Abstract
Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.
Highlights
Aspirin circulates transiently in blood, but the identity of the enzyme(s) that hydrolyzes its acetyl residue remains unknown
Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity
Not all individuals or populations are protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3
Summary
Aspirin circulates transiently in blood, but the identity of the enzyme(s) that hydrolyzes its acetyl residue remains unknown. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A2 with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. The physiologic basis for this aspirin resistance is undefined, and even whether the phenomenon is real or documentable is debated [17] Despite these clinical and laboratory issues, variation in the time aspirin circulates will affect its ability to inactivate cyclooxygenase 1 and inhibit platelet function. Plasma contains a uniquely selective deacetylase, plasma platelet-activating factor (PAF) acetylhydrolase (PLA2G7), which acts on the phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) [31] This phospholipase A2 circulates in association with low and high density lipoproteins. We purified erythrocyte aspirin hydrolase and found that the major hydrolytic activity in blood is type I PAF acetylhydrolase. We found that erythrocytes alter the suppression of aspirin on platelet function, and that the effectiveness of intracellular hydrolysis of aspirin varied among individuals
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