The role of neutrophil extracellular traps in thrombosis of paroxysmal nocturnal hemoglobinuria

Abstract

Abstract Background Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder caused by PIGA mutations, characterized by complement-mediated intravascular hemolysis and a prothrombotic state. Thrombotic events account for 40–67% of PNH-related mortality. While traditional models attribute thrombosis to nitric oxide (NO) depletion and platelet hyperactivation from hemolysis, emerging evidence highlights neutrophil extracellular traps (NETs) as key contributors. These DNA-histone-protease complexes, released during NETosis, promote coagulation through: (1) physical entrapment of platelets and coagulation factors via their fibrous structure, and (2) enzymatic activation of coagulation cascades by prothrombotic effectors like myeloperoxidase (MPO) and neutrophil elastase. This study investigates the mechanistic role of NETs in PNH-associated thrombosis. Methods 28 treatment-naïve PNH patients (8 with acute thrombosis, 20 without) and 17 healthy controls were enrolled. Plasma NET biomarkers (MPO-DNA complexes and citrullinated histone H3 [CitH3]) were quantified by ELISA. Circulating neutrophil-platelet aggregates (PNAs) were assessed via flow cytometry (CD45⁺CD11b⁺CD41a⁺ gating).Neutrophils isolated via density gradient centrifugation were stimulated with PMA (100–500 nM), C5a (100 ng/mL), C3a (10 μg/mL), or G-CSF (10 ng/mL) for 2–6 hours. NETosis was quantified using immunofluorescence (DAPI/CitH3/MPO) and flow cytometry. Results Thrombotic PNH patients showed elevated plasma MPO-DNA ([273.86±114.05] ng/mL vs. [198.47±79.34] ng/mL, P<0.05) and CitH3 ([74.99±35.56] ng/mL vs. [61.34±17.38] ng/mL, P＞0.05) versus non-thrombotic patients. PNAs were higher in PNH versus controls (P<0.01), peaking in thrombotic cases (P<0.05). GPI-deficient neutrophils (CD24⁻FLAER⁻) exhibited higher CD41a expression (P<0.01).Optimal NETosis (300 nM PMA, 4 hours) revealed greater eDNA release in PNH neutrophils versus controls (73.4%±5.2% vs. 52.7%±4.1%, P<0.05). Conclusion PNH is associated with elevated NETs and PNAs due to heightened neutrophil sensitivity to PMA. The correlation between NETs burden and thrombosis supports a model where complement-triggered NETosis and platelet-neutrophil interactions amplify thrombogenesis. These findings advance PNH pathophysiology understanding and position NETs as potential biomarkers for thrombosis risk and therapeutic targets for dual complement/NETs inhibition.