YS03
Gene Expression Profiling of Platelet RNA Identifies Differentially Expressed Genes Associated with Collagen Signalling Responses

Publication Summary

Platelet Glycoprotein VI (GPVI) is a lineage specific collagen signalling receptor that transmits outside‐in signalling via the FcR γ‐chain. Two GPVI haplotypes have been identified, ‘a’ and ‘b’, that differ by five amino acids. These substitutions have a profound effect on signalling downstream of GPVI and are also associated with variation in the expression level of GPVI on the platelet surface. The aim of this study was to screen the transcriptomes of platelets from ‘aa’ and ‘bb’ homozygous individuals and determine differences at the gene expression level between individuals with different haplotypes. Platelet RNA was isolated from leukocyte‐depleted apheresis concentrates obtained from eight ‘aa’ and eight ‘bb’ donors. Using a template‐switching PCR based amplification protocol the mRNA in 100 ng of total RNA was amplified and then labelled with either Cy3 or Cy5‐dCTP. The eight ‘aa’ and eight ‘bb’ samples were randomly paired and compared on a 15 552 feature DNA microarray. In total 32 competitive hybridizations were performed, with four replicates for each pairing to control for technical variability. The microarray data were analyzed using a combination of statistical approaches and a list of significantly differentially expressed (DE) genes was identified. The DE genes had small fold‐changes, typically two to three‐fold, however we were able to verify differential expression by qRT‐PCR. Of the 10 genes selected for validation, two were confirmed as significantly differentially expressed between the two haplotypes (TUBB1, P‐val 0.0004; VWF, P‐val 0.0126) while a further four showed a trend in the same direction as the microarray result. Three DE transcripts were not confirmed by qRT‐PCR and the final transcript could not be accurately quantified. The results of this study suggest that differences in GPVI signalling are associated with differential gene transcription. The strategy applied here will be used to compare the transcriptomes of platelets from individuals identified using functional tests as high and low responders to a number of agonists. The identification of differentially expressed genes and correlation with functional responses may identify critical proteins involved in determining the extent of platelet response in vivo.