Conformational Change In Antithrombin Research Articles

Efficacy and Safety of Antithrombin Supplementation in Neonates and Infants on a Continuous Heparin Infusion

Introduction: Antithrombin (AT) is an inhibitor of several coagulation proteins, including FIIa and FXa. Heparins accelerate/potentiate the interaction between AT and these factors through a conformational change in AT. Plasma AT concentrations are low in neonates, lower in sick premature neonates, and reach adult levels by 6 to 12 months of age. The developmental differences in plasma AT levels are an important consideration when treating an infant with heparin, as the activity of heparin depends on the presence of AT as a cofactor. As a result, AT supplementation is often considered in this cohort, particularly in the setting of heparin resistance. Objective: The objective of this study was to describe the use of AT supplementation at Boston Children's Hospital (BCH), a quaternary care, free-standing children's hospital, and investigate the safety and efficacy of AT supplementation in infants on a continuous heparin infusion. Methods: This study was approved by the Institutional Review Board at BCH. Data were abstracted for infants who received AT supplementation while on a therapeutic heparin infusion in the neonatal, cardiac, and medical intensive care units between January 2016 and October 2022. Infants on extracorporeal membrane oxygenation and continuous renal replacement therapy were excluded. The primary outcome was attainment of therapeutic anticoagulation within 48 hours after an AT course, defined as at least one occurrence of a partial thromboplastin time (PTT) level between 60 and 85 seconds and/or anti-factor Xa (anti-FXa) activity between 0.35 and 0.7 units/mL. Clinically relevant bleeding was defined as moderate/clinically relevant non-major bleeding or severe/major bleeding, as determined by either the Neonatal Bleeding Assessment Tool for infants with post-menstrual age (PMA) < 44 weeks or the International Society on Thrombosis and Haemostasis criteria for infants with PMA > 44 weeks, respectively. Data were summarized using descriptive statistics. Results: Over the 7-year period, 50 infants receiving 61 courses of AT supplementation were included. 12 patients received multiple AT doses within a course, resulting in the administration of 75 total AT doses. The median gestational age was 38 weeks (range 28-41 weeks), with a mean birth weight of 3.1 kg (range 0.8-4.5 kg). Median postnatal age at the time of the first AT dose was 19 days (range 3-341 days). The majority of heparin courses were indicated for thrombosis (49%) and post-operative cardiac surgery (40%). Median baseline AT activity was 37% (range 18-61%). The primary outcome was achieved in 90% AT courses. Of these, 73% were therapeutic only by PTT levels, 9% only by anti-FXa activity, and 18% by both. The benefit of AT administration was not sustained, as only 39% PTT levels and anti-FXa activity measured 48 hours after the AT course remained therapeutic. 41 infants had an AT level measured within 6 hours after the dose, and 61% (25/41) achieved a level between 80 and 120% of the calculated target. AT levels decreased over 48 hours, with an estimated half-life of 49 hours. 21% of AT courses had an increase in bleeding severity, with the majority being minor bleeds. Clinically relevant bleeding occurred after AT administration in only 8% (4/50) subjects. The relationship between simultaneously measured PTT levels and anti-FXa activity was investigated in a secondary analysis. Of the 250 paired levels, only one pair was therapeutic for both values ( Figure 1). Other than the single pair, in all other samples with a therapeutic anti-FXa, the simultaneously measured PTT was supratherapeutic. Three of the four patients with clinically relevant bleeding had at least one paired PTT level and anti-FXa activity available on the day of bleeding. Out of the seven paired samples, 86% of moderate bleeding occurred when the PTT level was supratherapeutic and the anti-FXa activity was subtherapeutic. Discussion: AT supplementation in infants may briefly increase the therapeutic efficacy of heparin, dependent on the monitoring parameter used, as PTT levels and anti-FXa activity poorly correlate. Additionally, permitting the PTT to increase over a certain threshold may increase the risk of bleeding. Further studies are needed to provide recommendations on heparin infusion titrations, AT dosing, and the monitoring of PTT levels and/or anti-FXa activity in the neonatal/infant populations on a continuous heparin infusion.

Positive Allosteric Modulation of Antithrombin's Inhibitory Activity by RNA Aptamers.

