CD40-CD40L inhibition attenuates platelet-neutrophil interaction and neutrophil extracellular trap release in primary antiphospholipid syndrome

OBJECTIVES: Neutrophil extracellular traps (NETs) are increasingly recognised for their role in primary antiphospholipid syndrome (PAPS) pathogenesis. Herein, we examined underlying mechanisms driving NET formation and the thrombogenic effects of NETs in patients with PAPS, along with potential inhibitors. METHODS: We examined NET release in PAPS, asymptomatic antiphospholipid autoantibodies (aPLs) carriers, and healthy controls (HCs) as well as NET-bound proteins by immunofluorescence, immunoblotting, quantitative polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). We assessed platelet-neutrophil aggregates by flow cytometry (CD61/CD66b staining) and autophagy using LC3B immunofluorescence and immunoblotting. Immunofluorescence was performed in paraffin-embedded kidney, skin ulcer, and endoarterial thrombus tissues from patients with PAPS. Suppression of NET release via CD40-CD40L inhibition was tested in in vitro and venous thrombosis mouse models. RESULTS: Neutrophils from patients with PAPS exhibited increased NET release compared with asymptomatic aPL carriers and HCs. NETs from patients with PAPS expressed tissue factor (TF) that induced thrombin generation in platelet-poor plasma from HCs. aPL induced in vitro intracellular TF expression in HC neutrophils. TF-expressing NETs were abundant in the kidney, skin ulcer, and endoarterial thrombus tissues from patients with PAPS, and colocalised with fibrinogen. NET release in PAPS neutrophils was driven by aPL-mediated platelet activation, subsequent platelet-neutrophil interaction, and autophagy induction. CD40-CD40L blockade reduced platelet activation, autophagy, and NET formation in patients with PAPS. In a mouse model, inhibition of CD40-CD40L reduced neutrophil-platelet aggregates and myeloperoxidase (MPO)-DNA complexes in mouse peripheral blood, and the presence of NETs in the formed thrombi. CONCLUSIONS: Targeting the platelet-neutrophil/autophagy/TF-expressing NETs axis by CD40-CD40L inhibition attenuates thromboinflammation in PAPS and should be explored as a potential therapeutic target.