Nuclear factor κB mediates a procoagulant response in monocytes during extracorporeal circulation

Complement is activated in blood during simulated extracorporeal circulation. Whole blood from healthy donors was circulated through a membrane oxygenator, as described in the “Material and Methods” section. At time points indicated on the x-axis, blood was withdrawn, and the serum fraction of each sample was assayed for C3a (A), C5a (B), or soluble C5b9 (C) by means of enzyme-linked immunosorbent assay. Each sample was measured in duplicate. Values on the y-axis represent the means ± SEM for 4 separate experiments.

Extracorporeal circulation induces procoagulant activity (PCA) in blood. A, After circulation of blood for various periods of time (x-axis), procoagulant activity, expressed as the length of time required for blood to clot, was determined in duplicate for each time point. Clotting times were determined in the presence (closed circles) or absence (open circles) of a saturating amount of anti-TF antibody. B, Drawn blood from healthy donors was treated with either MG132 (final concentration, 3 μmol/L) or an equal volume of vehicle alone. MG132-treated blood (filled bars) or vehicle control blood (shaded bars) was then circulated through the simulated extracorporeal circuit; procoagulant activity was determined at the various time points on the x-axis. Values (y-axis) represent duplicate determinations and the means ± SEM of 6 separate experiments.

MG132 inhibits TF expression on monocytes during extracorporeal circulation. Mononuclear phagocytes were isolated from circulated blood at various time points indicated on the y-axis. TF expression on surface membranes was determined by means of enzyme-linked immunosorbent assay, as described in the “Material and Methods” section. TF expression on monocytes from blood in the extracorporeal circuit was determined for blood treated with MG132 (3 μmol/L) before addition to the closed circuit (filled bars) or after treatment with vehicle alone (shaded bars). Each datum point was determined in duplicate and represents the mean ± SEM of 6 separate experiments.

NF-κB activation in monocytes during extracorporeal circulation is blocked by MG132. Mononuclear phagocytes were isolated from blood in the closed circuit at the time points indicated at the top of the lanes. Nuclear proteins were purified from these cells and probed by means of electrophoretic mobility shift assay for NF-κB translocation to the nucleus. Circulation of blood was carried out after treatment with MG132 (3 μmol/L) or MG132 vehicle. This figure is representative of 4 similar electrophoretic mobility shift assays.