Date of Award

2012

Degree Type

Dissertation

Department

Chemistry

First Advisor

McIntyre, Thomas

Subject Headings

Hydrolases, Aspirin, Proteins -- Purification, High performance liquid chromatography, Drugs -- Metabolism, Protein Purification, HPLC, Drug Metabolism, Enzyme, Aspirin, Platelets, Cardiovascular Disease, Personalized Medicine, Clinical Chemistry

Abstract

Aspirin prophylaxis suppresses major adverse cardiovascular events, but its turnover in blood is rapid, which limits its inhibitory effects on platelet cyclooxygenase and thrombosis. Inter-individual variability of platelet responses to aspirin clinically presents as treatment failure, and this increasing clinical phenomenon is frequently named aspirin "resistance". However, the molecular mechanisms behind this are unclear. Blood aspirin hydrolases are believed to control aspirin survival in vivo, but the identity of the circulating enzyme(s) that hydrolyzes aspirin remains unknown. In this thesis, blood aspirin hydrolases were identified and characterized. The relationship between blood aspirin hydrolases and aspirin efficacy was investigated. RP-HPLC analysis of the product salicylic acid with an internal standard showed plasma hydrolysis of aspirin varied 12-fold variation among 2,275 individuals. Genome-wide association analysis using serum aspirin hydrolytic activity from 2,275 individuals showed a genetic component to aspirin hydrolytic variation, and that only BChE significantly associated to aspirin variation. However, plasma from an individual with an inactivating point mutation in BChE effectively hydrolyzed aspirin. A non-BChE aspirin hydrolase was found in plasma, which can be distinguished from BChE by procainamide and oxidized ATP. Erythrocyte aspirin hydrolase was purified by 1400-fold and type I PAF acetylhydrolase was identified as a candidate aspirinase by mass spectrometry. Recombinant PAFAH1B2 hydrolyzed aspirin and aspirin was effectively hydrolyzed in cells ectopically expressing PAFAH1B2 and PAFAH1B3. Type I PAFAH also accounts for non-BChE plasma aspirin hydrolytic activity. Both plasma and erythrocytes were found modulated aspirin efficacy significantly, which varied by aspirin hydrolytic activity variation.I hope my work in this thesis will help to understand aspirin "resistance" and shed light on personalized medicine of aspirin

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