Residual cyclooxygenase activity of aspirin‐acetylated COX‐2 forms 15 R ‐prostaglandins that inhibit platelet aggregation

Aspirin (acetylsalicylic acid) inhibits prostaglandin (PG) synthesis by transfer of its acetyl group to a serine residue in the cyclooxygenase (COX) active site. Acetylation of Ser530 inhibits catalysis by preventing access of arachidonic acid substrate in the COX‐1 isoenzyme. Acetylated COX‐2, in contrast, gains a new catalytic activity and forms 15R hydroxy‐eicosatetraenoic acid (15R‐HETE) as alternate product. Here we show that acetylated COX‐2 also retains COX activity, forming predominantly 15R‐configuration PGs (70 or 62% 15 R , respectively, determined using radiolabeled substrate or LC‐MS analysis). Although the K m of arachidonic acid for acetylated COX‐2 was ~3‐fold lower than for uninhibited COX‐2, the catalytic efficiency for PG formation by the acetylated enzyme was reduced 10‐fold due to a concomitant decrease in V max . Aspirin increased 15 R ‐PGD 2 but not 15 R ‐PGE 2 in isolated human leukocytes activated with LPS to induce COX‐2.15 R ‐PGD 2 inhibited human platelet aggregation induced by the thromboxane receptor agonist U46,619, and this effect was abrogated by an antagonist of the DPI prostanoid receptor. We conclude that acetylation of Ser530 in COX‐2 not only triggers formation of 15R‐HETE but also allows oxygenation and cyclization of arachidonic acid to a 15 R ‐PG endoperoxide. 15 R ‐PGs are novel products of aspirin therapy via acetylation of COX‐2 and may contribute to its antiplatelet and other pharmacologic effects.—Giménez‐Bastida, J. A., Boeglin, W. E., Boutaud, O., Malkowski, M. G., Schneider, C. Residual cyclooxygenase activity of aspirin‐acetylated COX‐2 forms 15R‐prostaglandins that inhibit platelet aggregation. FASEB J. 33, 1033–1041 (2019). www.fasebj.org