Abstract 16834: Assessment of The Pharmacological Response to Aspirin by Quantitation of Platelet Cyclooxygenase Acetylation.
Aspirin inhibits platelet activity irreversibly by covalent acetylation of cyclooxygenase-1 (COX-1). Variable non-responsiveness to aspirin has been reported based on clinical outcome measures, platelet function tests ex vivo or the inhibition of the enzymatic capacity of platelet COX−1. Precise assessment of a patient's response to aspirin, however, would require information on whether the drug has indeed reached its target, platelet COX−1. We hypothesized that the quantification of COX-1 acetylation stoichiometry might provide novel insights into sources of variability in the detected aspirin response. We developed a stable isotope dilution liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) technique that allows the quantification of COX-1 acetylation at serine−529. COX-1 containing gel sections were isolated and proteolytically digested to yield the peptides 524-IGAPFSLK-531 or 524-IGAPFS(Ace)LK−531. These were quanitated by LC-MRM/MS using heavy isotope labeled standards. The average accuracy and precision of our method was 102.7±10.2% and 11.3±4.5%, respectively. Validation was performed on the recombinant protein, COX-1 expressing cells, human platelets treated with aspirin ex vivo and platelets isolated from volunteers who were administered aspirin. Complete acetylation of the recombinant protein in vitro was observed within 30 minutes of aspirin exposure (500 μM), which correlated highly with the reduction of COX activity. Human platelets contained on average 0.2 μg COX-1 per mg total protein. Administration of 325 mg of aspirin in an effervescent solution resulted in complete acetylation of platelet COX-1 within 30 minutes in volunteers. Interestingly, maximal acetylation of COX-1 in vitro and in vivo was 55%±3% of total COX-1 consistent with the notion that only one monomer of the COX-1 homodimer is acetylated by aspirin. Clinical studies of aspirin responsiveness using this method are ongoing. Exact quantitation of the pharmacological action of aspirin in vivo — acetylation of its target enzyme — will permit elucidation of factors that contribute to aspirin non-responsiveness in patients including non-compliance with drug intake and variation in aspirin pharmacokinetics.
- © 2010 by American Heart Association, Inc.