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

Abstract

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% 15R, respectively, determined using radiolabeled substrate or LC-MS analysis). Although the Km 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 Vmax. Aspirin increased 15R-PGD2 but not 15R-PGE2 in isolated humanleukocytes activated with LPS to induce COX-2. 15R-PGD2 inhibitedhumanplatelet aggregation inducedby the thromboxane receptor agonist U46,619, and this effect was abrogated by an antagonist of the DP1 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 15R-PG endoperoxide. 15R-PGs are novel products of aspirin therapy via acetylation of COX-2 and may contribute to its antiplatelet and other pharmacologic effects.