Platelet α_IIbβ₃ Receptor Antagonist Impedes the Hemostatic Efficacy of Platelet-Specific FVIII Gene Therapy in Hemophilia a Mice

Hemophilia A is a prime model for gene therapy. Data from the current human clinical trial using liver-specific AAV-mediated FVIII gene therapy are encouraging, but FVIII levels steadily decline, raising serious concerns about durability and safety. Furthermore, pediatric and adult patients who have severe liver disease, neutralizing antibodies to AAV, which are present in 30-50% of the population, are excluded from AAV liver gene therapy. Our previous studies have demonstrated that platelet-targeted FVIII expression under the control of the platelet-specific αIIb promoter (2bF8) is effective in rescuing the bleeding phenotype of hemophilia (FVIII^-/-) mice even in the presence of anti-FVIII inhibitory antibodies (inhibitors). Currently, our gene therapy approach is in Phase I clinical trial. Since antiplatelet drugs are commonly used in the clinic, it is important to investigate how platelet antagonists affect the clinical efficacy of platelet-derived FVIII. The α_IIbβ₃ complex is a receptor for fibrinogen and VWF, with a crucial role in platelet activation and platelet plug formation at the site of injury. The α_IIbβ₃ complex is the target of several drugs used in cardiovascular medicine. Thus, we propose to use an α_IIbβ₃ antibody to block the integrin α_IIbβ₃ receptor to investigate how platelet aggregation impacts the clinical efficacy of platelet-derived FVIII in FVIII^-/- mice.

We used anti-mouse integrin α_IIbβ₃ antibody (Leo.H4) to block the α_IIbβ₃ complex, inhibiting its binding to fibrinogen and therefore inhibiting platelet aggregation. Hemostatic properties of platelet-FVIII were assessed by ex vivo ROTEM and native whole blood TGA (nWB-TGA) analysis of whole blood and in vivo tail FeCl₃-induced carotid artery injury model and lateral tail vein transection (TVT) injury model.In 2bF8/FVIII^-/- mice, platelet-FVIII levels were 6.89±0.65 mU/10⁸ platelets, with all parameters from ROTEM analysis comparable to those in WT control mice. However, all parameters, including clotting time, clot formation time, maximal clot firmness, α-angle, and A10 were significantly impaired in the presence of Leo.H4 compared to native samples from 2bF8 mice (8.5±1.96 min, 3.1±0.41min, 67.25±2.63mm, 55.75±3.59°, 44.25±4.03mm in native samples versus 10.68±1.77min, 13.5±3.11min, 28.5±0.58mm, 33±2.83°, 18.25±1.26mm in those with Leo.H4, respectively). In contrast, in WT mice, only α-angle and A10 were significantly tempered in the presence of Leo.H4. Interestingly, in 2bF8/FVIII^-/- mice, peak thrombin and endogenous thrombin potential determined by TGA were significantly enhanced in the presence of Leo.H4 compared to native samples (58.4±23.72nM and 600.8±185.83nM vs. 97.93±13.89nM and 921.03±72.95nM, respectively), but there were no significant differences in other parameters. Leo.H4 did not impact the hemostatic parameters determined by nWB-TGA in WT mice. Complete occlusion occurred in the FeCl₃-induced carotid injury in all 2bF8 and WT mice, but none occluded when Leo.H4 was infused. In the TVT injury model, when Leo.H4 was infused, none of 2bF8/FVIII^-/- mice stopped bleeding in the 10-minute bleeding test, and animals lost a significant amount of blood (525.98 ± 92.06 μL). In contrast, when animals were infused with IgG isotype control, the bleeding time was 0.99 ± 0.05 min, and blood loss in the IgG control group was 17.05 ± 9.37 μL, which was 30-fold lower than in the Leo.H4 group. There were no significant differences in the bleeding time and blood loss between the 2bF8 infused with IgG and WT groups in the TVT test.

In conclusion, our results demonstrated that α_IIbβ₃-mediated platelet aggregation is critical in maintaining the hemostatic functions and clinical efficacy of platelet-derived FVIII in hemophilia A. Anti-αIIbβ3 antibody, Leo.H4, impedes the hemostatic efficacy of platelet-FVIII in FVIII^-/- mice.

No relevant conflicts of interest to declare.