Anti-β2glycoprotein i-induced neutrophil extracellular traps cause endothelial activation

Objective: Neutrophil extracellular traps (NETs) involvement in antiphospholipid syndrome (APS) pathogenesis is known, but the role of anti-β2glycoprotein I (aβ2GPI) antibodies-induced NETs in triggering a procoagulant and proinflammatory phenotype in endothelial cells (ECs) remains to be evaluated. This study investigated whether NET-aβ2GPI can activate ECs and whether NET-aβ2GPI and NET-phorbol myristate acetate (PMA) have different proteomic profiles. Methods: Healthy donor (HD) neutrophils were stimulated with APS-aβ2GPI, normal human IgG or PMA. NETs were stained with anti-neutrophil elastase and 4'6-diamidino-2-phenylindole (DAPI), and the ability of aβ2GPI to bind NETs and inhibit DNA degradation was investigated. Following aβ2GPI, NET-aβ2GPI and NET-PMA stimuli, we evaluated EC activation investigating intra-cellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and tissue factor (TF) expression using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and EC dysfunction analysing extracellular microvesicles (EMVs) release via flow cytometry and NanoSight analysis. Mass spectrometry-based proteomics was performed on NET-aβ2GPI and NET-PMA. Results: Unlike normal IgG, aβ2GPI induced NET formation and bound to NETs by colocalizing with the neutrophil elastase signal at 93.6% without preventing NET degradation. Compared with unstimulated EC, NET-aβ2GPI triggered higher mRNA and a robust expression of TF, VCAM and ICAM in EC with a change-fold median fluorescence intensity (MFI) of 6, 4.2 and 2.3. aβ2GPI induced a significant increase in EMVs compared with untreated samples and those treated with NETs. Fifty-six proteins were identified, seven resulted upregulated in NET-aβ2GPI and downregulated in PMA-induced ones. GO enrichment analysis revealed that proteins upregulated in NET-aβ2GPI were enriched for ubiquitin protein ligase binding and SLC2A4 translocation to the plasma membrane.