title: Proteomic signature of thrombin-activated platelets after in vivo nitric oxide-donor treatment: coordinated inhibition of signaling (phosphatidylinositol 3-kinase-γ, 14-3-3ζ, and growth factor receptor-bound protein 2) and cytoskeleton protein translocation.
abstract: OBJECTIVE
  Growing insight into the antiplatelet properties of new nitric oxide (NO) donors has expanded their potential use in cardiovascular diseases. As such, we reported that oral administration of a new exogenous NO donor (LA419) induced significant inhibition of platelet deposition on damaged vascular wall without provoking hypotension in an in vivo experimental model. Thrombin is one of the major triggers of platelet deposition and thrombosis on injured vessels; however, the effects of NO on thrombin-induced platelet activation are not fully known. Here, our aim was to investigate the inhibitory effects of exogenous NO administration on the major changes in platelet proteins induced by thrombin.
  
  
  RESULTS
  Platelets were obtained from a group of swine orally treated with LA419 (0.9 mg kg(-1)) or placebo for 8 days. Washed platelets were incubated with thrombin (0.4 NIH U/mL). Platelet proteins were then sequentially extracted based on differential solubility and studied by two-dimensional electrophoresis, mass spectrometry (matrix-assisted laser desorption ionization/time of flight), Western blot, and confocal immunofluorescence. NO treatment abrogated thrombin effects on 24 proteins involved in actin assembly, signaling, and metabolic activity. NO treatment prevented thrombin-induced translocation of gelsolin, filamin, 14-3-3ζ, phosphatidylinositol 3-kinase-γ isoform, and growth factor receptor-bound protein 2 (Grb2).
  
  
  CONCLUSIONS
  Our results show that exogenous NO donor treatment renders platelets less sensitive to thrombin activation and inhibits thrombosis by interfering with the platelet shape change machinery.
authors.affiliation: Cardiovascular Research Center, CSIC-ICCC, Biomedical Research Institute Sant-Pau, Barcelona, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Peña
authors.forename: Teresa
authors.initials: T
authors.lastname: Padro
authors.forename: Blanca
authors.initials: B
authors.lastname: Molins
authors.forename: Gemma
authors.initials: G
authors.lastname: Vilahur
authors.forename: Lina
authors.initials: L
authors.lastname: Badimon
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology; D003285:Contractile Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D064448:Filamins; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D018260:Gelsolin / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007548:Isosorbide Dinitrate / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D008840:Microfilament Proteins / Q000378:metabolism; D023421:Models, Animal; D020030:Nitric Oxide Donors / Q000008:administration & dosage / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D040901:Proteomics; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D064448:Filamins; D051380:GRB2 Adaptor Protein; D018260:Gelsolin; D007527:Isoenzymes; D007548:Isosorbide Dinitrate; D008840:Microfilament Proteins; D020030:Nitric Oxide Donors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013917:Thrombin; C503157:LA 419