Anti-β₂GPI peptide Abs activate endothelial cells. (A) IRAK phosphorylation was evaluated by Western blot analysis. Endothelial cells were incubated for 45 minutes with human purified endotoxin-free anti-β₂GPI peptide and, as controls, with anti-BSA Abs (100 μg/mL); E coli LPS (100 ng/mL); with purified IgG from an APS patient positive for the β₂GPI peptide (200 μg/mL); or with APS IgG (200 μg/mL) preabsorbed with β₂GPI peptide. (B) NF-κB activation was analyzed by treating endothelial cells for 45 minutes with human purified endotoxin-free anti-β₂GPI peptide and, as controls, with anti-BSA Abs (100 μg/mL); E coli LPS (100 ng/mL); APS IgG (200 μg/mL); or with APS IgG (200 μg/mL) preabsorbed with β₂GPI peptide. Values are the means ± SDs of 3 different experiments. Statistical analysis was performed by Student paired t test (P < .001). * indicates versus control. (C) VCAM expression on the cell surface was evaluated by flow cytometric analysis. Representative flow cytometry histogram plots show the fluorescence intensity of FITC-conjugated anti-VCAM mAbs after treatment of HUVECs with E coli LPS or endotoxin-free human purified anti-β₂GPI peptide Abs. Isotype control staining is represented by the dotted line; anti-VCAM-labeled cells are represented by the gray (untreated cells) and the black (treated cells) lines. Statistical differences between the peaks of cells were evaluated by the Kolmogorov-Smirnov test. D/s_(n) ratio > 15 for treated versus untreated cells.