Steven E McKenzie

Thomas Jefferson University, Filadelfia, Pennsylvania, United States

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Publications (92)448.99 Total impact


  • No preview · Article · Jan 2016 · Platelets
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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via FcγRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase (Syk) and leads to the generation of tissue factor and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases annexin and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.
    No preview · Article · Oct 2015 · Blood
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    ABSTRACT: FcγRIIA-mediated platelet activation is essential in heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. There is considerable inter-individual variation in platelet FcγRIIA activation, the reasons for which remain unclear. We hypothesized that genetic variations between FcγRIIA hyper- and hypo-responders regulate FcγRIIA-mediated platelet reactivity and influence HIT susceptibility. Using unbiased genome-wide expression profiling, we observed that human hypo-responders to FcγRIIA activation showed higher platelet T-cell ubiquitin ligand-2 (TULA-2) mRNA expression than hyper-responders. siRNA-mediated knockdown of TULA-2 resulted in hyper-phosphorylation of spleen tyrosine kinase (Syk) following FcγRIIA activation in HEL cells. Significantly, we found miR-148a-3p targeted and inhibited both human and mouse TULA-2 mRNA. Inhibition of miR-148a in FcγRIIA transgenic mice up-regulated the TULA-2 level and reduced FcγRIIA- and GPVI-mediated platelet αIIbβ3 activation and calcium mobilization. Anti-miR-148a also reduced thrombus formation following intravascular platelet activation via FcγRIIA. These results show that TULA-2 is a target of miR-148a-3p and TULA-2 serves as a negative regulator of FcγRIIA-mediated platelet activation. This is also the first study to show the effects of in vivo miRNA inhibition on platelet reactivity. Our work suggests that modulating miR-148a expression is a potential therapeutic approach for thrombosis.
    Full-text · Article · Oct 2015 · Blood
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    ABSTRACT: Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immuno-precipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unre-ported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage-and/or tissue-specific miRNAs that are uncharacterized. microRNAs | isomIRs | noncoding RNA | RNA sequencing | transcriptome
    Full-text · Article · Feb 2015 · Proceedings of the National Academy of Sciences
  • E. Dianne Pulte · Steven E. McKenzie · Jaime Caro · Samir K. Ballas
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    ABSTRACT: Previous studies have suggested that erythropoietin (Epo) levels may be inappropriately low in patients with sickle cell disease compared to the extent of the related anemia they demonstrate. Here, we evaluate Epo level vs. renal function, oxygenation, and markers of inflammation for patients treated for sickle cell disease at our institution. Blood was drawn from 54 patients with sickle cell disease during routine visits to the outpatient hematology office and analyzed for hemoglobin (Hb) level, Epo, markers of inflammation, oxygenation, and renal function. Erythropoietin levels were lower than expected for patients with sickle cell disease, compared to the degree of anemia demonstrated in these patients. In addition, a correlation between Hb level and Epo was not consistently observed. Higher Epo levels were seen in patients receiving hydroxyurea (HU), but no correlation with oxygenation, hemolysis, renal function, or inflammation was observed.
    No preview · Article · Nov 2014 · Hemoglobin
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    ABSTRACT: Objective: We previously determined that protein kinase C δ (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown. Approach and results: Using PKCδ(-/-) and wild-type littermate mice, we found that deficiency of PKCδ caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKCδ(-/-) mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKCδ deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKCδ(-/-) megakaryocytes compared with wild-type. Finally, PKCδ(-/-) mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. Conclusions: These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.
