Conformational Activation Of Antithrombin Research Articles

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

Antithrombin-mediated Anticoagulant Activity of Sulfated Polysaccharides: DIFFERENT MECHANISMS FOR HEPARIN AND SULFATED GALACTANS

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.

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.

Designing small, nonsugar activators of antithrombin using hydropathic interaction analyses.

Conformational activation of antithrombin is a critical mechanism for the inhibition of factor Xa, a proteinase of the blood coagulation cascade, and is typically achieved with heparin, a polyanionic polysaccharide clinically used for anticoagulation. Although numerous efforts have been directed toward the design of better activators, a fundamental tenet of these studies has been the assumed requirement of an oligo- or a polysaccharide backbone. We demonstrate here a concept that small nonsaccharidic nonpolymeric molecules may be rationally designed to interact with and activate antithrombin for enhanced inhibition of factor Xa. The rational design strategy is based on a study of complexes of natural and mutant antithrombins with heparin-based oligosaccharides using hydropathic interaction (HINT) technique, a quantitative computerized tool for analysis of molecular interactions. A linear correlation was observed between the free energy of binding for antithrombinminus signoligosaccharide complexes and the HINT score over a wide range of approximately 13 kcal/mol, indicating strong predictive capability of the HINT technique. Using this approach, a small, nonsugar, aromatic molecule, (minus sign)-epicatechin sulfate (ECS), was designed to mimic the nonreducing end trisaccharide unit DEF of the sequence specific heparin pentasaccharide DEFGH. HINT suggested a comparable antithrombin-binding geometry and interaction profile for ECS and trisaccharide DEF. Biochemical studies indicated that ECS binds antithrombin with equilibrium dissociation constants of 10.5 and 66 microM at pH 6.0, I 0.025, and pH 7.4, I 0.035, respectively, that compare favorably with 2 and 80 microM observed for the natural activator DEF. ECS accelerates the antithrombin inhibition of factor Xa nearly 8-fold demonstrating for the first time that conformational activation of antithrombin is feasible with appropriately designed small nonsugar organic molecules. The results present unique opportunities for de novo activator design based on this first-generation lead.

Critical Role of the Linker Region between Helix D and Strand 2A in Heparin Activation of Antithrombin

The binding of pentasaccharide heparin to antithrombin induces a conformational change that is transmitted to the reactive center loop and increases the rate of inhibition of factor Xa by approximately 300-fold. The mechanism of such transmission is not known. To test the role of residues 134-137, which link helix D to beta-sheet A, in this signal transduction, we created variant antithrombins in which we removed amino acids 134-137 stepwise and cumulatively. Although the deletions did not compromise the fundamental ability of antithrombin to bind to heparin or to inhibit target proteinases thrombin and factor Xa, they did largely decouple conformational changes in the heparin-binding site from conformational activation of the reactive center loop. Because the variant with only Ala(134) removed was as compromised as variants with larger deletions, yet the variant with Ser(137) removed was normal, we concluded that the length of the linker is less important than the precise interrelationship between residues in this region and other residues involved in conformational activation of antithrombin.

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.

Mechanism of Acceleration of Antithrombin-Proteinase Reactions by Low Affinity Heparin: ROLE OF THE ANTITHROMBIN BINDING PENTASACCHARIDE IN HEPARIN RATE ENHANCEMENT

The role of the sequence-specific pentasaccharide region of high affinity heparin (HAH) in heparin acceleration of antithrombin-proteinase reactions was elucidated by determining the accelerating mechanism of low affinity heparin (LAH) lacking this sequence. LAH was shown to be free of HAH (< 0.001%) from the lack of exchange of added fluorescein-labeled HAH into LAH after separating the polysaccharides by antithrombin-agarose chromatography. Fluorescence titrations showed that LAH bound to antithrombin with a 1000-fold weaker affinity (KD 19 +/- 6 microM) and 5-6-fold smaller fluorescence enhancement (8 +/- 3%) than HAH. LAH accelerated the antithrombin-thrombin reaction with a bell-shaped dependence on heparin concentration resembling that of HAH, but with the bell-shaped curve shifted to approximately 100-fold higher polysaccharide concentrations and with a approximately 100-fold reduced maximal accelerating effect. Rapid kinetic studies indicated these differences arose from a reverse order of assembly of an intermediate heparin-thrombin-antithrombin ternary complex and diminished ability of LAH to bridge antithrombin and thrombin in this complex, as compared to HAH. By contrast, LAH and HAH both accelerated the antithrombin-factor Xa reaction with a simple saturable dependence on heparin or inhibitor concentrations which paralleled the formation of an antithrombin-heparin binary complex. The maximal accelerations of the two heparins in this case correlated with the inhibitor fluorescence enhancements induced by the polysaccharides, consistent with the accelerations arising from conformational activation of antithrombin. 1H NMR difference spectroscopy of antithrombin complexes with LAH and HAH and competitive binding studies were consistent with LAH accelerating activity being mediated by binding to the same site on the inhibitor as HAH. These results demonstrate that LAH accelerates antithrombin-proteinase reactions by bridging and conformational activation mechanisms similar to those of HAH, with the reduced magnitude of LAH accelerations resulting both from a decreased antithrombin affinity and the inability to induce a full activating conformational change in the inhibitor.