New direct and indirect methods for the detection of cyclooxygenase 1 acetylation by aspirin; the lack of aspirin resistance among healthy individuals

Background: Aspirin is widely used in the prevention of acute atherothrombotic complications. It acetylates Ser529 residue in cyclooxygenase-1 (COX-1) and prevents thromboxane A2 (TXA2) formation from arachidonic acid (AA) in platelets. Laboratory methods used for the detection of aspirin effect provide inconsistent results.

Methods: Two new methods were developed for the direct and indirect detection of COX-1 acetylation by aspirin in 108 healthy volunteers treated daily with 100mg enteric-coated aspirin for 7days. Monoclonal antibodies were raised against acetylated and non-acetylated nonapeptides corresponding to the amino acid sequence of human COX-1 525-533 residues. Using Western blotting technique the antibodies clearly distinguished between acetylated and non-acetylated COX-1 in platelet lysate. The second method measures AA-induced TXB2 production of platelets in diluted platelet rich plasma.

Results: No acetylated COX-1 was detected in platelets before aspirin treatment. At the same time antibodies raised against non-acetylated peptide gave intense reaction with COX-1 on the Western blot. In contrast, after 7days of aspirin treatment, with a single exception, only acetylated COX-1 could be detected in the platelet lysate. The non-responding volunteer showed full response to aspirin after controlled drug intake. In parallel experiments aspirin treatment for 7days practically completely inhibited AA-induced TXB2 production by platelets.

Conclusions: Chemical ("true") aspirin resistance, if it exists, must be a rarity among healthy individuals. The new methods could be used for detecting the acetylation of COX-1 by aspirin in patients on preventive aspirin therapy and for evaluating methods routinely used for such purpose.