An Inhibitory Single-Domain Antibody against Protein Z-Dependent Protease Inhibitor Promotes Thrombin Generation in Severe Hemophilia A and FXI Deficiency

Claire Auditeau; Tung-Son Nguyen; Floriane Devaux; François Saller; Ivan Peyron; Adeline Blandinières; Christelle Repérant; Sadyo Daramé; Cécile V. Denis; Peter Lenting; Delphine Borgel; Elsa P. Bianchini

doi:10.1055/a-2373-2829

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2025; 125(03): 207-217
DOI: 10.1055/a-2373-2829

Coagulation and Fibrinolysis

An Inhibitory Single-Domain Antibody against Protein Z-Dependent Protease Inhibitor Promotes Thrombin Generation in Severe Hemophilia A and FXI Deficiency

Claire Auditeau

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

²Service d'Hématologie Biologique, Hôpital Necker Enfants Malades, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France

,

Tung-Son Nguyen

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Floriane Devaux

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

François Saller

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Ivan Peyron

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Adeline Blandinières

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

³Laboratoire d'Hématologie Biologique, Hôpital Bicêtre, Assistance Publique-Hôpitaux de Paris (AP-HP), Faculté de médecine, université Paris Saclay, Le Kremlin-Bicêtre, France

,

Christelle Repérant

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Sadyo Daramé

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Cécile V. Denis

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Peter Lenting

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

,

Delphine Borgel

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

²Service d'Hématologie Biologique, Hôpital Necker Enfants Malades, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France

,

Elsa P. Bianchini

¹INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France

› Author Affiliations

Abstract

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 in normal plasma, factor VIII- 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.

Keywords

ZPI - SERPINA10 - single-domain antibody - hemophilia - factor XI deficiency - rebalancing therapy

Full Text

References

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Supplementary Material

Supplementary Material