title: Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods.
abstract: BACKGROUND
  Aspirin, a commonly used antiplatelet agent, blocks platelet thromboxane A₂ (TXA₂) formation from arachidonic acid (AA) by acetylating platelet cyclooxygenase-1 (COX-1). Laboratory methods currently used to detect this antiplatelet effect of aspirin provide variable results. We have reported three methods that assess platelet COX-1 acetylation (inactivation) by aspirin and its direct consequences. The first and second assays use monoclonal anti-human-COX-1 antibodies that only detect acetylated (inactivated) COX-1 and active (non-acetylated) COX-1, respectively. The third method measures platelet production of TXB₂ (the stable metabolite of TXA₂) in vitro in response to AA. We compared the results of these three reference methods with other routinely used methods for assessing the functional consequences aspirin treatment.
  
  
  METHODS
  108 healthy volunteers were treated with low-dose aspirin for 7 days. On day 7 following aspirin treatment COX-1 in the platelets was fully acetylated whereas only non-acetylated COX-1 was present in the day 0 platelets. Further, TXB2 production by day 7 platelets was completely blocked. The following tests were performed on the samples obtained from study participants before and after seven days of aspirin treatment: PFA-100 closure time with collagen/epinephrine cartridge, VerifyNow (VN) Aspirin Assay, platelet aggregation and ATP secretion using AA, ADP, epinephrine and collagen as agonists.
  
  
  RESULTS
  Comparing the pre-treatment and day 7 values, methods that use AA as platelet agonist (AA-induced platelet aggregation/secretion and VN Aspirin Assay) showed high discriminative power. In contrast, results of the other tests showed considerable overlap between day 7 and day 0 values.
  
  
  CONCLUSIONS
  Only assays that clearly distinguish between acetylated and non-acetylated platelet COX-1 are useful for establishing the antiplatelet effect of aspirin. The other tests are not suitable for this purpose.
authors.affiliation: Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: Emese G
authors.initials: EG
authors.lastname: Kovács
authors.affiliation: Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Katona
authors.affiliation: Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Bereczky
authors.affiliation: Institute of Cardiology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: Nóra
authors.initials: N
authors.lastname: Homoródi
authors.affiliation: Institute of Cardiology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Balogh
authors.affiliation: Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Tóth
authors.affiliation: Diagnosticum Co., Research Laboratory, Budapest, Hungary.
authors.forename: Hajna
authors.initials: H
authors.lastname: Péterfy
authors.affiliation: State Health Center, Department of Cardiology, Budapest, Hungary.
authors.forename: Róbert G
authors.initials: RG
authors.lastname: Kiss
authors.affiliation: Institute of Cardiology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Édes
authors.affiliation: Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; Thrombosis, Haemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary. Electronic address: muszbek@med.unideb.hu.
authors.forename: László
authors.initials: L
authors.lastname: Muszbek
mesh_terms: D000328:Adult; D001241:Aspirin / Q000008:administration & dosage / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology; D051545:Cyclooxygenase 1 / Q000097:blood; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007755:Laboratories, Hospital; D008297:Male; D008875:Middle Aged; D010975:Platelet Aggregation Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D012016:Reference Values; D055815:Young Adult
chemical_list: D010975:Platelet Aggregation Inhibitors; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D001241:Aspirin
keywords: aspirin; aspirin resistance; platelet aggregation; platelet secretion; reference method; thromboxane