Integration of Proteomics and Genomics in Platelets

Abstract

Platelets, while anucleate, contain RNA, some of which is translated into protein upon activation. Hypothesising that the platelet proteome is reflected in the transcriptome, we identified 82 proteins secreted from activated platelets and compared these, as well as published proteomic data, to the transcriptional profile. We also compared the transcriptome of platelets to other tissues to identify platelet-specific genes and used ontology to determine gene categories over-represented in platelets. RNA was isolated from highly pure platelet preparations for hybridization to Affymetrix oligonucleotide arrays. We identified 2,928 distinct messages as being present in platelets. The platelet transcriptome was compared with the proteome by relating both to UniGene clusters. Platelet proteomic data correlated well with the transcriptome, with 69% of secreted proteins detectable at the mRNA level, and similar concordance was obtained using two published datasets. While many of the most abundant mRNAs are for known platelet proteins, messages were detected for proteins not previously reported in platelets. Some of these may represent residual megakaryocyte messages; however, proteomic analysis confirmed the expression of many previously unreported genes in platelets. Transcripts for well-described platelet proteins are among the most platelet-specific messages. Ontological categories related to signal transduction, receptors, ion channels, and membranes are over-represented in platelets, while categories involved in protein synthesis are depleted. Despite the absence of gene transcription, the platelet proteome is mirrored in the transcriptome. Conversely, transcriptional analysis predicts the presence of novel proteins in the platelet. Transcriptional analysis is relevant to platelet biology, providing insights into platelet function and the mechanisms of platelet disorders.

Recent advances in blood‐related proteomics

“…With more complex proteomic approaches (multidimensional chromatography followed by digestion with trypsin, strong cation exchange, RP chromatography, and IT MS), more than 300 proteins were isolated, 82 of which repeatedly; 40% were known secreted proteins, the remaining not being known to be present in platelets or secreted by them. Interesting recent studies provided evidence that the platelet transcriptome correlated quite well with the platelet proteome, and that proteomic analysis revealed the expression of many previously unreported genes in platelets [131][132][133]. Proteomic technologies applied to platelet research have been recently reviewed [134].…”

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

“…RNA Isolation and Purification from Platelets-1 ml of Tri reagent was added to 10 8 platelets from a highly purified platelet preparation (45) and allowed to lyse on ice for 15 min to 1 h. 1-Bromo-3-chloropropane (Sigma) was then added (0.1 ml/ml Tri reagent), and samples were shaken vigorously for 15 s then allowed to stand for 15 min. Platelets were then centrifuged for 15 min at 12,000 ϫ g at 4°C.…”

“…ICln RT-PCR from Platelet RNA-The purification of the platelet RNA was verified as previously described (45). Platelet RNA (1 g) was denatured at 65°C for 10 min and incubated at 37°C overnight in the presence of 1ϫ reverse transcription buffer, 1 l of Moloney murine leukemia virus reverse transcriptase, 25 M deoxycholate triphosphates (dNTPs), and 1 l of random hexamers in a final volume of 8 l. The reaction was stopped by heating at 95°C for 5 min.…”

“…The preparation of pure platelet RNA is not an insignificant task in platelets, because, being non-nucleated, their RNA content is considered vestigial and is consequently very low. Gel-filtered platelets were stringently prepared by repeated differential centrifugation and analyzed for white cell contamination by estimating T-cell receptor and CD45 (leucocyte common antigen) positivity by RT-PCR assays (45). The presence of intact platelet RNA is estimated by PCR analysis of mRNA for glycoprotein Ib␣.…”

Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues

“…Transcript profiling was used to assess normal variability in gene expression levels of mouse liver, kidney, and testis (11) and to analyze changes in expression patterns during embryonic and fetal liver development (12). So far, comparative transcriptome and proteome analyses in complex organisms are very limited and have been performed in human platelets (13) and heart tissue (14), and in the Anopheles and Culex salivary glands (15,16). In rodents, the proteome of mouse primary islet cells was correlated with RNA expression data of purified primary rat beta cells, suggesting a close correlation between mRNA and protein expression (17).…”