Involvement of superoxide generated by NADPH oxidase in the shedding of procoagulant vesicles from human monocytic cells exposed to bupivacaine

It is known that a variety of sized procoagulant vesicles that express tissue factor are released from several types of cells including monocytes by mechanisms related to the induction of apoptosis, while it has not yet been evaluated whether superoxide is involved in the production of such vesicles. Here, we report that a local anesthetic bupivacaine induces apoptosis in human monocytic cells THP-1 within a short observation period, where the shedding of procoagulant vesicles is associated. The property as procoagulant vesicles was evaluated using flow cytometry by the binding of FITC-conjugated fibrinogen to vesicles in the presence of fresh frozen plasma and the suppression of this binding by heparin. Bupivacaine (1 mg/ml) increased the apoptotic cells and procoagulant vesicles. LY294002 (100 µM), that inhibits the recruiting of intracellular component of NADPH oxidase to construct the activated form of this enzyme complex, or superoxide dismutase (1500 unit/ml) suppressed bupivacaine-provoked induction of apoptosis and the increase of procoagulant vesicles. We suggest that this simple experimental system is useful to explore the molecular mechanisms of action of superoxide in the shedding of procoagulant vesicles from human monocytic cells.