Abstract
Platelet activation triggers thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies still fail to prevent thrombotic events in numerous patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how megakaryocyte-derived mRNAs are regulated in these anucleate cell fragments. Proteogenomics was applied here to investigate this phenomeon in platelets activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining proteomics and transcriptomics allowed in depth platelet proteome characterization, revealing a significant effect of either stimulus on proteome composition. In silico analysis revealed the presence of resident immature RNAs in resting platelets, characterized by retained introns, while unbiased proteogenomics correlated intron removal by RNA splicing with changes on proteome composition upon activation. This allowed identification of a set of transcripts undergoing maturation by intron removal during activation and resulting in accumulation of the corresponding peptides at exon-exon junctions. These results indicate that RNA splicing events occur in platelets during activation and that maturation of specific pre-mRNAs is part of the activation cascade, contributing to a dynamic fine-tuning of the transcriptome.
Highlights
Better understanding of the processes that characterize platelet activation, and early markers of these processes, are still much sought after as they may be of great clinical relevance[6]
Searching the mass spectrometry (MS) data for correlations between extent of intron removal and abundance of the resulting exon/exon junction peptide, we found that COLL activation induced both events on clathrin heavy chain (CLTC) (Clathrin Heavy Chain) and ATP2C1 (ATPase Secretory Pathway Ca2+ Transporting 1), Thrombin Receptor Activating Peptide (TRAP) on FAM160B1 (Family With Sequence Similarity 160 Member B1) and B-cell scaffold protein with ankyrin repeats 1 (BANK1) (B-Cell Scaffold Protein With Ankyrin Repeats 1) mRNAs and either stimulus on transmembrane 9 superfamily member 2 (TM9SF2) (Transmembrane 9 Superfamily Member 2), IQGAP2 (IQ Motif Containing GTPase Activating Protein 2) and MSN (Moesin) gene products (Fig. 5B,C)
We were able to provide a proof of concept that in several cases this may lead to protein accumulation, as we found inverse correlation between IR and protein, or even exon/exon junction peptide, concentration
Summary
Better understanding of the processes that characterize platelet activation, and early markers of these processes, are still much sought after as they may be of great clinical relevance[6]. The present study aimed at investigating on a global scale whether changes in the maturation of specific RNAs by non-canonical splicing events occur in platelets upon activation by physiological stimuli, and if this may result in modulation of protein expression To this end, we performed an in-depth proteome analysis of resting platelets using peptide level high-resolution isoelectric focusing and reverse phase fractionation[23], followed by mass spectrometry (MS) analysis (HiRIEF LC-MS/MS). RNA-Seq provided a catalog of partially spliced mRNAs in resting platelets and showed how retained introns are removed upon activation, to generate mature mRNA molecules This last result was reinforced by comparison of proteomics and transcriptomics data, showing coherence between intron removal events and accumulation of peptides mapping on the corresponding exon/exon junctions. We propose that maturation of activation-modulated transcripts represent a novel mechanism for control of platelet functions and may be used as early marker of their activation in thrombotic diseases
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