One or more free thiols generated within TRX-1–treated β2GPI undergoes S-nitrosylation. (A) nβ2GPI (1μM) pretreated with DTT (20μM) activated TRX-1 was then incubated with EAhy926 cells for 20 minutes at 37°C. Equal amounts of supernatant were then labeled with MPB and probed for the presence of S-nitrosocysteines. S-nitrosocysteine formation within TRX-1–treated nβ2GPI is detected only after incubation with endothelial cells. (B) nβ2GPI (1μM) pretreated with TRX-R/NADPH–activated TRX-1 was incubated for 20 minutes at 37°C with unstimulated HUVECs, HUVECs pretreated with lipopolysaccharide (LPS; 100 ng/mL for 20 hours at 37°C) or with empty wells. S-nitrosocysteine formation was detected only in TRX-1–treated β2GPI and only after incubation with preactivated HUVECs. (C) rβ2GPI (1μM) pretreated with DTT-activated TRX-1 was then incubated with S-nitrosogluathione (GSNO). Free thiols were blocked with N-ethylmaleimide (NEM), and nitrosylated cysteine thiols then were exposed by degradation with ascorbic acid and labeled with MPB. Only TRX-1–treated rβ2GPI was treated with GSNO and ascorbate label with MPB. The above data are representative of 3 independent experiments for all panels.