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Thromb Haemost
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
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
2   Service d'Hématologie Biologique, Hôpital Necker Enfants Malades, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France
,
Tung-Son Nguyen
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Floriane Devaux
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
François Saller
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Ivan Peyron
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Adeline Blandinières
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
3   Laboratoire d'Hématologie Biologique, Hôpital Bicêtre, Assistance Publique-Hôpitaux de Paris (AP-HP), Faculté de médecine, université Paris Saclay, Le Kremlin-Bicêtre, France
,
Christelle Repérant
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Sadyo Daramé
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Cécile V. Denis
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Peter Lenting
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Delphine Borgel
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
2   Service d'Hématologie Biologique, Hôpital Necker Enfants Malades, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France
,
Elsa P. Bianchini 
1   INSERM, Hémostase inflammation thrombose HITh U1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
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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

Supplementary Material



Publication History

Received: 15 February 2024

Accepted: 24 July 2024

Accepted Manuscript online:
25 July 2024

Article published online:
12 August 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

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