Elimination of P1 Arginine 393 Interaction with Underlying Glutamic Acid 255 Partially Activates Antithrombin III for Thrombin Inhibition but Not Factor Xa Inhibition

Abstract

The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu 255 on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg 393 -Glu 255 hydrogen bond and ionic interactions were eliminated by converting Glu 255 to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu 255 -mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wildtype parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000-and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although "approximation" is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all. Antithrombin III (ATIII)1 is a key inhibitor of blood coagulation enzymes and belongs to the serpin family of protease inhibitors whose members employ a common inhibition mechanism. However, the rate at which ATIII inhibits its target enzymes is abnormally low for a serpin, and activation by heparin or vascular wall heparan sulfate proteoglycans (HSPGs) is necessary for it to achieve the high inhibition rates characteristic of other serpins. The physiological importance of ATIII-heparin/HSPG interactions is supported by the occurrence of thrombosis in individuals carrying ATIII molecules with defects in heparin binding or activation, and by the clinical use of pharmaceutical heparin as an anticoagulant in patients with hereditary and acquired thrombosis.Because of the extensive use of heparin as a pharmaceutical, the mechanism for heparin activation of ATIII anticoagulant activity has been extensively investigated. These studies show that dramatically increased rates of thrombin and factor Xa inhibition can be achieved upon binding of heparin. Heparin binding to antithrombin is a two-step process consisting of an initial low affinity interaction that induces a large scale protein conformational change, which leads to high affinity binding and ATIII activation (1). The rate enhancement for the inhibition of thrombin results from an approximation (bridging) mechanism in which long, high affinity heparin chains (Ͼ18 sugars) facilitate a Ͼ1000-fold increased rate of association between thrombin and ATIII (2). In contrast, a specific anticoagulantly activ...

Residues Tyr253 and Glu255 in Strand 3 of β-Sheet C of Antithrombin Are Key Determinants of an Exosite Made Accessible by Heparin Activation to Promote Rapid Inhibition of Factors Xa and IXa

“…Since factor Xa, like thrombin, is reactive with the native conformation of antithrombin, which is most sensitive to the surface charge distribution (31), similar context-dependent effects of mutations may account for some of the observed effects on antithrombin reactivity with factor Xa in the absence of heparin, such as the larger than expected effect of the single Y253A mutation. (35). Notably, replacement of Tyr 253 with Ala greatly perturbed both factor Xa and factor IXa exosite interactions, replacement with Lys only perturbed the factor IXa and not the factor Xa exosite interaction, and replacement with Phe had minimal effects on either exosite interaction.…”

Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation

“…Stoichiometries, Affinities, and Ionic Strength Dependence of Heparin Binding-Stoichiometries and dissociation equilibrium constants (K d ) for pentasaccharide and full-length heparin binding to ATIII variants were determined by titrations monitored by the tryptophan fluorescence enhancement that accompanies the binding interaction as previously described (22,26). Stoichiometric titrations were performed with full-length heparin at an ionic strength of 0.10, using antithrombin concentrations based on absorbance measurements that were more than 10 times the K d .…”

“…Human factor Xa was purchased from Enzyme Research Laboratories. High affinity heparin (molecular mass, ϳ20,000 Da) was purified in our laboratory as previously described (22). High affinity heparin and pentasaccharide concentrations were determined by stoichiometric fluorescence titrations versus plasma antithrombin.…”

Disruption of a Tight Cluster Surrounding Tyrosine 131 in the Native Conformation of Antithrombin III Activates It for Factor Xa Inhibition

“…Human factor Xa was from Enzyme Research Laboratories. Full-length high affinity heparin (HAH) (molecular mass, ϳ20,000 Da) was purified as described previously (13). High affinity heparin and pentasaccharide concentrations were determined by stoichiometric fluorescence titrations versus freshly purified human plasma antithrombin.…”

“…Thrombin and fXa Inhibition-Apparent second order rate constants (k app ) for ATIII inhibition of thrombin and fXa and the stoichiometries of inhibition (SI) were measured in the absence or presence of pentasaccharide or full-length high affinity heparin (HAH), as described previously (7,13). Association rate constants (k assoc ) were obtained by correcting k app values for the content of inactive material in the sample and/or substrate pathway partitioning by multiplying the average of 2-3 k app measurements by the SI value obtained for the same ATIII-target enzyme-(heparin) combination.…”