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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.
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.
Given the shortcomings of current factor-based, nonfactor-based, and AAV gene-based therapies, the recent advent of RNA-based therapeutics for hemophilia is changing the fundamental approach to hemophilia management. From siRNA therapeutics that knockdown clot regulators antithrombin, protein S, and heparin cofactor II, to CRISPR/Cas9 gene editing that may personalize treatment, improved technologies have the potential to reduce bleeds and factor use and avoid inhibitor formation. These novel agents, some in preclinical studies and others in early phase trials, have the potential to simplify treatment and improve hemostasis and quality of life. Further, as these therapies arise from manipulation of the coagulation cascade and thrombin generation and its regulation, they will enhance our understanding of hemostasis and thrombosis, and ultimately lead to better therapies for children and adults with inherited. bleeding disorders. What does the future hold? With the development of novel preclinical technologies at the bench, there will be fewer joint bleeds, debilitating joint disease, orthopedic surgery, and improved physical and mental health, not previously possible. In this review, we will identify current limitations of treatment and progress in the development of novel RNA therapeutics, including mRNA nanoparticle delivery and gene editing for treatment of hemophilia.
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