The Oligosaccharide Side Chain on Asn-135 of α-Antithrombin, Absent in β-Antithrombin, Decreases the Heparin Affinity of the Inhibitor by Affecting the Heparin-Induced Conformational Change

Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a twostep mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHGH). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.The anticoagulant effect of the widely prescribed drug heparin is mediated by its binding to and activation of the serpin antithrombin against the final two proteinase in the blood coagulation cascade, factor Xa and thrombin. Specific binding to heparin is due to the presence of a unique pentasaccharide that is found in about a third of the heparin chains contained in commercial heparin preparations. Pentasaccharide binding by antithrombin is a two-step process where, after the rapid formation of an initial, weak complex, antithrombin changes conformation to form a tight complex (1). The conformational change in antithrombin also affects the reactive center loop and releases constraints on the P1 arginine residue that can then be recognized by target proteinases (2), thus resulting in a 300-fold acceleration in the rate of factor Xa inactivation, whereas full-length heparin is required for acceleration of the inactivation of thrombin since thrombin must also bind heparin via its exosite 2 (3, 4). Due to the detrimental side effects of long chain therapeutic heparin preparations, drug design has focused on maximization of the anti-factor Xa activity of heparin and minimization of the anti-thrombin activity.It has been proposed that the mechanism by which pentasaccharide binding affects conformational change in the reactive center loop of antithrombin involves binding to and elongation of the C terminus of helix D (5). The recently solved structure of antithrombin in complex with a high affinity form of the pentasaccharide (6) indicated that the C terminus of helix D was not directly involved in pentasaccharide binding and was more likely involved in the binding of the non-reducing-e...

Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

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The Effect of a Reducing-end Extension on Pentasaccharide Binding by Antithrombin

Belzar¹,

Dafforn²,

Petitou³

et al. 2000

“…The cDNA for N135Q antithrombin was used as the template for mutating reactive center loop residues to eliminate glycosylation heterogeneity resulting from incomplete glycosylation at Asn-135 and to mimic the high heparin affinity ␤-form of plasma antithrombin that lacks the Asn-135 carbohydrate chain (15,16). Site-directed mutagenesis of human antithrombin cDNA was carried out in the singlestranded M13mp19 vector as described (7) using an antisense oligonucleotide encoding the desired mutation.…”

Section: Construction Expression and Purification Of Antithrombin Vmentioning

confidence: 99%

Heparin Enhances the Specificity of Antithrombin for Thrombin and Factor Xa Independent of the Reactive Center Loop Sequence

Chuang

Swanson

Raja

et al. 2001

Self Cite

148

130

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6 -P3 proteinase binding sequence excluding P1-P1 to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2 residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6 -P3 residues flanking the primary P1-P1 recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6 -P3 region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.Antithrombin is a member of the serpin family of serine proteinase inhibitors whose primary physiologic function is to inhibit and thereby regulate several serine proteinases in the blood coagulation pathway (1, 2). Inhibition of these proteinases results from the serpin trapping the enzymes as stable acyl-intermediate complexes of a regular substrate reaction through a major conformational change. A unique feature of antithrombin is that it requires activation by the polysaccharide, heparin, to inhibit its main target enzymes, thrombin and factor Xa, at a physiologically significant rate. Heparin activates antithrombin through two distinct mechanisms whose relative contribution depends on the proteinase inhibited (3, 4). In both mechanisms, antithrombin binds to a sequence-specific pentasaccharide in heparin (5) which induces an activating conformational change in the reactive center loop of the serpin (6 -9). Such conformational changes are alone sufficient to accelerate antithrombin inactivation of factor Xa through an allosteric mechanism. By contrast, the conformational changes negligibly affect the rate of thrombin inhibition, heparin a...

“…The naturally occurring ␤-isoform of ATIII lacks carbohydrate on asparagine 135 (30) because its NXS type consensus sequence is inefficiently glycosylated (31). The ␤-isoform binds heparin more tightly than does ␣-isoform due to effects of the Asn 135 oligosaccharide on the heparin-induced conformational change which accompanies formation of the high affinity heparin-ATIII complex (59). Affinity of antithrombin for heparin is also affected by characteristics of attached carbohydrates.…”

mentioning

confidence: 99%

Identification of the Antithrombin III Heparin Binding Site

Ersdal-Badju

Zuo

et al. 1997

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.