An inhibitory single-domain antibody against protein Z-dependent protease inhibitor promotes thrombin generation in hemophilia A and FXI deficiency.

BACKGROUND

Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia.

AIMS

To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity.

METHODS

We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay (TGA) in normal plasma, factor VIII (FVIII)- and FXI-deficient plasma.

RESULTS

Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency.

CONCLUSION

ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders.

Informations de copyright

Thieme. All rights reserved.

Déclaration de conflit d'intérêts

The authors declare that they have no conflict of interest.