    No preview · Article · Oct 2014 · Arteriosclerosis Thrombosis and Vascular Biology
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    ABSTRACT: IntroductionPlatelet activation via the Fcγ receptor IIa (FcγRIIa) is implicated in the pathogenesis of immune complex (IC)-mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or β2 Glycoprotein I (β2GPI) induce ITT in mice transgenic for human FcγRIIa (hFcR) but not wild-type controls (which lack FcγRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG-GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs.Methods Pre-formed anti-CD40L or anti-β2GPI ICs were injected into hFcR/Caldaggef1+/+ or hFcR/Caldaggef1-/- mice, with or without clopidogrel pre-treatment. Animals were observed for symptoms of shock for 30 minutes, during which time core body temperature was monitored. Platelet counts were obtained before and 30 minutes after IC injection. Lungs were assessed for thrombosis by histology or near-infrared imaging.ResultsBoth CD40L and β2GPI ICs rapidly induced severe thrombocytopenia, shock and a reduction in body temperature in hFcR/Caldaggef1+/+ mice. hFcR/Caldaggef1-/- mice were protected from CD40L and β2GPI IC-induced thrombocytopenia and shock, whereas P2Y12 inhibition had only a modest effect on IC-induced ITT. Consistent with these findings, IC-induced integrin activation in vitro and the accumulation of activated platelets in the lungs of IC-challenged mice was strongly dependent on CalDAG-GEFI.Conclusions Our studies demonstrate that CalDAG-GEFI plays a critical role in platelet activation, thrombocytopenia and thrombosis induced by clinically relevant ICs in mice. Thus, CalDAG-GEFI may be a promising target for the intervention of IC-associated, FcγRIIa-mediated thrombotic conditions.This article is protected by copyright. All rights reserved.
    No preview · Article · Oct 2014 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis which is often life-threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. Here we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacological inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. 12-LOX was shown to be essential for FcγRIIa-induced PLCγ2 activity leading to activation of calcium mobilization, Rap1 and PKC activation, and subsequent activation of the integrin αIIbβ3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbβ3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identify 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.
    Preview · Article · Aug 2014 · Blood
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    ABSTRACT: 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.
    Full-text · Article · Jul 2014 · PLoS ONE
  • Steven E McKenzie · Bruce S Sachais
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    ABSTRACT: Purpose of review: To review the recent developments in understanding the pathophysiology of heparin-induced thrombocytopenia (HIT) and in applying this knowledge to the treatment of patients with suspected and proven HIT. Recent findings: HIT pathophysiology is dynamic and complex. HIT pathophysiology is initiated by four essential components--heparin (Hep), platelet factor 4 (PF4), IgG antibodies against the Hep-PF4 complex, and platelet FcγRIIa. HIT is propagated by activated platelets, monocytes, endothelial cells, and coagulation proteins. Insights into the unique HIT antibody response continue to emerge, but without consensus as to the relative roles of B cells, T cells, and antigen-presenting cells. Platelet activation via FcγRIIa, the sine qua non of HIT, has become much better appreciated. Therapy remains challenging for several reasons. Suspected HIT is more frequent than proven HIT, because of the widespread use of Hep and the inadequacies of current diagnostic tests and scoring systems. In proven HIT, approved treatments reduce but do not eliminate thrombosis, and have substantial bleeding risk. Rational novel therapeutic strategies, directed at the initiating steps in HIT pathophysiology and with potential combinations staged over time, are in various phases of development. Summary: Progress continues in understanding the breadth of molecular and cellular players in HIT. Translation to improved diagnosis and treatment is needed.
    No preview · Article · Jul 2014 · Current Opinion in Hematology
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    ABSTRACT: For 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. We 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 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) yet significant (P = 5.0E-16) connection. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified gene groups corresponding to distinct cellular processes. The results of our analyses provide novel insights for platelet biology, 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 by: This article was reviewed by Dr Mikhail Dozmorov (nominated by Dr Yuri Gusev), Dr Neil Smalheiser and Dr Eugene Koonin.
    Full-text · Article · Feb 2014 · Biology Direct
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    ABSTRACT: There is little data considering relationships among human RNA, demographic variables and primary human cell physiology. The Platelet RNA And eXpression-1 (PRAX1) study measured platelet aggregation to arachidonic acid, ADP, PAR1-AP and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. 5911 uniquely mapped mRNAs and 181 miRNAs were expressed commonly and validated in a separate cohort. 129 mRNAs and 15 miRNAs were differentially expressed (DE) by age, and putative targets of these miRNAs were over-represented among these mRNAs. 54 mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship between the RNA types in these "pairs" suggests miRNAs regulate mRNA levels upon 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 association studies between RNAs and clinical end points or ex vivo platelet function must account for age and gender.
    Full-text · Article · Feb 2014 · Blood
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    Full-text · Dataset · Sep 2013
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    ABSTRACT: Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Y323, Y352 and Y525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Y525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases (SFKs). Phosphorylation of Lat Y191 and PLCγ2 Y759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-induced Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Since potentiation of Syk phosphorylation is not observed in murine platelets, PKC deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyper-phosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk kinase regulation by PKCβ in human platelets.
