FACS analysis showing ROS-induced increases in VWF on HUVECs, and in P-selectin and GPIIbIIIa on mouse platelets. (A-E) Adhesion molecules, activation markers, and cell-surface VWF were analyzed by flow cytometry in cultured HUVECs. Some cells were treated with hematoporphyrin (1mM, HP, as a source of O₂⁻) for 20 minutes and then stimulated with laser irradiation to induce ROS production. Other cells were treated with 1mM H₂O₂ and/or 100μM NAC for 20 minutes before the incubation with antibodies. Some HUVECs were treated with si-RNA targeting TNF-R1 (si-TNF-R1) or with control scrambled si-RNA (si-CTRL) 48 hours before the experiments. Black lines denote vehicle-treated control cells (CTRL); red lines, H₂O₂ or hematoporphyrin treatment; blue lines, ROS source plus NAC; green lines, si-CTRL and ROS; and purple lines, si-TNF-R1 and ROS. Note that O₂⁻ markedly increased cell surface expression of VWF on HUVECs, which was completely blocked by NAC, and diminished in si-TNF-R1 cells. Expression of P-selectin in ECs was also increased by ROS, without affecting E-selectin expression. ICAM-1 expression was moderately increased by O₂⁻. (F-G) Washed platelets from 12-week-old male wild-type C57BL/6J, TNF-α KO and TNF-R1 KO mice were collected and analyzed after ROS stimulation as in the HUVEC experiments. Black lines denote vehicle-treated control cells from wild-type mice (CTRL); red lines, H₂O₂ or hematoporphyrin treatment; blue lines, ROS source plus NAC; green lines, ROS plus TNF-α KO platelets; and purple lines, ROS plus TNF-R1 KO platelets. Note that expression of P-selectin, and GPIIbIIIa was increased by ROS treatment, and that these effects were inhibited by NAC.