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Characterization of Protein Z-Dependent Protease Inhibitor/Antithrombin Chimeras Provides Insight into the Serpin Specificity of Coagulation Proteases.

ABSTRACT: Protein Z (PZ)-dependent protease inhibitor (ZPI) and antithrombin (AT) are two physiological serpin inhibitors involved in the regulation of proteolytic activities of the blood coagulation cascade. ZPI has restricted protease specificity capable of inhibiting factors Xa (FXa) and XIa (FXIa) but exhibiting no reactivity with other coagulation proteases. Unlike ZPI, AT is a general inhibitor of all coagulation proteases and the only physiological inhibitor of factor IXa (FIXa). To understand the molecular determinants of protease specificity of the two serpins, we engineered two ZPI mutants in which the P12-P3' residues of the reactive center loop of ZPI were replaced with either P12-P3' or P12-P7' residues of AT (ZPI-AT^P12-P3' and ZPI-AT^P12-P7'). The reactivity of chimeras with FXa was improved ∼4-25-fold in the absence of PZ. Both chimeras inhibited FIXa with rate constants that were ∼2 orders of magnitude higher than the rate of the AT inhibition of the protease. PZ improved the reactivity of chimeras with FIXa by another 2 orders of magnitude, rendering the chimeras potent inhibitors of FIXa so that the PZ-mediated inhibitory activity of the ZPI-AT chimeras toward FIXa was ∼20-fold higher than that of the fondaparinux-catalyzed inhibition of FIXa by AT. Further studies revealed that the substitution of P1-Tyr of ZPI with an Arg is sufficient to convert the serpin to an effective inhibitor of FIXa. The potential therapeutic utility of the serpin chimeras as specific inhibitors of FIXa was diminished by findings that the chimeras function as effective substrates for other coagulation proteases.

SUBMITTER: Yang L

PROVIDER: S-EPMC5537704 | biostudies-literature |

REPOSITORIES: biostudies-literature

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