Impact of antiplatelet therapy on the hemostatic efficacy of platelet-targeted FVIII gene therapy in hemophilia A mice

Platelet-targeted factor VIII (FVIII) (2bF8) gene therapy offers promising hemostatic correction in hemophilia A (HA), even in the presence of FVIII inhibitors. Because platelet-derived FVIII is released from platelet α-granules upon activation at injury sites, it is essential to evaluate whether antiplatelet agents would compromise its therapeutic efficacy. Here, we investigated the effect of antiplatelet agents on the efficacy of platelet-FVIII in HA mice. 2bF8Tg mice express FVIII in platelets controlled by the αIIb promoter and were treated with aspirin, clopidogrel, or the αIIbβ3-blocking antibody Leo.H4. Hemostatic function was evaluated through tail-bleeding models and in vitro assays. We found that all antiplatelet agents impaired platelet-FVIII-mediated hemostasis in vivo. Leo.H4 treatment caused severe bleeding in both the tail tip and tail vein transection injury models, consistent with its inhibition of platelet aggregation. Clopidogrel-treated 2bF8Tg mice exhibited 10 to 14 times more blood loss than controls, with extended bleeding; however, they still showed improvement compared to FVIIInull controls. Notably, platelet-FVIII retained superior efficacy compared to plasma-derived FVIII in clopidogrel-treated mice, despite having only 24% of normal FVIII activity. Aspirin also impaired hemostasis but to a lesser extent. In vitro rotational thromboelastometry and whole-blood thrombin generation assays showed no significant differences between aspirin- or clopidogrel-treated and control mice, suggesting that these assays may not fully capture platelet-FVIII dynamics. Our results indicate that platelet aggregation is crucial for the efficacy of platelet-FVIII. Although antiplatelet agents hinder its function, platelet-FVIII remains preferable to plasma-FVIII, highlighting its clinical potential and the importance of considering concomitant therapies in future applications.