Platelet RNA-Seq Identifies Genes Expressed Differentially After Clopidogrel Treatment

Clopidogrel is an antiplatelet agent that acts through direct inhibition of ADP-dependent platelet activation. Several studies have also found an inhibitory effect of clopidogrel on inflammation. Although platelets are known to participate in inflammatory processes, the mechanism(s) through which clopidogrel might modify platelet-mediated inflammation is unknown. We examined the platelet transcriptome to gain insight into the mechanism(s) through which clopidogrel modulates inflammation.

Three individuals with subclinical atherosclerosis and family history of early onset coronary artery disease were enrolled. Blood was collected for RNA-seq at baseline and one-week after clopidogrel 75mg daily. RNA was extracted from leukocyte-depleted platelet-rich plasma and approximately 70 million 100bp paired-end reads per sample were obtained using the Illumina HiSeq 2500. RNA-seq analysis was performed using Bowtie/TopHat/Cufflinks software pipeline. Genes with expression ≥ 0.3 fragments per kilobase of transcript per million reads (FPKM) were considered to be expressed. We set the threshold for significant differential expression as ≥ 2-fold change in gene expression after clopidogrel therapy with a false discovery rate of 0.05.

The study sample consisted of 2 women and 1 man. Among the 12,297 expressed genes, the most highly expressed nonubiquitous genes included PPBP, PF4, and TUBB1, confirming platelets as the source of the RNA. Comparing samples before and after clopidogrel, treatment was associated with up-regulation of 733 genes and down-regulation of 387 genes. Pathway analyses found that genes related to platelet activation, degranulation, and aggregation were up-regulated, while genes related to immune system processes, such as B- and T-cell activation and innate immune response, were down-regulated. Examples of down-regulated genes include the toll-like receptor family genes, TLR2, TLR4, TLR7, and TLR8, the chemokine receptor genes CCR2, CCR5,CCR6, and CCR7, and the interleukin receptor genes, IL4R, IL7R, and IL13RA.

One week of clopidogrel therapy up-regulates transcription of pro-thrombotic platelet genes and down regulates transcription of inflammation-related platelet genes. These data suggest that clopidogrel may exert a direct anti-inflammatory action by inhibiting the ability of platelets to participate in inflammatory processes. The relationship of variability in clopidogrel-induced modulation of these pathways to clinical outcomes is unknown.

No relevant conflicts of interest to declare.