**5.2** *In vitro* **studies (characterization of EVs released by aPL stimulated cells)**

One mechanism by which aPL promote thromboses is through their binding to endothelial cells causing the activation of endothelial cells [98, 99] which in response, release EVs that might modulate the activation of other adjacent cells [87, 100]. These effects were investigated on endothelial cells [87, 100–102] and placental explants [103] involving both small EVs and medium/large EVs (**Table 3**). A study by Dignat-George et al., showed a significant 4-fold increase in endothelial EVs with procoagulant activity after stimulation of human umbilical vein endothelial cells (HUVEC) with plasma of APS patients [87]. Only a moderate, non-significant increase was observed after HUVEC stimulation with the plasma from HBDs. In addition, endothelial EVs released after HUVEC stimulation with APS plasma, significantly reduced the normalized clotting time ratio. Wu et al. showed data where stimulation of HUVEC with anti-β2GPI caused the formation of an endothelial cell inflammasome and the release of EVs that were enriched in mature IL-1β, with a distinct mIR profile and caused endothelial activation [101]. However, activation of HUVEC does not appear to involve IL-1β receptor, but most likely follows the TLR/myd88-IRAK4 signaling pathway. Pericleous et al. [102] investigated the effect of purified polyclonal IgG from patients with APS (APS-IgG) and healthy controls (HC-IgG) on HUVEC [102]. HUVEC exposed to APS-IgG, produced significantly more endothelial EVs than those exposed to HC-IgG and a larger proportion of these EVs carried surface E-selectin. Levels of ICAM-1+, endoglin+ and VE-cadherin+ EVs did not differ from the ones stimulated with HC-IgG. VCAM-1+ and TF+ endothelial EVs could not be detected. Later Betapudi et al., also observed a 2-fold increase in levels of endothelial EVs released from HUVEC stimulated with anti-β2GPI [100]. EVs in obstetric APS patients



### *Extracellular Vesicles: Intercellular Communication Mediators in Antiphospholipid Syndrome DOI: http://dx.doi.org/10.5772/intechopen.97412*

were studied by Tong et al. [103], whereby exposure of first trimester human placental explants to monoclonal anti-β2GPI and IgG fractions from five anti-β2GPI positive APS patients did not affect the number or size of EVs. However, an increase in levels of mitochondrial DNA was observed in these vesicles that activated endothelial cells through a TLR-9-mediated pathway. This is supporting the idea that EV-associated mitochondrial DNA could be pathological in pregnant women with aPL.
