Role of the Reactive Center Loop of Protein Z Dependent Protease Inhibitor in Determining Specificity of the Protease Reaction

Abstract

Antithrombin (AT) and protein Z‐dependent protease inhibitor (ZPI) are two serpin inhibitors in plasma that contribute to regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has a narrower specificity, thus being capable of inhibiting only factors Xa (fXa) and XIa. To investigate the contribution of residues of the reactive center loop (RCL) to restricting the specificity of ZPI, we engineered two AT mutants in which the P1‐Arg of the RCL was replaced with P1‐Tyr of ZPI (AT‐R393Y) in one construct and the P12‐P3’ residues were replaced with the corresponding residues of ZPI in the other. The mutants were characterized with respect to their ability to react with coagulation proteases, chymotrypsin and a fXa mutant containing a Ser substitution for Asp‐189 at its primary specificity pocket (D189S). The reactivity of AT‐R393Y with all coagulation proteases including fXa was dramatically impaired, however, the serpin mutant inhibited chymotrypsin with >four orders of magnitude higher specificity. On the other hand, the P12‐P3’ mutant of AT did not exhibit improved reactivity with chymotrypsin, but reacted with a normal reactivity with fXa, though the reaction was not enhanced by heparin cofactors. The fXa‐D189S mutant exibited similar poor reactivity with both AT and AT‐R393Y, however, the protease mutant exhibited normal or improved reactivity with ZPI and AT‐P12‐P3’. These results suggest that P1‐Tyr as well as other unique structural features within the ZPI RCL are responsible for restricting the protease specificity of the serpin.