Genetic and Dietary Predictors of Anti-platelet Response

Purpose: In this aim, the investigators will test the hypothesis that n-3 PUFAs modify aspirin anti-platelet response, by measuring and comparing three different indicators of aspirin response in individuals with extremely high and low RBC n-3 PUFA levels, under a basal state and after 1-week of once a day aspirin treatment. The three indicators are: 1) platelet TXB2 and 20-HETE concentrations; 2) platelet aggregation ex vivo (in response to external stimuli); and 3) aspirin covalent adduct of COX-1. The investigators will also test whether or not common variants in the CYP4A11, CYP4F2, CYP4F11, PEAR1 and ACTN1 genes are associated with basal and aspirin-induced changes in platelet aggregation and, in an exploratory analysis, modify the effects of n-3 PUFAs on aspirin response.

Procedures and Population: Investigators from the University of Montana, Southcentral Foundation and Oregon Health \& Science University will recruit a total of 150 individuals (50 at each site) to participate in the study. At each performance site, the participants recruited will represent 25 individuals with high and 25 individuals with low RBC n-3 PUFA concentrations. After providing written consent, each study participant will be screened for medical history and clinical lab testing to ensure good health and good liver, kidney and blood test parameters. If found to be eligible, each participant will provide 12-hour fasted (overnight) blood samples for isolation of plasma, platelets, lymphocytes, and red blood cells. A separate blood sample will be collected into a citrate treated tube for platelet aggregation testing using the PFA-100 platform. Isolated blood fractions will be stored at -80°C.

After baseline sampling, each participant will be given six doses of aspirin (80 mg/day for 2 days) and instructed to take one dose daily at approximately 9 am. On the day after the last aspirin dose, the participant will provide blood samples for repeated platelet aggregation testing, platelet lipid analysis and measurement of aspirin covalent adduct to COX-1, and a spot urine sample to confirm aspirin consumption by testing for salicylate. For 24 hours prior to platelet aggregation testing, study participants will be asked to avoid foods that could lead to spurious test results (eg, chocolate, tea, coffee, cola, red wine, beer, tomato, grapes, grape juice, orange and cranberry juices, as well as traditional wild foods that include berries, plant roots, plant greens etc).

Analytical Methods: Whole blood, point of care, platelet aggregation testing will be performed using the automated PFA-100 instrument. Performance by research personnel will be compared head-head to clinical testing services at each site, as a quality control measure. In each test setting, the investigators will obtain results in duplicate for both collagen/epinephrine and collagen/ADP cartridges. The investigators will also measure platelet TXB2, 20-HETE and AA concentrations, by LC-MS/MS. In addition, the investigators will develop and employ a method to quantify theaspirin covalent adduct of COX-1 in isolated platelets from study participants.

Statistical Methods: For each study population, and subgroups with extreme RBC n-3 PUFA phenotypes, the investigators will use linear regression models with robust standard errors to identify significant differences in the mean absolute platelet aggregation test results under basal and aspirin-treated conditions (primary outcome), as well as the post-treatment-to-baseline change in platelet aggregation. Heterogeneity between pairs of extreme phenotypes subgroups will be accounted for by including sex, age, BMI and platelet count as covariates in the regression model. Sample size calculation for each performance site (n = 25 per n-3 PUFA subgroup) was based on having power of at least 0.8 for detecting a difference in mean platelet aggregation between the two extreme (0-10 and 90-100 percentiles) n-3 PUFA subgroups under basal conditions, at a significance level of 0.05. As a secondary level of analysis, for each subgroup of the extreme RBC n-3 PUFA phenotypes, the investigators will also compare mean platelet TXB2 and 20-HETE concentrations under basal and aspirin-treated conditions, as well as the post-treatment-to-baseline change. Finally, a third level of analysis will involve testing each study population for a difference in the extent of aspirin adduct to COX-1 between groups with extreme RBC n-3 PUFA phenotypes. The investigators will test for associations between platelet aggregation test results and biochemical measures of platelet activation, each study population separately and in aggregate.

The investigators will use linear regression models to test for an association between common CYP4A11,CYP4F2, CYP4F11, PEAR1 and ACTN1 genotypes and the mean absolute platelet aggregation test results under basal and aspirin-treated conditions, as well as the post-treatment-to-baseline change in platelet aggregation. In an exploratory analysis, the investigators will apply Linear Mixed Effects (LME) models to the combined pre- and post-treatment dataset to test for associations with n-3 PUFAs group and drug effects, as well as to identify any modifications of those effects by CYP4A11, CYP4F2, CYP4F11, PEAR1 and ACTN1 variation by including and testing for significant interaction effects in the models. The investigators will include random effects in the LME to account for potential heterogeneity in each of the populations.