Leonard C. Edelstein

The complex transcriptional landscape of the anucleate human platelet

BackgroundHuman blood platelets are essential to maintaining normal hemostasis, and platelet dysfunction often causes bleeding or thrombosis. Estimates of genome-wide platelet RNA expression using microarrays have provided insights to the platelet transcriptome but were limited by the number of known transcripts. The goal of this effort was to deep-sequence RNA from leukocyte-depleted platelets to capture the complex profile of all expressed transcripts.ResultsFrom each of four healthy individuals we generated long RNA (≥40 nucleotides) profiles from total and ribosomal-RNA depleted RNA preparations, as well as short RNA (<40 nucleotides) profiles. Analysis of ~1 billion reads revealed that coding and non-coding platelet transcripts span a very wide dynamic range (≥16 PCR cycles beyond β-actin), a result we validated through qRT-PCR on many dozens of platelet messenger RNAs. Surprisingly, ribosomal-RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts. Of the known protein-coding loci, ~9,500 are present in human platelets. We observed a strong correlation between mRNAs identified by RNA-seq and microarray for well-expressed mRNAs, but RNASeq identified many more transcripts of lower abundance and permitted discovery of novel transcripts.ConclusionsOur analyses revealed diverse classes of non-coding RNAs, including: pervasive antisense transcripts to protein-coding loci; numerous, previously unreported and abundant microRNAs; retrotransposons; and thousands of novel un-annotated long and short intronic transcripts, an intriguing finding considering the anucleate nature of platelets. The data are available through a local mirror of the UCSC genome browser and can be accessed at:http://cm.jefferson.edu/platelets_2012/.

Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs.

MicroRNAs in platelet production and activation

MicroRNAs are small RNA molecules that modulate protein expression by degrading mRNA or repressing translation. They have been shown to play important roles in hematopoiesis, including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. miR-150 and miR-155 play divergent roles in megakaryocytopoiesis, with the former promoting development of megakaryocytes at the expense of erythrocytes and the latter causing a reduction in megakaryocyte colony formation. Platelets also contain fully functional miRNA machinery, and certain miRNA levels in platelets have been found to coordinate with reactivity to specific agonists and to pathologic states. This review will cover the current state of knowledge of miRNAs in megakaryocytes and platelets and the exciting possibilities for future research.

Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics.

There is little data considering relationships among human RNA, demographic variables, and primary human cell physiology. The platelet RNA and expression-1 study measured platelet aggregation to arachidonic acid, ADP, protease-activated receptor (PAR) 1 activation peptide (PAR1-AP), and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. A total of 5911 uniquely mapped mRNAs and 181 miRNAs were commonly expressed and validated in a separate cohort. One hundred twenty-nine mRNAs and 15 miRNAs were differentially expressed (DE) by age, and targets of these miRNAs were over-represented among these mRNAs. Fifty-four mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship in these RNA pairs suggests miRNAs regulate mRNA levels on aging and between genders. A simple, interactive public web tool (www.plateletomics.com) was developed that permits queries of RNA levels and associations among RNA, platelet aggregation and demographic variables. Access to these data will facilitate discovery of mechanisms of miRNA regulation of gene expression. These results provide new insights into aging and gender, and future platelet RNA association studies must account for age and gender.

Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists.

The human platelet: strong transcriptome correlations among individuals associate weakly with the platelet proteome

BackgroundFor the anucleate platelet it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA profiling platforms, and what the transcriptomes’ relationship is with the platelet proteome. We profiled the platelet transcriptome of 10 healthy young males (5 white and 5 black) with no notable clinical history using RNA sequencing and by Affymetrix microarray.ResultsWe found that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, independently of race and of the employed technology. Our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. Pseudogenes represented a notable exception by exhibiting a difference in expression by race. Comparison of our mRNA signatures to a publicly available quantitative platelet proteome showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. However, a high number of mRNAs that were present in the transcriptomes of all 10 individuals had no representation in the proteome. Spearman correlations of the relative abundances for those genes represented by both an mRNA and a protein showed a weak (~0.3) connection. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified gene groups corresponding to distinct cellular processes.ConclusionsThe results of our analyses provide novel insights for platelet biology, show only a weak connection between the platelet transcriptome and proteome, and indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease.Reviewed byThis article was reviewed by Dr Mikhail Dozmorov (nominated by Dr Yuri Gusev), Dr Neil Smalheiser and Dr Eugene Koonin.

Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4+ T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

MicroRNA Expression Differences in Human Hematopoietic Cell Lineages Enable Regulated Transgene Expression.

Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.

Mechanism of Race-Dependent Platelet Activation Through the Protease-Activated Receptor-4 and Gq Signaling Axis

Objective— Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies. Approach and Results— Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the Gq pathway compared with platelets from white donors. Differences in signaling included increased Ca2+ mobilization, Rap1 (Ras-related protein 1) activation, and integrin &agr;IIb&bgr;3 activation with no observed difference in platelet protein expression between the groups tested. Conclusions— Our study is the first to demonstrate that the Gq pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites.