Chemiluminescent Analysis of Reactive Oxygen Species Synthesis by Platelets from Patients with Coronary Heart Disease
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Bulletin of Experimental Biology and Medicine, Vol. 169, No. 4, August, 2020 METHODS
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Chemiluminescent Analysis of Reactive Oxygen Species Synthesis by Platelets from Patients with Coronary Heart Disease A. A. Savchenko1, M. D. Goncharov2,3, Yu. I. Grinsthein2, I. I. Gvozdev1, T. S. Mongush2,3, and A. A. Kosinova2
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 169, No. 4, pp. 525-528, April, 2020 Original article submitted October 24, 2019 A novel chemiluminescent method was developed to evaluate ROS generation by platelets. This method allows measuring activities of NADPH oxidase (NOX2) and enzymes synthesizing secondary ROS (superoxide dismutase, catalase, etc.) in resting and ADP-activated platelets (inductor of platelet aggregation and ROS generation) using a small number of cells. The method was tested in the examination of patients with coronary heart disease. It was found that platelets from patients with coronary heart disease were characterized by NOX2 activation, while cell metabolism is tuned for a long-term intensive production of superoxide anion radical. The enzymes synthesizing secondary ROS were also activated, but cell metabolism could not maintain their enhanced activity for a long time. Key Words: platelets; chemiluminescent activity; reactive oxygen species; coronary heart disease; NADPH oxidase Functional activity of platelets is an important factor of cardiovascular pathologies. In activated platelets, ROS are intensively synthesized by enzymes located both on the membrane and inside of the cell (particularly, in mitochondria) [2,12]. The key stage of ROS synthesis, generation of superoxide anion radical (primary ROS), is catalyzed by NADPH oxidase (NOX) enzyme complex primarily located on the outer membrane. Of 7 isoforms of mammal NOX differing by cell specificity, subunit composition, and some other parameters, NOX2 is the most prevalent type of Nox in human platelets [7,13]. Inhibition of NOX2 reduces platelet aggregation, while high activity of this enzyme is associated with high level platelet of aggregation Research Institute of Medical Problems of the North — Separate Division of Federal Research Center Krasnoyarsk Research Center, Siberian Division of Russian Academy of Sciences; 2V. F. VoynoYasenetsky Krasnoyarsk State Medical University, Ministry of Health of the Russian Federation; 3Federal Center of Cardiovascular Surgery, Ministry of Health of the Russian Federation, Krasnoyarsk, Russia. Address for correspondence: [email protected]. M. D. Goncharov 1
and sometimes is observed in patients with coronary heart disease (CHD) [9,14]. Dismutation of superoxide radical by superoxide dismutase (SOD) yields H2O2 that can oxidize SH groups and peroxidize unsaturated fatty acids [9]. In platelets, two SOD isoforms are synthesized: cytoplasmic SOD1 and mitochondrial SOD2. Generation of H2O2 triggers a cascade of reactions resulting in the synthesis of secondary ROS. Catalase, cyclooxygenase, and glutathione peroxidase also play an essential role in ROS generat
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