The Antithrombin P1 Residue Is Important for Target Proteinase Specificity but Not for Heparin Activation of the Serpin. Characterization of P1 Antithrombin Variants with Altered Proteinase Specificity but Normal Heparin Activation

Abstract

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.

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

“…It has been postulated that the RCL of ATIII changes conformation upon binding H5 to a "canonical" conformation similar to that seen in the crystal structure of the related serpin, ␣ 1 -antitrypsin (8,7,40). Recent evidence suggests that although P1 Arg of ATIII may indeed reorientate upon binding H5, this alone does not explain the increased rate of interaction between the serpin and fXa (41,42).…”

Localization of an Antithrombin Exosite That Promotes Rapid Inhibition of Factors Xa and IXa Dependent on Heparin Activation of the Serpin

“…The N-terminal hinge residues of the antithrombin reactive loop are thus buried in ␤-sheet A in the free serpin structure but are expelled from sheet A in the heparin-complexed serpin, resulting in exposure of the loop in a manner typical of other serpin structures (21). Additionally, the x-ray structures together with biochemical and mutagenesis data have suggested that the primary specificity-determining residue of the loop, the P1 Arg-393 residue, makes a weak interaction with Glu-255 of the serpin body in the native partially buried loop conformation, which is then disrupted in the activated expelled loop state (22)(23)(24). However, mutagenesis of the reactive loop P6 -P3Ј residues that comprise the protease binding site has shown that conformational activation of antithrombin does not simply involve making the reactive loop more accessible to target proteases.…”

“…However, mutagenesis of the reactive loop P6 -P3Ј residues that comprise the protease binding site has shown that conformational activation of antithrombin does not simply involve making the reactive loop more accessible to target proteases. Thus, whereas the reactivity of unactivated antithrombin with proteases is sensitive to such reactive loop mutations, the heparin enhancement in reactivity is not affected (5,22,25,26). These findings have suggested that exosites outside of the P6 -P3Ј reactive loop region are responsible for the heparin enhancement of antithrombin's reactivity with its target proteases.…”

“…These findings have suggested that exosites outside of the P6 -P3Ј reactive loop region are responsible for the heparin enhancement of antithrombin's reactivity with its target proteases. Whereas exosites clearly exist on heparin to enable a full-length heparin to promote antithrombin-protease reactions by bridging the inhibitor and protease in a ternary complex, additional exosites must also exist on antithrombin to explain how conformational activation by the pentasaccharide produces several hundred-fold enhancements in antithrombin reactivity with two target proteases, factor Xa and factor IXa (5,22,25,26).…”

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

“…FVIIa can also be inhibited by the serpin (serine protease inhibitor) ATIII (antithrombin III), and the inhibition is accelerated many folds by sulfated glycosaminoglycans lining the vascular wall [4]. Interactions taking place at regions remote from the active-site cleft, at so-called exosites, also influence the rate of TFPI inhibition of the FVIIa-TF complex and FXa [5], whereas the direct interaction between ATIII and its target proteases is mediated primarily through active-site interactions and few exosite interactions [6].…”