Yun Wang1, Ya-Ping Wu2, Ji-Ju Han1, Mao-Qing Zhang3, Chen-Xi Yang4, Peng Jiao1, Hua Tian1, Chao Zhu5, Shu-Cun Qin1, Xue-Jun Sun6, Han-Ting Zhang7, Xiao-Min Zhao8. 1. Key Laboratory of Atherosclerosis in Universities of Shandong, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271000, PR China. 2. Province Key Laboratory of Oral and Maxillofacial, Head and Neck Medical Biology Laboratory, Liaocheng People's Hospital, Shandong First Medical University & Shandong Academy of Medical Sciences, Liaocheng 252000, PR China. 3. Division of Health Education, Taian Maternity and Child Health Care Hospital, Taian 271000, PR China. 4. Division of Quality Management, Taian Red Cross Central Blood Station, Taian 271000, PR China. 5. Institute of Pharmacology, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271016, PR China. 6. Department of Navy Aeromedicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, PR China. 7. Department of Behavioral Medicine & Psychiatry and Physiology, Pharmacology & Neuroscience, Rockefeller Neurosciences Institute, West Virginia University Health Sciences Center, Morgantown, USA. 8. Institute of Pharmacology, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271016, PR China. Electronic address: xmzhao@sdfmu.edu.cn.
Abstract
AIMS: Hydrogen (H2) has antioxidant effects. The pharmacologic function of H2 in platelets is not yet clear. Therefore, in this study we sought to investigate the inhibitory effects of H2 on in vitro platelet activation and in vivo prevention of thrombus formation. MAIN METHODS: After platelets were incubated with H2-rich saline (HRS), platelet adhesion in whole human blood was assessed in fibrinogen-coated perfusion chambers, while rat platelet aggregation induced by ADP, collagen and H2O2 was detected through light transmission aggregometry. The level of P-selectin, thromboxane B2, nitric oxide (NO), malondialdehyde, reactive oxygen species (ROS), cGMP, extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), and fibrinogen binding to platelets were evaluated in vitro. Besides, the in vivo effects were examined in arterio-venous shunt thrombosis, FeCl3-induced artery thrombus formation, and tail bleeding time in mice and rats. KEY FINDINGS: HRS prolonged tail bleeding time in mice and rats, decreased thrombus weight and prolonged the time to occlusion in rat and mouse thrombosis models in vivo and inhibited platelet adhesion as well as aggregation in vitro. Additionally, HRS decreased P-selectin expression, release of thromboxane B2, ROS, and fibrinogen binding, but enhanced NO levels in H2O2-exposed platelets. HRS also decreased malondialdehyde levels in plasma of the rat arterial thrombosis or H2O2-exposed platelet model. Moreover, HRS increased cGMP level, decreased p-ERK1/2 (diminished with KT5823) in the platelets stimulated by H2O2. SIGNIFICANCE: These results suggest that H2 has antithrombotic effects, which may be due to its antioxidant property and subsequent inhibition of platelet activation via NO/cGMP/PKG/ERK pathway.
AIMS: Hydrogen (H2) has antioxidant effects. The pharmacologic function of H2 in platelets is not yet clear. Therefore, in this study we sought to investigate the inhibitory effects of H2 on in vitro platelet activation and in vivo prevention of thrombus formation. MAIN METHODS: After platelets were incubated with H2-rich saline (HRS), platelet adhesion in whole human blood was assessed in fibrinogen-coated perfusion chambers, while ratplatelet aggregation induced by ADP, collagen and H2O2 was detected through light transmission aggregometry. The level of P-selectin, thromboxane B2, nitric oxide (NO), malondialdehyde, reactive oxygen species (ROS), cGMP, extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), and fibrinogen binding to platelets were evaluated in vitro. Besides, the in vivo effects were examined in arterio-venous shunt thrombosis, FeCl3-induced artery thrombus formation, and tail bleeding time in mice and rats. KEY FINDINGS: HRS prolonged tail bleeding time in mice and rats, decreased thrombus weight and prolonged the time to occlusion in rat and mousethrombosis models in vivo and inhibited platelet adhesion as well as aggregation in vitro. Additionally, HRS decreased P-selectin expression, release of thromboxane B2, ROS, and fibrinogen binding, but enhanced NO levels in H2O2-exposed platelets. HRS also decreased malondialdehyde levels in plasma of the rat arterial thrombosis or H2O2-exposed platelet model. Moreover, HRS increased cGMP level, decreased p-ERK1/2 (diminished with KT5823) in the platelets stimulated by H2O2. SIGNIFICANCE: These results suggest that H2 has antithrombotic effects, which may be due to its antioxidant property and subsequent inhibition of platelet activation via NO/cGMP/PKG/ERK pathway.