Proteomic approaches to dissect platelet function: half the story

Abstract

Platelets play critical roles in diverse hemostatic and pathologic disorders and are broadly implicated in various biological processes that include inflammation, wound healing, and thrombosis. Recent progress in high-throughput mRNA and protein profiling techniques has advanced our understanding of the biological functions of platelets. Platelet proteomics has been adopted to decode the complex processes that underlie platelet function by identifying novel platelet-expressed proteins, dissecting mechanisms of signal or metabolic pathways, and analyzing functional changes of the platelet proteome in normal and pathologic states. The integration of transcriptomics and proteomics, coupled with progress in bioinformatics, provides novel tools for dissecting platelet biology. In this review, we focus on current advances in platelet proteomic studies, with emphasis on the IntroductionHuman blood platelets play important roles in fundamental biological processes, including thrombosis, inflammation, wound repair, and stroke. Although they are anucleate and lack nuclear DNA, platelets retain small amounts of megakaryocyte-derived mRNA. 1,2 Platelets also contain rough endoplasmic reticulum and polyribosomes, thus retaining the capacity for protein biosynthesis from cytoplasmic mRNA. 3 Quiescent platelets display minimal translational activity, although platelet activation leads to the rapid translation of preexisting mRNA, 4 with the release or derivation of platelet-secreted proteins, cytokines, exosomes, and microparticles.The traditional paradigm that platelet mRNA content is invariant and gradually declines with cell senescence was challenged when signal-dependent pre-mRNA splicing was identified in platelets. 5 Signal-dependent splicing provides a mechanism for altering the repertoire of translatable messages in response to cellular activation/stimulation. Furthermore, platelets have essential components of a functional spliceosome and selected unspliced pre-mRNAs. These spliceosomes retain a unique ability to splice pre-mRNA in the cytoplasm (as opposed to the typical nuclear location), a capability not described in any other mammalian cell. This discovery emphasizes that the molecular mechanisms of platelet function cannot be optimally dissected without accurate platelet transcript profiling.Modern postgenomic, high-throughput approaches allow integrated studies of molecular components (at the RNA and the protein levels) involved in cell function. Platelets represent an attractive, simplified model for these studies because they lack nuclear DNA and because their genome consists of a small subset of megakaryocyte-derived mRNA transcripts. This complete pool of platelet RNAs is significantly smaller than the transcriptome of a nucleated cell. 6 The entire pool of platelet proteins constitutes the platelet proteome: the initially static, but functionally dynamic, protein interactions that occur with platelet activation. In this review, we focus on recent applications of proteomic and transcriptomic technologies to...

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

“…Platelets were isolated immediately after blood donation to avoid changes in their physiological state and viability. Only the upper third of the platelet rich plasma was isolated to avoid contamination from other blood cells such as erythrocytes, leukocytes and plasma proteins [15,28]. Platelets also underwent additional centrifugation steps to minimise potential contamination with plasma proteins normally present in platelet rich plasma, which could influence the outcome of the experiment [14].…”

Class prediction models of thrombocytosis using genetic biomarkers

“…After removal of duplicates, the final list contained 432 genes, which cosegregated by cell type (platelet vs leukocyte) as determined by in silico analysis. 32 The relative gene-expression patterns comprising the gene list were found to logically follow a Poisson distribution (not shown). Several Arabidopsis probe elements were included to serve as normalization controls and as quantitative measures of interslide and intraslide variability 33 ; for all genes, 70-mer oligonucleotides were designed and synthesized based on the Ensembl (www.ensembl.org) Human 13.31 Database; all probe sets were spotted in quadruplicate to provide replicates and statistical robustness.…”

What Can Proteomics Tell Us About Platelets?

“…ACSA indicates acetyl-coenzyme A synthetase; DTS, dense tubular system; ER, endoplasmic reticulum; FG, fibrinogen, FN, fibronectin; GPVI, platelet glycoprotein VI; LAMP, lysosome-associated membrane glycoprotein; LDH, L-lactate dehydrogenase; MYH, myosin; PAR, proteinase-activated receptor; PDI, platelet disulfide isomerase; PF-4, platelet factor 4; SERCA, sarcoplasmic endoplasmic reticulum Ca human genes are expressed in platelets at the messenger RNA level. 68,69 Several studies focused on analysis of the platelet transcriptome [68][69][70][71][72] and some of these compared protein and transcript levels, deducing a certain degree of correlation. 68,70 A comprehensive comparison between our quantitative proteome data and published transcriptome data revealed only weak correlation.…”