Abstract Number: OC 14.2
Meeting: ISTH 2021 Congress
Theme: Coagulation and Natural Anticoagulants » Regulation of Coagulation
Background: An emerging strategy in the treatment of hemophilia is to inhibit natural anticoagulants to restore hemostasis. Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin targeting factor Xa (FXa) in the presence of Protein Z (PZ) as cofactor and factor XIa (FXIa) in a cofactor-independent manner. PZ or ZPI gene invalidation is associated with a mitigation of the bleeding phenotype in factor VIII deficient mice, and pharmacological inhibition of PZ-mediated anti-FXa activity of ZPI improves thrombin generation in plasma from patients with hemophilia.
Aims: Here, we propose to develop an innovative approach to inhibit ZPI anticoagulant activity by using a single-domain antibody (sdAb).
Methods: A llama-derived immune library of sdAbs was screened by phage display to identify sdAbs recognizing ZPI. The selected sdAbs were expressed in bacteria and purified for further characterization. Binding of sdAbs to ZPI or other serpins was evaluated by ELISA and ZPI inhibition was measured in anti-FXa or anti-FXIa chromogenic assay. Finally, the procoagulant activity of the sdAbs was assessed in a thrombin generation assay (TGA) in normal or hemophilic plasma.
Results: Among four sdAbs selected for their ability to bind ZPI, one, named ZPI-sdAb, specifically recognized ZPI and did not cross-react with other plasma serpins like antithrombin or α2-antitrypsin. ZPI-sdAb dose-dependently inhibited the anti-FXa and anti-FXIa activities of ZPI, with a median inhibitory concentration of 3.1 ± 0.4 and 1.5 ± 0.4 µM, respectively. In addition, ZPI-sdAb significantly promoted thrombin generation in plasma from healthy donors or from patients with severe hemophilia A or B and shifted the hemophilia phenotype from severe to moderate, according to the measurement of the TGA parameters.
| Hemophilia A patient plasma | Hemophilia B patient plasma | |||||
| ctrl | + ZPI-sdAb | + FVIII | ctrl | + ZPI-sdAb | + FIX | |
| Lag time (min) | 10,7 ± 0,88 | 9,58 ± 0,77 | 14,9 ± 1,6* | 11,4 ± 1,0 | 8,51 ± 0,81 | 14,9 ± 1,3 |
| ETP (nM.min) | 149 ± 14 | 250 ± 15* | 293 ± 40*,$ | 104 ± 14 | 259 ± 33* | 357 ± 68*,$ |
| Peak high (nM) | 7,5 ± 0,79 | 12,6 ± 0,99* | 16,7 ± 2,4*,$ | 3,94 ± 0,55 | 10,2 ± 1,1* | 22,0 ± 4,9* |
| Time to peak (min) | 26,5 ± 1,3 | 25,1 ± 1,3 | 28,8 ± 2,0$ | 29,9 ± 1,5 | 25,3 ± 1,7 | 23,5 ± 1,8$ |
Thrombin generation assay parameters measured in plasma from patients with hemophilia: Plasma were spiked with ZPI-sdAb (10 µM final), factor VIII (FVIII for hemophilia A, 0.01 UI/mL final), factor IX (FIX for hemophilia B, 0.01 UI/mL final), or vehicle as control (ctrl) and thrombin generation was triggered with tissue factor (1 pM) in the presence of phospholipid vesicles (4µM). TGA parameters were measured using the Thrombinoscope software (Stago). Statistical analysis was performed using a one-way ANOVA with Tukey’s correction for multiple comparison. *indicates that the mean is significantly different from the corresponding mean in the control group (p < 0.05), and $ indicates that the mean is not statistically different from the corresponding mean in the group with ZPI-sdAb (p>0,05).
Conclusions: ZPI-sdAb is the first reported specific and direct ZPI inhibitor exhibiting procoagulant activity in plasma. ZPI-sdAb could therefore present a potential interest in the treatment of hemophilia or other bleeding disorders.
To cite this abstract in AMA style:
Auditeau C, Nguyen TS, Repérant C, Daramé S, Saller F, Borgel D, Bianchini E. A Single-domain Antibody Anti-protein Z Dependent Protease Inhibitor Improves Thrombin Generation in Hemophilia [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/a-single-domain-antibody-anti-protein-z-dependent-protease-inhibitor-improves-thrombin-generation-in-hemophilia/. Accessed November 29, 2023.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/a-single-domain-antibody-anti-protein-z-dependent-protease-inhibitor-improves-thrombin-generation-in-hemophilia/