The leading cause of death in adults in the United States is cardiovascular disease, with mortality and morbidity mainly attributed to thromboembolism. Heparin is the most common therapy used for treating venous and arterial thrombosis. Heparin effectively accelerates the inhibition of coagulation proteases thrombin and factor Xa through the serine protease inhibitor (serpin) antithrombin (AT). Heparin is an essential therapeutic anticoagulant because of its effectiveness and the availability of protamine sulfate as an antidote. However, heparin therapy has several limitations. Thus, new anticoagulants, including direct thrombin inhibitors (ie, argatroban) and low-molecular-weight heparins (ie, fondaparinux), are used to treat some thromboembolic disorders. We developed and characterized a family of novel RNA-based aptamers that bind AT using two novel selection schemes. One of the aptamers, AT-16, accelerates factor Xa inhibition by AT in the absence of heparin. AT-16's effect on thrombin inhibition by AT is less effective compared to factor Xa. AT-16 induces a conformational change in AT that is different from that induced by heparin. This study demonstrates that an AT-specific RNA aptamer, AT-16, exhibits a positive allosteric modulator effect on AT's inhibition of factor Xa.

Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-Å structure of the ternary complex

Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-A crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on beta-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.

Heparin Binding Induces a Conformational Change in Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.

Antithrombin–S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.

Clinical use of enoxaparin in the management of non-ST segment elevation acute coronary syndromes

Enoxaparin (Lovenox®; Roule-Poulenc Rorer, Inc.), a low molecular weight heparin (LMWH), is commonly used in the management of non-ST-segment elevation acute coronary syndromes (NSTE ACS) based on clinical trial outcomes. It is one of a group of glycosaminoglycan compounds that accelerate the inactivation of factor Xa by inducing a conformational change in antithrombin. In contrast to unfractionated heparin (UFH), LMWH have greater bioavailability, a more predictable anticoagulant response, longer half-life and a higher proportion of anti-factor Xa to anti-factor IIa activity. As a consequence, laboratory monitoring of the anticoagulant effect is typically unnecessary. Antithrombin therapy with LMWH or UFH has the highest-level recommendation (IA) in the 2002 professional guidelines for the management of unstable angina and non-ST-elevation myocardial infarction, where enoxaparin has a IIA recommendation over UFH unless early coronary artery bypass surgery is planned. In a recent systematic overview of > 20,000 patients with NSTE ACS from six clinical trials, including conservative and invasively managed patients, enoxaparin provided a statistically significant reduction in 30-day death or nonfatal myocardial infarction (MI) compared with UFH with no significant excess in transfusions, or major bleeding. These data support the role of enoxaparin as an anti-coagulant in patients with NSTE ACS.

Effect of Covalently-Bound Heparin on Insertion of the Reactive Centre Loop in Antithrombin.

Heparin exerts its anticoagulant effects through its interaction with antithrombin (AT), leading to the inhibition of thrombin, a major player in the coagulation cascade. AT exists in equilibrium between a latent and an active state. In latent AT, the reactive centre loop (RCL) of AT is inserted into the central b-sheet (A-Sheet), forming a six-stranded A-Sheet. In active AT, residues P10-P14 of the RCL of AT are inserted into the A-sheet, retaining inhibitory activity. We have produced a molecule of covalently linked AT and heparin (ATH). Intrinsic fluorescence studies of ATH have shown that the AT is permanently activated in the covalent molecule. This study was designed to examine degree of the insertion of the RCL into the A-sheet of ATH. The tetradecapeptide P1-P14 was used to compete with the RCL of AT for insertion into the A-sheet. Peptides corresponding to the RCL residues P7-14, P3-P14 and P1-P14 of human AT were radiolabelled using reductive alkylation with formaldehyde + [3H]-NaBH4. Varying concentrations of radiolabelled peptide (0-3000 nM) were incubated with 1000 nM AT in the presence or absence of unfractionated heparin (UFH), and with ATH in 0.05 M Tris HCl buffer (pH 7.3) at 37°C. A 24-hour equilibrium dialysis against 0.05 M Tris HCl buffer followed. The amount of peptide bound to the AT or ATH was quantified following dialysis using liquid scintillation counting of samples taken from inside and outside the dialysis tubing. Results were expressed as bound vs. free peptide in moles. No binding to AT in the presence or absence of UFH, nor to ATH was seen using the P7-P14 peptide, while some binding to AT was seen for both the P3-P14 and P1-P14 peptides. There was a trend towards increased binding with AT in the presence of UFH over AT alone or ATH with the P3-P14 peptide. Binding to AT was lost in the presence of UFH, and no binding was seen with ATH in the P1-P14 condition, consistent with the literature. Furthermore, binding to AT was greatly reduced compared to that seen with the P3-P14 peptide. These data suggest that binding of heparin creates a conformational change in AT preventing the insertion of the RCL peptide into the molecule. Furthermore, results with the P3-P14 peptide may infer that it is the P1-P2 region that is most affected by the conformational change. This is consistent with the AT being in a partially activated conformation in the covalently linked ATH molecule.

Crystal structure of antithrombin in a heparin-bound intermediate state.

