APL activation of PP2A underlies eNOS antagonism in endothelial cells. (A) HAECs were exposed to aPLs from 4 different APS patients or NHIgG (100 µg/mL) for 90 minutes, and PP2A activity was measured in cell lysates; N = 6. (B) PP2A activity was determined in HAECs exposed to anti-β2GPI monoclonal antibody (3F8, 10 µg/mL), its isotype-matched control (BBG, 10 µg/mL), NHIgG, or aPLs. Additional HAECs were incubated with C2-ceramide (30 µM) for 4 hours to provide a positive control; N = 4. (C) HAECs were transfected with control RNAi or RNAi targeting the PP2A A subunit (PP2A-A), and 24 hours later treated with aPLs or NHIgG followed by vehicle (phosphate-buffered saline [PBS]) or VEGF (100 ng/mL) for 30 minutes. Cell lysates were immunoblotted for phosphorylated-eNOS S1177 (peNOS), total eNOS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Findings shown were confirmed in 3 independent experiments. (D) In HAECs transfected and treated as in panel C, VEGF (100 ng/mL) activation of NOS activity was assessed in intact cells by measuring ¹⁴C-arginine conversion to ¹⁴C-citrulline; N = 6. (E-G) HAECs were incubated with the inactive analog 1,4-dimethyl-endothall (Dm-endo, 10 µM) vs endothall (Endo, 10µM) for 4 hours (E,G), or with vehicle (Veh, methanol) vs Fostriecin (Fostri, 10 µM) for 4 hours (F-G), and with aPLs vs NHIgG, and vehicle vs VEGF. In cell lysates, PP2A activity was quantified (E-F, n = 7) and immunoblotting for peNOS, total eNOS, and GAPDH was performed (G, findings were confirmed in 3 independent experiments). (H-I) In studies paralleling those in panels E-G, VEGF stimulation of NOS activity was evaluated in intact cells; N = 6. In graphs, values are mean ± SEM, ****P < .0001, ***P < .001, and **P < .01. (J) Summary of findings in the figure.