Thubthed R.Praneetponkang R.Sittipaisankul P.Thiengtavor C.Chumpuchanaphai S.Paiboonsukwong K.Fucharoen S.Pattanapanyasat K.Vadolas J.Smith D.R.Svasti S.Chaichompoo P.Mahidol University2026-06-062026-06-062026-01-01British Journal of Haematology (2026)00071048https://repository.li.mahidol.ac.th/handle/123456789/117116The hypercoagulable state is a major contributor to thromboembolic events and mortality in β-thalassaemia. The mechanisms underlying platelet-induced neutrophil activation leading to immunothrombosis remain poorly understood. Three-dimensional confocal microscopy demonstrated that platelets induced neutrophil extracellular trap (NET) formation through P-selectin- or high mobility group box 1 protein (HMGB1)-mediated binding to P-selectin glycoprotein ligand-1 (PSGL-1) or receptor for advanced glycation end products (RAGE), respectively, on neutrophils. Molecular signalling pathways involved in NETs—focusing on mitogen-activated protein kinase kinase (MAPKK) or mitogen-activated protein kinase kinase 1/2 (MAP2K) (MEK)/extracellular signal–regulated kinase (ERK), reactive oxygen species (ROS), ofnicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 2 (NOX2), myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4)—were investigated in neutrophils from β-thalassaemia/haemoglobin E (HbE) patients (splenectomy and non-splenectomy) and from normal subjects by priming neutrophils with specific inhibitors before treatment with either platelets, recombinant P-selectin or disulphide HMGB1. In splenectomised patients, neutrophils primed with U0126, but not an ERK inhibitor, exhibited reduced web-like NETs and cell aggregation, associated with antioxidant effects. In non-splenectomised patients and normal subjects, P-selectin activated MEK/ERK, NOX2, MPO and PAD4 pathways, promoting web-like NETs and cell aggregation. HMGB1 activated neutrophils via MEK/ERK, NOX2, MPO and PAD4 pathways, in all groups, resulting in NETs without aggregation-associated NET morphology. These findings indicate that splenectomy alters P-selectin and HMGB1 expression on platelets, leading to altered signalling dynamics in neutrophils and promoting neutrophil–platelet aggregation. ROS pathway could be a key regulator of NET-driven thrombosis in splenectomised β-thalassaemia/HbE disease and highlight its potential as a therapeutic target.MedicineArticleSCOPUS10.1111/bjh.705772-s2.0-10504035910413652141