Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods

Thromb Res. 2014 May;133(5):811-6. doi: 10.1016/j.thromres.2013.10.008. Epub 2013 Oct 16.


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.

Keywords: aspirin; aspirin resistance; platelet aggregation; platelet secretion; reference method; thromboxane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aspirin / administration & dosage
  • Aspirin / pharmacology*
  • Blood Platelets / drug effects
  • Blood Platelets / enzymology
  • Cyclooxygenase 1 / blood
  • Dose-Response Relationship, Drug
  • Female
  • Humans
  • Laboratories, Hospital
  • Male
  • Middle Aged
  • Platelet Aggregation Inhibitors / administration & dosage
  • Platelet Aggregation Inhibitors / pharmacology*
  • Reference Values
  • Young Adult


  • Platelet Aggregation Inhibitors
  • Cyclooxygenase 1
  • PTGS1 protein, human
  • Aspirin