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

Abstract

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...

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

“…Studies of FXa have demonstrated that the P2 glycine preference does not solely determine the specificity towards ATIII indicated by the findings that interactions at secondary sites outside the P6-P3 region also influence the inhibition of FXa by ATIII [35]. In addition, an exosite within strand three of β-sheet C of ATIII has been identified and has been shown to be important in the reaction of ATIII with FIXa and FXa [36].…”

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

“…Theunissen et al (43) showed that substitutions at the P1Ј and P3Ј positions of the RCL of ATIII markedly decreased the interaction of ATIII with thrombin in the presence of heparin but had only moderate effects on fXa interactions with ATIII in the presence of heparin. A more recent study by Chuang et al (44) found that the RCL residues P6-P3Ј of ATIII did not contribute substantially to the increase in association seen with H5-activated ATIII and fXa, although it must be noted that no substitutions were made at P1Ј position. Rezaie (45) recently showed that a mutant of ATIII that had substituted the P4 -P4Ј region of ATIII with the prothrombin activation sequence (IEGR-IVEG) still had normal if not increased acceleration of its interaction with human fXa in the presence of H5, whereas its interaction with human thrombin was markedly decreased.…”

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

“…Early models based on x-ray structures of native and heparin-activated antithrombin suggested the importance of releasing the reactive center protease binding loop (RCL) 2 from a constrained interaction with sheet A in the native state, first as a means of making the critical P1 Arg recognition determinant accessible to target proteases (11) and later as a means to gain access to exosites outside the RCL to augment the protease interaction (12)(13)(14)(15). Although a critical role for exosites in augmenting the antithrombin RCL interaction with factors Xa/IXa in the allosterically activated state is now well established, a role for RCL expulsion from sheet A in making the exosites available for interaction with protease is not supported by much available data.…”