Background:Patients with immune thrombocytopenia (ITP) are at increased risk of vascular events (VE), but the precise mechanisms of this prothrombotic state remain largely unknown. Neutrophil extracellular traps (NETs) are DNA‐containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non‐immune disorders. However, the contribution of NETs in the prothrombotic state in ITP patients is largely unknown.Aims:To determine if ITP patients have enhanced intrinsic potential for NET formation and to evaluate their prothrombotic role in ITP, their correlation with platelet activation, and also with specific therapeutic approaches.Methods:Sixty‐three ITP patients at different stages of disease and 30 healthy controls were included. NETs were evaluated by quantifying cell free DNA (cfDNA) using SYTOX Green, and by assessing the levels of Citrullinated Histone H3 complexed to DNA (H3Cit‐DNA) via a sandwich ELISA. Platelet and white blood cell (WBC) counts were assessed by a cell counter (Sysmex), and platelet glycoproteins, size, and degranulation were evaluated by flow cytometry.Results:The levels of cfDNA and H3Cit‐DNA in the peripheral blood of ITP patients were higher than in controls (p = 0.006, and p = 0.001, respectively). The percentage of patients with detectable H3Cit‐DNA complexes (optical density >0.199) was significantly different to that of controls (26.2% vs. 6.7%; p = 0.029). In patients, positivity for H3Cit‐DNA correlated with higher cfDNA (Pearson correlation: R = 0.407; p = 0.01). No significant differences in the levels of cfDNA or H3Cit‐DNA were detected between the patients in remission (n = 24) and those with active ITP (n = 39). Seven ITP patients (11.1%) had experienced previous VE and presented with increased cfDNA (0.207 vs. 0.152 ng/ml; p = 0.034), WBC count (9.6 vs. 8.0 x109/L; p = 0.037), and neutrophil count (5.4 vs. 4.3x109/L; p = 0.027) compared to patients with no history of thrombotic events. Only one of the patients with VE was under low dose prednisone. Previous thrombosis was neither associated to H3Cit‐cfDNA positivity, platelet counts, mean fluorescence intensity of platelet glycoprotein expression (GPIba, aIIb), size (FSC), nor percentage of degranulated platelets (CD62, CD63). When we evaluated whether other acquired risk factors correlated with cfDNA, neither previous splenectomy, hypertension, hypercholesterolemia, smoking, age, nor diabetes correlated with cfDNA. Notably, treatment with thrombopoietin receptor agonists, corticosteroids, or immunosuppresants at sampling were neither related to circulating NETs cfDNA or H3Cit‐DNA) nor to platelet activation.Summary/Conclusion:Our results show that plasma NET levels are elevated in ITP patients, and that this increase is especially marked in patients who have suffered from a vascular event. The fact that patients with previous thrombosis had increased WBC and neutrophil counts suggests that there may be more NETosis in these individuals, probably indicating a dysregulation between production and clearance. Other factors (platelet activation, acquired thrombotic risk factors or current therapy) did not seem to influence NET formation or platelet degradation. Similar to patients with cancer and myeloproliferative disorders, where leukocytosis has been associated with increased thromboembolic risk, we propose an association between NET generation and neutrophil counts leading to increased risk of thrombosis in ITP, which may provide new therapeutic targets to combat VE in ITP.Funding: ISCIII and FEDER [PI17/00051].
