Induction of endothelial cell tissue factor expression. (A) Effect of human anti-β2GPI, human anti–A2 IgG, and rabbit anti–A2 IgG. As described in “Patients, materials, and methods,” human umbilical vein endothelial cells were incubated with either medium alone (Medium), tumor necrosis factor α (TNF-α, 10 ng/mL), or patient IgG containing anti-β2GPI (Hu > β2GPI, 200 μg/mL). Cells were also incubated with increasing concentrations of patient anti–A2 IgG that was antiphospholipid antibody- and endotoxin-free (Hu > A2), patient IgG lacking anti-A2 antiphospholipid antibodies (Hu-NI), rabbit anti–human A2 IgG (RAH-A2), or rabbit preimmune IgG (RNI; 0.2, 20, and 200 μg/mL; white, gray, and black bars, respectively). Bars represent normalized mean values (± SEM, n = 2-4). (B) Immunodepletion of patient IgG. As described in “Patients, materials, and methods,” whole IgG from a representative patient, who was positive for anti-A2 antibodies but lacked anti-β2GPI and ACL antibodies, was applied to an A2-sepharose immunoaffinity column, eluted with glycine buffer (pH 2.8), and immediately neutralized with Tris base (pH 8.0). Both affinity-purified IgG (Hu > A2 AP) and immunodepleted IgG (Hu > A2 ID) were extensively dialyzed against PBS (pH 7.2) and tested by immunoblot analysis against rA2 (250 ng/lane). (C) Effect of A2 immunodepletion on tissue factor induction. HUVECs were incubated alone (Medium), with whole anti-A2–containing IgG (Hu > A2) at 200 μg/mL (▪) or 20 μg/mL (▦), with A2-immunodepleted IgG from the same patient (Hu > A2 ID) at 200 μg/mL, or with human anti-β2GPI at 200 μg/mL (▪) or 20 μg/mL (▦). Shown are mean A₆₅₀ nm values for (± SEM, n = 3).