Validation of PON1 as a Biomarker of Clopidogrel Resistance in Caribbean Hispanics

Abstract

Background : Clopidogrel is an antiplatelet drug used to prevent heart attack and strokes in cardiovascular patients. Pharmacological studies of clopidogrel have been mainly conducted in individuals of European ancestry, therefore missing genetic diversity and enhancing disparities of care. Since admixed Caribbean Hispanics are among those often underrepresented, we want to identify predictive or risk biomarkers of the response to clopidogrel within this population. Preliminary data using Tandem Mass Tag (TMT) coupled with mass spectrometry revealed that paraoxonase-1 (PON1) is downregulated in patients treated with clopidogrel (-49.50) when compared to other cardiovascular patients. PON1 is an HDL-associated protein that protects against accumulation of oxidized lipids and prevent cardiovascular disease. Our aim is to validate the low abundance of PON1 by western blot analysis. Methods : All patients signed an informed consent approved by the Institutional Review Board (IRB-protocol number A4070417). Blood was collected and a platelet reactivity test was performed using the VerifyNow® P2Y12 assay to determine if the patient is a poor responder (PRU≥230). Plasma was separated by centrifugation and stored at -80°C. For Western Blot analysis, 25µg of plasma proteins were run on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the healthy (n=6), cardiovascular (n=6), normal responders (n=14), and poor responders (n=10) groups. Proteins were electro-transferred from the gel onto polyvinylidene membrane. The membranes were incubated with PON1 (4G8D3) and goat anti-mouse IRDye 680RD antibodies, followed by an incubation with a loading control, anti-transferrin (101) rabbit monoclonal antibody and goat anti-rabbit IRDye 680RD antibody. The membranes were scanned in the Odyssey® CLx Imager in AUTO (680 nm and 800 nm channels) to reveal the immunofluorescent bands that were quantified by the Image StudioTM Software. Statistical analysis was carried out by Graphpad Prism 6.01 for Windows. After determining the normality of the data by Shapiro Wilkins Test, a one-way ANOVA was run as a variance test. Results : The total PON1 intensity signal was detected in healthy (1.11±0.34) and cardiovascular controls (1.00±0.00), normal (1.06±0.45) and poor responders (0.89±0.34), with a lower mean value in the latter but no statistical differences among the groups (p>0.05). Conclusions : PON1 could not be validated as a downregulated protein in poor responders when compared to other cardiovascular groups by western blotting.