CO084 | NEW INSIGHTS INTO HEMOPHILIA A: FACTOR VIII–INTEGRIN Β1 PATHWAY AS KEY REGULATOR OF ENDOTHELIAL CELL FUNCTION

A. Cucci¹, C. Olgasi², G. Venturin¹, S. Assanelli¹, C. Sgromo¹, S. Reano², M. Manfredi², A. Follenzi^1|3 | ¹Dip. di Scienze della Salute, Università del Piemonte Orientale, Novara, Italy; ²Dip. di Medicina Traslazionale, Università del Piemonte Orientale, Novara, Italy; ³Dip. Attività Integrate Ricerca Innovazione, AOU SS. Antonio e Biagio e C. Arrigo, Alessandria, Italy

Background and Aims. Factor VIII (FVIII) is a well-known coagulation factor, and its deficiency causes hemophilia A (HA). Beyond its canonical role in coagulation, FVIII has emerged as a regulator of endothelial cell (EC) biology. However, the molecular mechanisms by which FVIII exert direct effects on EC function remain poorly defined, and the endothelial receptor mediating FVIII-dependent signaling, vascular integrity, and metabolism are still under investigation. The aim of the study is to identify the endothelial surface receptor that binds FVIII and to map the downstream signaling pathways regulating EC function and vascular homeostasis.
Methods. Blood outgrowth ECs (BOECs) from HA patients and healthy donors were isolated. FVIII expression in HA-BOECs was restored either by lentiviral vector transduction with a B-domain deleted FVIII (LV-FVIII) or by recombinant FVIII (rhFVIII) treatment. Proteomic profiling of healthy, HA, and LV-FVIII HA-BOECs was assessed. Ligand-receptor interactions were investigated using Flow-TriCEPS technology, co-immunoprecipitation, and immunofluorescence. Functional assays included angiogenesis, migration, permeability, 3D vessel-on-chip models, mitochondrial respirometry, and proliferation. The role of integrin β1 signaling was assessed by shRNA-mediated knockdown and downstream FAK inhibition.
Results. Co-immunoprecipitation, colocalization, and shRNA-mediated knockdown studies identified integrin β1 as a key endothelial surface receptor mediating FVIII binding. Engagement of the FVIII–integrin β1 activated intracellular FAK/Src signaling, which was essential for FVIII-driven angiogenesis, migration, and barrier stabilization. In 3D vessel-on-chip models, FVIII-integrin β1 signaling restored vascular integrity and network formation in HA BOECs, whereas integrin β1 silencing abolished these effects. Proteomic profiling revealed dysregulation of pathways related to angiogenesis, extracellular matrix organization, and mitochondrial metabolism in HA-BOECs, all normalized by FVIII reintroduction. Consistently, HA BOECs displayed impaired mitochondrial respiration, ATP production, and reserve respiratory capacity, which were rescued by FVIII in an integrin β1-dependent manner. FVIII deficiency significantly impaired endothelial proliferation, which was restored by either FVIII replacement therapy or gene transfer. However, FVIII signaling was not rescued in HA-BOECs following integrin β1 silencing, supporting the existence of a functional FVIII–integrin β1 signaling axis.
Conclusions. FVIII functions as a ligand for integrin β1 on ECs, activating FAK-dependent signaling that governs vascular integrity, proliferation, angiogenesis, and mitochondrial activity. This newly defined FVIII–integrin β1 axis is critical for endothelial homeostasis and provides a mechanistic explanation for endothelial dysfunction in hemophilia A, highlighting novel therapeutic opportunities beyond hemostasis.