ABSTRACT
Objective To detect the levels of neutrophil extracellular traps (NETs) in patients with antiphospholipid syndrome (APS) and to preliminarily explore its formation mechanism.Methods Plasma samples from 27 APS patients and 30 healthy controls were collected.The circulating free DNA (cf-DNA) in plasma was detected by the PicoGreen nucleic acid quantitative assay kit,and the concentration of citrulline histone 3 (CitH3) was analyzed by enzyme-linked immuno sorbent assay (ELISA).The association of cf-DNA/NETs with thrombotic events in APS patients was further analyzed.The neutrophils in healthy controls were separated by density gradient centrifugation and stimulated with anti-β2GPl/β2GPI complex (100 μg/mL) for 4 h,and the cf-DNA/NETs in the culture supernatant was determined.TLR-4 inhibitor-TAK242 (5 μmol/L) was further used to observe whether the stimulation of the anti-β2GPI/β2GPI complex on cells could be intervened.The differences between groups were analyzed by analysis of variance (ANOVA) or rank sum test,Sidak or Dunnett's test were used to compare the mean of multiple samples and the correlation between variables was analyzed by Spearman's correlation test.Results The concentration of cf-DNA/NETs and CitH3 were significantly increased in plasma of APS patients compared with that in healthy controls [175.7(70.6,205.7) ng/ml vs 29.8(7.6,115.7) ng/ml,Z=-3.654,P＜0.05;19.5(7.8,26.4) ng/ml vs 3.3(0.84,10.3) ng/ml,Z=-3.932,P＜0.05],and there was a significant positive correlation between the cf-DNA/NETs and CitH3 (r=0.447,P=0.019).In the APS group,there was no significant difference in cf-DNA/NETs between patients with arterial thrombosis and those with venous thrombosis [177.1(67.8,297.2) ng/ml vs 184.7(82.4,233.9) ng/ml,Z=-0.301,P=0.786],whereas cf-DNA/NETs in the patients who experienced a new thrombotic event in 1 month was significantly higher than those with a history of thrombosis [192.1(83.6,328.8) ng/ml vs 90.0(42.8,184.7) ng/ml,Z=-2.006,P=0.046].In vitro,anti-β2GPI/β2GPI complex (100 μg/ml) stimulated the release of cf-DNA/NETs from neutrophils,which was significantly increased compared with the control group (t=10.39,P＜0.05),while TAK242 significantly inhibited the stimulating effects of anti-β2GPI/β2GPI complex on cells (t=4.22,P＜0.05).Conclusion The level of cf-DNA/NETs in peripheral blood of APS patients is significantly increased,which may play an important role in APS thrombosis.Anti-β2GPI/β2GPI complex induces the formation of cf-DNA/NETs through TLR4 and participates in the pathological process of APS.