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Engineering a protein Z-dependent protease inhibitor (ZPI) mutant as a novel antagonist of ZPI anticoagulant function for hemophilia treatment.

ABSTRACT:

Background

Protein Z-dependent protease inhibitor (ZPI), is an important anticoagulant protein in plasma that functions in complex with its cofactor, protein Z (PZ) to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. Recent studies suggest that the ZPI-PZ anticoagulant complex is a promising target for restoring hemostasis in hemophilia (Girard, et al, J Thromb Haemost, 2019, 17, 149-156).

Objective

Engineering a ZPI mutant as a novel antagonist of ZPI anticoagulant function.

Methods

We engineered two alanine mutations in human ZPI, one in the reactive loop P1 Y387 residue to inactivate the FXa/FXIa inhibitory function, and the second in the K239 binding interface residue to enhance the affinity of the inactive ZPI for PZ. The mutant was expressed, purified, and characterized by in vitro and plasma assays.

Results

The mutant, Y387A/K239A (ZPI-2A), bound PZ >20-fold tighter than WT ZPI or a PZ antibody (PZAb). FXa inhibition assays showed that ZPI-2A effectively neutralized ZPI/PZ anti-FXa activity with a ~three-fold molar excess over wild type ZPI (WT ZPI) whether FXa was bound to FVa in prothrombinase or unbound. Thrombin generation assays in a purified system or in normal/hemophilia plasmas showed that ZPI/PZ activity was reversed by ZPI-2A in a dose-dependent manner, with a three-fold molar excess sufficient to fully reverse ZPI/PZ inhibition of thrombin generation.

Conclusions

ZPI-2A is a potent antagonist of ZPI/PZ anticoagulant function, capable of fully blocking the anti-FXa activity of plasma levels of ZPI/PZ at significantly lower doses than a PZAb and thus a promising prophylactic agent for treating hemophilia.

SUBMITTER: Huang X

PROVIDER: S-EPMC8112298 | biostudies-literature |

REPOSITORIES: biostudies-literature

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