The Effect of Dual Anti-Platelet Therapy on Platelet Gene Expression

M Hayman1, M Chan1, N Kirkby1,2, R Knowles1, P Munroe1, T Warner1. 1William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London, UK, 2National Heart and Lung Institute, Imperial College London, London, UK

Introduction: Thrombosis linked to cardiovascular disease remains a major global health burden despite the existence of effective pharmacological and surgical strategies. Combination of aspirin and a P2Y₁₂ receptor blocker, such as prasugrel, known as dual anti-platelet therapy (DAPT), inhibits platelet activation and slows the progression of thrombus formation. At present little is known about whether the changes in platelet function caused by DAPT are in any way dependent upon platelet gene expression.

Methods: 8 healthy Caucasian males, aged 18-40 received DAPT (aspirin, 75 mg plus prasugrel, 10 mg) once a day for 7 days. Blood was taken before and after DAPT administration. The study was approved by NHS St. Thomas’ Hospital Research Ethics Committee (Ref. 07/Q0702/24). Platelet rich plasma (PRP) was obtained by centrifugation and the Optimul assay was used to validate the known effects of DAPT on platelet aggregation in response to adenosine diphosphate (ADP: 0.005-40 μM), arachidonic acid (AA: 0.03-40 μM), Horm collagen (0.01-40 μg/ml), epinephrine (0.0004-10 μM), ristocetin (0.14-4 mg/ml), TRAP-6 amide (0.03-40 μM) and U46619 (0.005-40 μM). Data was analysed using a two-way ANOVA. Purified platelet pellets were obtained from PRP by magnetic MACS® cell separation (Miltenyi Biotec, Surrey, UK) using anti-CD45 microbeads to remove leukocytes and resuspended in lysis buffer. RNA (RNeasy Mini Kit, Qiagen, Manchester, UK) and cDNA (Life Technologies, Paisley, UK) was extracted or made according to manufacturer’s instructions. Quantitative RT-PCR was performed using TaqMan probes for genes of interest related to platelet signalling, aggregation and drug pathways (e.g ADRA2A, PEAR1, PTGS1 ect.). Data was analysed using the 2(-Delta Delta C(T)) method with a one sample T-test was used to determine significance.

Results: There was significant reduction (p<0.05) in aggregation in response to all agonists except ristocetin after DAPT. There was no significant (p>0.05) change in any genes studied.

Conclusion: The ex vivo effects on functional platelet assays following from one week of DAPT were not mirrored by changes in the expression of genes known to be important to molecular events. These findings, however, may warrant further investigation into novel gene changes by the use of full transcriptomic analysis of the platelet genome to look for the broad range of platelet effectors that may be altered by DAPT.

Reference:

1. Chan, M. V, & Warner, T. D. (2012). Standardised optical multichannel (optimul) platelet aggregometry using high-speed shaking and fixed time point readings. Platelets, 23(5), 404–408.