Annexin A2, TLR4, calreticulin, and nucleolin are necessary for activation of ECs by β₂GPI and anti-β₂GPI Abs. ECs were either not pretreated or were pretreated with control RNA of random sequence but identical composition as specific siRNAs or with siRNAs specific for annexin A2, TLR4, calreticulin, or nucleolin. Twenty-four hours later, cells were replated in 6- or 96-well plates, incubated for an additional 24 hours, and then harvested and analyzed for protein expression (A) or assessed for cell-surface E-selectin expression in response to β₂GPI and either control IgG or anti-β₂GPI Abs (B). Error bars represent the means ± SD of quadruplicate points. Decreases in E-selectin expression in cells pretreated with control versus specific siRNA before incubation with β₂GPI and anti-β₂GPI Abs were highly significant (P < .0001 by ANOVA). The results shown are from 1 representative experiment of 3.