Antithrombin is activated as an inhibitor of the coagulation proteases through its specific interaction with a heparin pentasaccharide. The binding of heparin induces a global conformational change in antithrombin which results in the freeing of its reactive center loop for interaction with target proteases and a 1000-fold increase in heparin affinity. The allosteric mechanism by which the properties of antithrombin are altered by its interactions with the specific pentasaccharide sequence of heparin is of great interest to the medical and protein biochemistry communities. Heparin binding has previously been characterized as a two-step, three-state mechanism where, after an initial weak interaction, antithrombin undergoes a conformational change to its high-affinity state. Although the native and heparin-activated states have been determined through protein crystallography, the number and magnitude of conformational changes render problematic the task of determining which account for the improved heparin affinity and how the heparin binding region is linked to the expulsion of the reactive center loop. Here we present the structure of an intermediate pentasaccharide-bound conformation of antithrombin which has undergone all of the conformational changes associated with activation except loop expulsion and helix D elongation. We conclude that the basis of the high-affinity state is not improved interaction with the pentasaccharide but a lowering of the global free energy due to conformational changes elsewhere in antithrombin. We suggest a mechanism in which the role of helix D elongation is to lock antithrombin in the five-stranded fully activated conformation.

Molecular Determinants of the Mechanism Underlying Acceleration of the Interaction between Antithrombin and Factor Xa by Heparin Pentasaccharide

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.

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.

Role of Arginine 132 and Lysine 133 in Heparin Binding to and Activation of Antithrombin

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.

Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction.

In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E+I-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin-AT reaction. On the basis of these results, a modified model for thrombin-AT interaction is proposed.

Conformational Change in Antithrombin Induced by Heparin, Probed with a Monoclonal Antibody against the 1C/4B Region

A murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47-->Cys or His, Leu99-->Phe, Arg129-->Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393-->Cys) or resulting in substrate-variant antithrombin (Ala384-->Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402-->Cys, Phe402-->Ser, Phe402-->Leu, Ala404-->Thr, Pro407-->Thr) in the 402-407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429-->Leu also blocks recognition [Olds et al. (1992) Blood 79, 1206-1212], and this residue is believed to be spatially approximated to strand 1C.(ABSTRACT TRUNCATED AT 250 WORDS)

Defining the heparin-binding domain of antithrombin

Antithrombin is a serine protease inhibitor that participates in the inactivation and removal from the circulation of thrombin and a variety of other procoagulant serine proteases. Antithrombin is also the major plasma cofactor of heparin which exerts its therapeutic effect primarily through its ability to substantially increase the rate of inactivation by antithrombin of the procoagulant serine proteases. Binding of heparin to antithrombin is thus believed to be a prerequisite for this rate enhancement effect. Heparin binding to antithrombin is mediated by a well-defined unique heparin pentasaccharide sequence. Interaction between this pentasaccharide sequence and antithrombin induces a conformational change in antithrombin, an alteration that appears to be sufficient to explain the enhanced ability of antithrombin to inhibit factor Xa and related serine proteases, but not thrombin. Heparin species with longer polysaccharide chains appear to be required in order to enhance the inhibition of thrombin by antithrombin. This may be because the enhancement of this reaction requires that heparin interacts simultaneously with both the antithrombin and the thrombin molecules. This review describes the interactions between heparin and antithrombin, focusing on the antithrombin residues which are involved in the binding of heparin. The role of the heparin-induced conformational change in enhancing serine protease inhibition by antithrombin is also explored. Then, based on available data, an hypothesis is proposed to explain the mechanisms by which heparin accelerates the rate of inactivation by antithrombin of the various serine proteases.

Transmission of conformational change from the heparin binding site to the reactive center of antithrombin

Heparin greatly increases the rates at which antithrombin inhibits target proteinases. An important part of this rate acceleration is a heparin-induced conformational change in antithrombin. To answer the question of whether or not this change is transmitted to the reactive center, we have prepared a recombinant P1 mutant of antithrombin, R393C, labeled the cysteine with nitrobenzofuran (NBD) fluorophore, and examined the perturbation of NBD fluorescence intensity as a function of bound sulfated oligosaccharide. Two high-affinity heparins, low-affinity heparin, and dextran sulfate were used. We found (i) that binding to antithrombin of all these oligosaccharides resulted in transmission of conformational change to P1 in the reactive center, (ii) that these oligosaccharides all gave enhancements of the rate of inhibition of factor Xa beyond any contribution from surface approximation, and (iii) that the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approximation.

