Antioxidative treatment inhibits the release of thrombogenic tissue factor from irradiation- and cytokine-induced endothelial cells

OBJECTIVES: The aim of this study was to investigate the effect of the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) on the ionizing radiation (IR)- and tumor necrosis factor-alpha (TNF-alpha) induced tissue factor (TF) expression and its release from human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were irradiated with a single dose of either 5 Gy or 10 Gy and stimulated with TNF-alpha (10 ng/mL) in the presence or absence of PDTC and NAC, respectively. Quantitative real-time PCR, ELISA, and TF activity measurements were performed, including TF activity in the supernatant. Apoptosis was detected by flow cytometric active caspase-3 measurement and formation of reactive oxygen species (ROS) by chemiluminescence. RESULTS: We demonstrated a thus far uninvestigated persistent induction of TF expression in HUVECs after treatment with IR and TNF-alpha. Combined stimulation with IR and TNF-alpha led to an immense shedding of microparticle-associated TF which was positively correlated with apoptosis and ROS formation. Antioxidative pre-treatment reduced not only apoptosis and ROS formation, but also the release of thrombogenic microparticles. CONCLUSIONS: Antioxidative treatment inhibited apoptosis and shedding of microparticles, thereby reducing thrombogenicity. Thus, antioxidants may help to prevent late thrombosis after antiproliferative treatment when used in combination with anticoagulants.