Anti-β2glycoprotein I-induced neutrophil extracellular TRAPS cause endothelial activation.

Objective: NETs involvement in antiphospholipid syndrome (APS) pathogenesis is known, but the role of anti-β2glycoprotein I antibodies (aβ2GPI)-induced NETs in triggering a procoagulant and proinflammatory phenotype in endothelial cells (EC) remains to be evaluated. This study investigated whether NET-aβ2GPI can activate ECs and whether NET-aβ2GPI and NET-PMA have different proteomic profiles.

Methods: Healthy donors (HD) neutrophils were stimulated with APS-aβ2GPI, normal human IgG or PMA. NETs were stained with anti-neutrophil elastase and 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 ICAM, VCAM, and TF expression using flow cytometry and RT-qPCR; and EC dysfunction analyzing 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 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, 7 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.

Conclusions: aβ2GPI-induced NETs can cause EC activation and TF expression.