Binding of Heparin to Human Antithrombin III Activates Selective Chemical Modification at Lysine 236: Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of antithrombin III

A new water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC), was used to identify lysine residues of antithrombin III which participate in the binding of heparin. Antithrombin, modified with S-DABITC in the presence and absence of low molecular weight heparin (Mr 5000) was reduced, carboxymethylated, and digested with trypsin. The digest was analyzed by high-performance liquid chromatography and monitored at 465 nm. In the absence of heparin, four major colored peptides (T1, T2, T3, and T4) were identified. When antithrombin was preincubated with heparin (2-fold by weight), followed by S-DABITC modification, the recovery of peptide T4 remained unchanged, but the recoveries of T1, T2, and T3 were reduced by 93, 86, and 98%, respectively. In addition, a new colored peptide, TA, appeared. Amino acid sequencing of peptides T1, T2, T3, and TA localized S-DABITC modification sites as Lys-136, Lys-125, Lys-107, and Lys-236, respectively. Thus, binding of heparin to human antithrombin diminished S-DABITC modification at Lys-107, Lys-125, and Lys-136, but at the same time enhanced S-DABITC modification at Lys-236. This phenomenon was further characterized by varying the molar ratio of heparin/antithrombin (from 0.04 to 20). The shielding of Lys-125 and Lys-136 was inversely proportional to the activation of Lys-236. At a heparin/antithrombin molar ratio of 1, the extent of shielding of Lys-125 and Lys-136 and the unmasking of Lys-236 were 25-33%. This shielding-unmasking effect correlated with enhanced antithrombin inhibition of thrombin. We conclude that Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of human antithrombin and that binding of heparin to antithrombin causes a conformational change of antithrombin that leads to the exposure of Lys-236 for S-DABITC modification.

Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III.

The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin conformation and the catalytic role of heparin. II. Is the heparin-induced conformational change in antithrombin required for rapid inactivation of thrombin?

The role of antithrombin conformation in heparin-catalyzed inhibition of thrombin was investigated using antithrombins modified with the tryptophan reagent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB). Affinity fractionation of HNB-labeled antithrombin (0.6-0.7 mol of HNB/mol of protein) on heparin-Sepharose using a linear salt gradient allowed separation of three singly labeled protein species and a fourth HNB-antithrombin species which co-eluted with unlabeled protein. Conformational alterations induced by heparin binding to each of the labeled antithrombins were assessed by spectroscopic techniques, including protein fluorescence, difference spectroscopy in the ultraviolet-visible range, and circular dichroism. Comparison of spectra of the labeled proteins in the presence and absence of added heparin indicated changes to occur in protein conformation at the sites of the bound HNB moieties and at aromatic amino acid residues within the protein matrix. These spectroscopic alterations mimicked changes induced by heparin in the native protein, but were reduced in magnitude. Rates of thrombin inactivation by the labeled antithrombins were measured over a wide range in both heparin concentration and inhibitor concentration to determine maximal rates of protease inactivation. The kinetic analysis indicated that each of these HNB-antithrombin derivatives, which undergo the heparin-induced changes to varying extents, can react with thrombin at the same maximal rate. Thus, this series of chemically modified antithrombin species demonstrated that the conformational change which is induced in antithrombin by heparin does not render the protein intrinsically more reactive toward thrombin.

Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction.

The kinetics of high affinity heparin binding to human antithrombin III has been studied by stopped flow fluorimetry, using the 40% antithrombin fluorescence enhancement resulting from this interaction. At mu 0.15, pH 7.4, and 25 degrees C, the observed pseudo-first order rate constant varies hyperbolically with heparin concentration with a limiting rate constant of 440 +/- 90 s-1, demonstrating that heparin binding is a two-step process involving a conformational change in antithrombin III. An identical dependence is produced when antithrombin is varied, consistent with a symmetrical mechanism in which heparin binding induces a conformational change in antithrombin rather than perturbing an equilibrium between two conformational states of the protein. The rate constant for dissociation of the antithrombin-heparin complex is 1.1-1.5 s-1 at mu 0.15, as determined from the ordinate intercept at low heparin concentrations or by dissociation of the antithrombin-heparin complex with iodide. Observation of a single pseudo-first order binding rates over a 400-fold heparin concentration range with no detectable lags is compatible with the initial binding step being in rapid equilibrium with a KD of 4.3 +/- 1.3 X 10(-5) M at mu 0.15. Variation in ionic strength primarily affects the KD for the initial binding step with little effect on the conformational change rate constants, implying that binding involves ionic interactions. Calculation of the overall dissociation equilibrium constant from these rate parameters agrees with the directly determined value of 7.2 +/- 1.9 X 10(-8) M at mu 0.15. A major function of the conformational change is, thus, to increase the affinity of heparin for antithrombin III greater than 300-fold. The implications of these findings for the mechanism of the heparin-catalyzed inhibition of coagulation proteases by antithrombin III are discussed.