Engineering functional antithrombin exosites in α 1- proteinase inhibitor that specifically promote the inhibition of factor Xa and factor IXa

Abstract

We have previously shown that residues Tyr-253 and Glu-255 in the serpin antithrombin function as exosites to promote the inhibition of factor Xa and factor IXa when the serpin is conformationally activated by heparin. Here we show that functional exosites can be engineered at homologous positions in a P1 Arg variant of the serpin α 1-proteinase inhibitor (α 1PI) that does not require heparin for activation. The combined effect of the two exosites increased the association rate constant for the reactions of α 1PI with factors Xa and IXa 11-14-fold, comparable with their rate-enhancing effects on the reactions of heparin-activated antithrombin with these proteases. The effects of the engineered exosites were specific, α 1PI inhibitor reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in factor Xa, which interacts with the exosite residues in heparin-activated antithrombin, abrogated the ability of the engineered exosites in α 1PI to promote factor Xa inhibition. Binding studies showed that the exosites enhance the Michaelis complex interaction of α 1PI with S195A factor Xa as they do with the heparin-activated antithrombin interaction. Replacement of the P4-P2AIP reactive loop residues in the α 1PI exosite variant with a preferred IEG substrate sequence for factor Xa modestly enhanced the reactivity of the exosite mutant inhibitor with factor Xa by ∼2-fold but greatly increased the selectivity of α 1PI for inhibiting factor Xa over thrombin by ∼ 1000-fold. Together, these results show that a specific and selective inhibitor offactor Xa can be engineered by incorporating factor Xa exosite and reactive site recognition determinants in a serpin. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.