title: Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.
abstract: OBJECTIVE
  To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO).
  
  
  METHODS
  Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100).
  
  
  RESULTS
  ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786) → C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)(1177), an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177) phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser(1177). On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177). During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets.
  
  
  CONCLUSIONS
  Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177).
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Modrego
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain ; Hemodynamic Unit, Cardiology Department, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Azcona
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Naiara
authors.initials: N
authors.lastname: Martín-Palacios
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: José J
authors.initials: JJ
authors.lastname: Zamorano-León
authors.affiliation: Health Science Institute, Talavera de la Reina, Toledo, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Segura
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Rodríguez
authors.affiliation: Hemodynamic Unit, Cardiology Department, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Reddy
authors.initials: R
authors.lastname: Guerra
authors.affiliation: Pharmacology Department, School of Medicine, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Tamargo
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain ; Hemodynamic Unit, Cardiology Department, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Macaya
authors.affiliation: Cardiovascular Research Unit, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: López-Farré
mesh_terms: D000368:Aged; D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood; D001241:Aspirin / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D015153:Blotting, Western; D003094:Collagen / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008027:Light; D008297:Male; D009154:Mutation / Q000235:genetics; D009566:Nitrates / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*
chemical_list: D009566:Nitrates; D009573:Nitrites; D010768:Phosphoserine; C018524:N,N-dimethylarginine; D003094:Collagen; D001120:Arginine; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D001241:Aspirin