title: Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.
abstract: BACKGROUND
  CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation.
  
  
  METHODS
  The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study.
  
  
  RESULTS
  CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation.
  
  
  CONCLUSIONS
  This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and MAPKs), ultimately inhibiting platelet activation. This novel role of CME-1 indicates that CME-1 exhibits high potential for application in treating and preventing CVDs.
authors.affiliation: Department of Pharmacology and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Wan-Jung
authors.initials: WJ
authors.lastname: Lu
authors.affiliation: Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Nen-Chung
authors.initials: NC
authors.lastname: Chang
authors.affiliation: Department of Pharmacology and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Thanasekaran
authors.initials: T
authors.lastname: Jayakumar
authors.affiliation: Department of Pharmacology and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Jiun-Cheng
authors.initials: JC
authors.lastname: Liao
authors.affiliation: Department of Pharmacology and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Mei-Jiun
authors.initials: MJ
authors.lastname: Lin
authors.affiliation: Core Facility Center, Office of Research and Development, Taipei Medical University, Taipei, Taiwan.
authors.forename: Shwu-Huey
authors.initials: SH
authors.lastname: Wang
authors.affiliation: Department of Pharmacology and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Duen-Suey
authors.initials: DS
authors.lastname: Chou
authors.affiliation: Department of Microbiology, Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli, Tamil Nadu, India.
authors.forename: Philip Aloysius
authors.initials: PA
authors.lastname: Thomas
authors.affiliation: Department of Pharmacology and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan. Electronic address: sheujr@tmu.edu.tw.
authors.forename: Joen-Rong
authors.initials: JR
authors.lastname: Sheu
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D032661:Cordyceps / Q000737:chemistry*; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D062610:Fungal Polysaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D025282:Mycelium / Q000737:chemistry; D015539:Platelet Activation / Q000187:drug effects* / Q000502:physiology; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013927:Thrombosis / Q000188:drug therapy* / Q000503:physiopathology; D016896:Treatment Outcome
chemical_list: C000599929:CME-1 polysaccharide, Cordyceps sinensis; D062610:Fungal Polysaccharides; D010975:Platelet Aggregation Inhibitors; D011134:Polysaccharides; D000242:Cyclic AMP; D000262:Adenylyl Cyclases
keywords: CME-1; Cordyceps sinensis; Cyclic AMP; Platelet activation; Polysaccharide