Annexin A2, TLR4, calreticulin, and nucleolin promote EC activation in response to β₂GPI and anti-β₂GPI Abs through regulation of mRNA levels. ECs were pretreated with either control RNA of random sequence but identical composition as specific siRNA or with specific siRNA against annexin A2, TLR4, calreticulin, or nucleolin. Twenty-four hours later, cells were replated in 6-well microplates, incubated for an additional 24 hours, and then either harvested and analyzed for expression of the targeted protein (not shown) or incubated with β₂GPI and anti-β₂GPI Abs or TNFα. The content of specific mRNAs was then analyzed by qPCR. Graphs depict the levels of E-selectin (A), ICAM-1 (B), VCAM-1 (C), or TF (D) mRNA. In all cases, specific siRNAs significantly reduced the levels of mRNA for each of the cell-adhesion molecules in response to β₂GPI and anti-β₂GPI Abs, but not in response to TNFα. Error bars represent the means ± SD of quadruplicate points. The results shown are from 1 representative experiment of 3.