombin-agarose showed that their proportions of chains with no affinity for antithrombin (NA-HS chains) ranged from 36 to 71%. There was a reciprocal relationship between the proportion of NA-HS chains in each HA-HSPG subfraction and the degree of affinity for antithrombin of the rest of its chains (assessed relative to 3H-labelled HA-heparin chains with which they were co-chromatographed). Similar characteristics of antithrombin-binding-site distribution apply to HA-heparin proteoglycans from rat skin studied previously [Horner (1987) Biochem. J. 244, 693-698]. The data suggest that the sites at which 3-O-sulphation of some glucosamine N-sulphate residues occurs in the Golgi complex of brain cells (probably endothelial cells) which synthesize HA-HSPGs (as in mast cells, which synthesize HA-heparin PGs) are distributed sparsely but not randomly.

Animals↗

PubMed2004

Decreased concentration of antithrombin after preoperative therapeutic heparin does not cause heparin resistance during cardiopulmonary bypass.

OBJECTIVE: To determine if preoperative heparin therapy causes an increase in the incidence of intraoperative heparin resistance by reducing the concentration of antithrombin in plasma. DESIGN: Prospective laboratory investigation of clinical samples. SETTING: Public tertiary care hospital and public pathology service. PARTICIPANTS: Forty-six patients undergoing cardiac surgery involving cardiopulmonary bypass. INTERVENTIONS: Fourteen patients received preoperative heparin therapy (POHI group) and 32 patients were controls (CONT group). MEASUREMENTS AND MAIN RESULTS: The concentration of antithrombin, activated coagulation time (ACT), and clinical parameters were measured at intervals. More POHI patients had on-bypass heparin resistance than CONT (43% and 3%, respectively, p < 0.01). The POHI group had a lower concentration of antithrombin than the CONT group before (80.9% and 92.6%, respectively, p < 0.01) and while on cardiopulmonary bypass (51.6% and 57.5%, respectively, p = 0.04). Comparison of heparin-resistant and heparin-responsive POHI patients showed that the concentration of antithrombin did not differ before bypass (82.4% and 79.8%, respectively, p = 0.53) or during bypass (51.8% and 51.4%, respectively, p = 0.91). In fact, antithrombin concentrations were slightly higher in the heparin-resistant POHI patients (not significant). POHI patients received more heparin than CONT patients (medians 787 U/kg and 600 U/kg, respectively, p = 0.01) and were transfused with more fresh frozen plasma on bypass (p = 0.03). CONCLUSIONS: Preoperative heparin causes an increased incidence of heparin resistance and reduced antithrombin concentrations. However, heparin resistance was not causally related to reduced antithrombin because antithrombin concentrations were not different between heparin-resistant and heparin-responsive patients in the POHI group.

Anticoagulants↗

PubMed2002

Pharmacokinetic behaviour of antithrombin III following intravenous infusion in healthy volunteers.

Antithrombin III (CAS 52014-67-2) is produced from plasma of healthy donors and is purified from any detectable agent of transmissible infection. This drug is used in therapy by i.v. route to manage acute thrombotic episodes and to treat patients suffering from its deficiency. The present trial was conducted with the aim to evaluate the pharmacokinetics and the safety of a new preparation of antithrombin III, which in the purification procedure has included a nanofiltration step to increase the safety against the transmission of any agent of infection. The trial was conducted in twelve healthy volunteers of either sex. All the ethics procedures were fully respected, namely the approval from Ethics Committee, the notification to the central regulatory authority, the approval of consent form in writing. Volunteers were hospitalized about 36 h before drug treatment. The baseline situation was investigated throughout the day before the treatment (day-1). On day 1, the drug was infused i.v. for 20 min. Twenty blood samples were drawn from each volunteer, in baseline situation, during and after the infusion. Functional antithrombin III, antithrombin III antigen, prothrombin time, partial thromboplastin time were evaluated in all plasma samples. Both functional antithrombin III and antithrombin III antigen produced well defined plasma concentration-time profiles after the infusion, which allowed net values of these two analytes to be obtained subtracting baseline from post-infusion concentrations. Net pharmacokinetic parameters of functional antithrombin III and antithrombin III antigen proved to be almost superimposable. A comparison of data obtained in this trial on healthy volunteers with those previously obtained by other authors on target population demonstrates similar and fully comparable results. The authors conclude that the nanofiltration step neither affects at all the pharmacokinetics/pharmacodynamics nor the safety of the formulation investigated.

Adult↗

PubMed1979

Structure of the antithrombin-binding site in heparin.

Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on immobilized antithrombin. Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-affinity heparin; furthermore, this component was concentrated in the antithrombin-binding regions of the high-affinity heparin molecules, amounting to approximately one residue per binding site. It is suggested that nonsulfated L-iduronic acid is essential for the anticoagulant activity of heparin. The location of the non-sulfated uronic acid in the antithrombin-binding site was determined by periodate oxidation of antithrombin-binding fragments containing a terminal 2,5-anhydro-D-[1-3H]mannitol unit. Tentative structures for antithrombin-binding sequences in heparin are proposed, including some structural variants believed to be compatible with, but not required for, activity.

Animals↗

PubMed1999

Importance of the P2 glycine of antithrombin in target proteinase specificity, heparin activation, and the efficiency of proteinase trapping as revealed by a P2 Gly --> Pro mutation.

A sequence-specific heparin pentasaccharide activates the serpin, antithrombin, to inhibit factor Xa through an allosteric mechanism, whereas full-length heparin chains containing this sequence further activate the serpin to inhibit thrombin by an alternative bridging mechanism. To test whether the factor Xa specificity of allosterically activated antithrombin is encoded in the serpin reactive center loop, we mutated the factor Xa-preferred P2 Gly to the thrombin-preferred P2 Pro. Kinetic studies revealed that the mutation maximally enhanced the reactivity of antithrombin with thrombin 15-fold and decreased its reactivity toward factor Xa 2-fold when the serpin was activated by heparin pentasaccharide, thereby transforming antithrombin into an allosterically activated inhibitor of both factor Xa and thrombin. Surprisingly, the enhanced thrombin specificity of the mutant antithrombin was attenuated when a full-length bridging heparin was the activator, due both to a reduced rate of covalent reaction of the mutant serpin and thrombin and preferred reaction of the mutant serpin as a substrate. These results demonstrate that the reactive center loop sequence determines the specificity of allosterically activated antithrombin for factor Xa and that the conformational flexibility of the P2 Gly may be critical for optimal bridging of antithrombin and thrombin by physiologic heparin and for preventing antithrombin from reacting as a substrate in the bridging complex.

Amino Acid Substitution↗