Antiphospholipid antibodies induce endothelial procoagulant activity and release of extracellular vesicles independently of a second hit.

Introduction: Antiphospholipid antibodies (aPLs) can promote thrombosis Methods: Human umbilical vein endothelial cells (HUVECs) were stimulated with IgG from patients with vascular and obstetric APS (IgG APS) or IgG purified from the serum of healthy women with proven gestational success (IgG healthy control -HC-). IgG binding to HUVECs, expression of tissue factor, and the procoagulant activity of the endothelial surface were evaluated. EVs were isolated from the supernatants and characterized by nanoparticle tracking analysis, cryo-transmission electron microscopy, flow cytometry, and Western blotting. Results: Compared to IgG HC, IgG APS showed increased binding to the endothelial surface upon prior and concomitant stimulus with LPS (HC 10.92; APS 81.61 mean fluorescence intensity -MFI-, p < 0.01). This enhanced binding capacity of IgG APS to HUVECs was preserved even in the absence of LPS (HC 12.23; APS 84.26 MFI, p < 0.05). Additionally, IgG APS enhanced the platelet-rich plasma-dependent procoagulant activity of the endothelial surface (HC 0.031; APS 0.098 clot density; p < 0.05), and the release of large EVs (HC 7.3 x 10 Conclusion: Our findings suggest that IgG APS can trigger second hit-independent procoagulant mechanisms in HUVECs and induce the release of lEVs that subsequently display surface-bound IgG, highlighting a potential role for endothelial EVs in APS pathophysiology.