Acetylation of prostaglandin H2 synthases by aspirin is inhibited by redox cycling of the peroxidase

Abstract

Aspirin exerts its unique pharmacological effects by irreversibly acetylating a serine residue in the cyclooxygenase site of prostaglandin-H 2 -synthases (PGHSs). Despite the irreversibility of the inhibition, the potency of aspirin varies remarkably between cell types, suggesting that molecular determinants could contribute to cellular selectivity. Using purified enzymes, we found no evidence that aspirin is selective for either of the two PGHS isoforms, and we showed that hydroperoxide substrates of the PGHS peroxidase inhibited the rate of acetylation of PGHS-1 by 68%. Using PGHS-1 reconstituted with cobalt protoporphyrin, a heme devoid of peroxidase activity, we demonstrated that reversal by hydroperoxides of the aspirin-mediated acetylation depends upon the catalytic activity of the PGHS peroxidase. We demonstrated that inhibition of PGHS-2 by aspirin in cells in culture is reversed by 12-hydroperoxyeicosatetraenoic acid dose-dependently (ED 50 = 0.58 ± 0.15 μM) and that in cells with high levels of hydroperoxy-fatty acids (RAW264.7) the efficacy of aspirin is markedly decreased as compared to cells with low levels of hydroperoxides (A549; IC 50 s = 256 ± 22 μM and 11.0 ± 0.9 μM, respectively). Together, these findings indicate that acetylation of the PGHSs by aspirin is regulated by the catalytic activity of the peroxidase which yields a higher oxidative state of the enzyme.

Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2

“…from (endogenous) arachidonic substrate already before addition of the drugs. Furthermore, aspirin showed only modest effi cacy in reducing eicosanoid formation, consistent with previous fi ndings that a high cellular redox state in activated RAW264.7 cells impedes aspirin's ability to covalently modify COX enzymes ( 20 ).…”

“…Treatment of CT26 cells with aspirin prior to incubation with 5 S -HETE enhanced the biosynthesis of 5,15-diHETE, consistent with the fi ndings using recombinant COX-2 enzyme. Aspirin, even at a high concentration, did not show this effect in RAW264.7 cells, most likely due to lesser effi cacy for covalent modifi cation of the COX enzyme in cells with a highly oxidative tone ( 20,33 ). 5 S ,11 R -diHETE and 5 S ,15 R -diHETE have not been described as metabolites of arachidonic acid before, although there are two reports published more than 20 years ago that implicate the possibility of COX-dependent biosynthesis of diHETEs in human umbilical arteries ( 34,35 ).…”

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy

“…43 From a direct comparison of the results obtained with aspirin versus NS-398 in vitro, we can conclude that there is a fraction of enzymatically active COX-1 in platelets isolated from ET patients taking 100 mg of aspirin once daily, and this pool of unacetylated enzyme is normally sensitive to aspirin. We observed a weak positive correlation (r ϭ 0.31) between the platelet count and whole blood TXB 2 production.…”

Coxibs interfere with the action of aspirin by binding tightly to one monomer of cyclooxygenase-1

“…Native ovCOX-1 (5-10 μM) was incubated with 1-mM [1-14 C-acetyl]salicylate for different times at 37°C essentially as described by Bala et al (36). Further details are provided in SI Text.…”