Vol.:(0123456789) 1 3 J Thromb Thrombolysis DOI 10.1007/s11239-017-1531-z Involvement of superoxide generated by NADPH oxidase in the shedding of procoagulant vesicles from human monocytic cells exposed to bupivacaine Toshiharu Azma 1,2  · Saori Ogawa 1,3  · Akira Nishioka 2  · Hiroyuki Kinoshita 4  · Shinji Kawahito 5  · Hiroshi Nagasaka 1  · Nobuyuki Matsumoto 1   © Springer Science+Business Media, LLC 2017 that this simple experimental system is useful to explore the molecular mechanisms of action of superoxide in the shed- ding of procoagulant vesicles from human monocytic cells. Keywords Monocytes · Superoxide · Tissue factor · Apoptotic vesicles · Procoagulant vesicles · Bupivacaine Introduction Superoxide and its partly reduced oxygen metabolites, hydro- gen peroxide (H 2 O 2 ) or hydroxyl radical yielded by chemical reactions under some conditions (e.g., Haber–Weiss cycle), react non-specifcally with a variety of biomolecules (e.g., proteins, lipids, or DNA), implicating thus in cell death and cellular dysfunction in several pathophysiological conditions [1]. Superoxide dismutase (SOD) is an enzyme that cata- lyzes the conversion of two superoxide anion molecules to oxygen and H 2 O 2 [2]. Experimental evidence from animals that lack the gene expression for certain isoforms of SOD demonstrated that SOD1 or SOD2, located in cytosol or in mitochondria, respectively [3], plays pivotal roles in diseases such as macular degeneration [4], osteoporosis [5], Alzhei- mer disease [6], and cardiomyopathy [7]. By contrast, SOD3, expressed in the extracellular space, appeared not to be involved in severe pathophysiological disorders from animal studies using SOD3 knockout mice [8]. Several studies including ours using electron spin reso- nance spectrometry demonstrated that SOD increases the generation of highly reactive oxygen species hydroxyl radi- cal even in the presence of transition metals at a trace level in cell-free superoxide generating conditions [9]. Although it has been implicated that reactive oxygen species (ROS) cause apoptotic cell death, a number of studies suggested the involvement of H 2 O 2 or hydroxyl radical, rather than Abstract 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 evalu- ated whether superoxide is involved in the production of such vesicles. Here, we report that a local anesthetic bupiv- acaine induces apoptosis in human monocytic cells THP-1 within a short observation period, where the shedding of procoagulant vesicles is associated. The property as pro- coagulant vesicles was evaluated using fow cytometry by the binding of FITC-conjugated fbrinogen 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 com- ponent of NADPH oxidase to construct the activated form of this enzyme complex, or superoxide dismutase (1500 unit/ ml) suppressed bupivacaine-provoked induction of apopto- sis and the increase of procoagulant vesicles. We suggest * Toshiharu Azma azmacci@nifty.com 1 Department of Anesthesiology, Saitama Medical University Hospital, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan 2 Department of Anesthesiology & Pain Medicine, Kohnodai Hospital, National Center for Global Health and Medicine, Kohnodai 1-7-1, Ichikawa, Chiba 272-8516, Japan 3 Department of Dental Anesthesiology, Matsumoto Dental University, Hirooka Goubara 1780, Shiojiri, Nagano 399-0781, Japan 4 Department of Anesthesiology, Aichi Medical University, Yazako Karimata 1-1, Nagakute, Aichi 480-1195, Japan 5 Department of Anesthesiology, Tokushima University Hospital, Kuramoto 3-18-15, Tokushima 770-8503, Japan