    Full-text · Article · Aug 2013 · Journal of Biological Chemistry
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    ABSTRACT: Recent work by the Encyclopedia of DNA Elements project showed that non-protein-coding RNAs account for an unexpectedly large proportion of the human genome. Among these non-coding RNAs are microRNAs (miRNAs), which are small RNA molecules that modulate protein expression by degrading mRNA or repressing mRNA translation. MiRNAs have been shown to play important roles in hematopoiesis including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. Additionally, disordered miRNA biogenesis and quantitative or qualitative alterations in miRNAs and their targets are associated with hematological pathologies. Platelets contain machinery to process pre-miRNAs into mature miRNAs, and specific platelet miRNA levels have been found to correlate with platelet reactivity. This review summarizes the current state of knowledge of miRNAs in megakaryocytes and platelets, and the exciting possibilities for future megakaryocyte–platelet transcriptome research.
    Full-text · Article · Jun 2013 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: 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/.
    Full-text · Article · Jan 2013 · BMC Genomics
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    ABSTRACT: Next generation sequencing of RNA (RNA-seq) is an emerging technology that has so far been used successfully to profile the transcriptomes of several cell types and cell states. For the platelet transcriptome, RNA-seq descriptions exist for only a few subjects. Additionally, there have been no studies of the same individual's transcriptome using two different technologies. As such, it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA platforms, and what the transcriptomes' relationship is with the platelet proteome. We generated RNA-seq profiles of the long RNA transcriptomes from the platelets of 10 healthy young males (5 white and 5 black). In addition to RNA-seq, we profiled the platelet messenger RNAs of the same 10 individuals using the Affymetrix GeneChip System. We observed that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, a finding that was independent of race and of the employed technology. Additionally, our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. However, there was a notable exception: the category of pseudogenes exhibited a clear difference in expression by race. Comparison of our mRNA signatures with the only publicly available quantitative platelet proteome data showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. Interestingly, there was also a high number of mRNAs that were present in the transcriptomes of all 10 individuals but had no representation in the proteome. Spearman correlation of the relative abundances for those platelet genes that were represented by both an mRNA and a protein, revealed an unexpectedly weak correlation between the transcriptome and the proteome. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified groups of genes with very distinct characteristics. Gene Ontology analysis of the respective gene identifiers revealed that the gene groups corresponded to distinct cellular processes, an interesting finding that provides novel insights for platelet biology. The very high inter-individual correlations of the transcriptome signatures across 10 different subjects representing two races together with the results of our analyses 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. DisclosuresNo relevant conflicts of interest to declare.
    Full-text · Article · Jan 2013 · Blood
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    ABSTRACT: The integrin family is comprised of a series of 24 αβ heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand binding-induced signals into cells. In human platelets, FcγRIIa has been identified as an ITAM-bearing transmembrane receptor responsible for mediating "outside-in" signaling through αIIbβ3 - the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and PLCγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for α(IIb)β(3)-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.
    Full-text · Article · Dec 2012 · Blood
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    Steven E McKenzie
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    ABSTRACT: In this issue of Blood,Kasthuri and colleagues have examined the role of Fcγ receptors and signaling molecules in monocytes in HIT.
    Preview · Article · May 2012 · Blood
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    ABSTRACT: Abstract 3298 The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. While the repertoire of platelet transcripts is a reflection of the megakaryocyte at the time of platelet differentiation, post-transcriptional events are known to occur. Furthermore, a strong correlation between the expressed mRNAs and proteome has been identified. Having a complete understanding of the platelet transcriptome is important for generating insights into the genetic basis of platelet disease traits. To capture the complexity of the platelet transcriptome, we performed RNA sequencing (RNA-seq) in leukocyte-depleted platelets from 10 males, with median age of 24.5 yrs and unremarkable medical history. Their short and long RNA platelet transcriptomes were analyzed on the SOLiD 5500xl sequencing platform. We generated [~]3.5 billion sequence reads [~]40% of which could be mapped uniquely to the human genome. Our analysis revealed that [~]9,000 distinct protein-coding mRNAs and [~]800 microRNAs (miRNAs) were present in the transcriptome of each of the 10 sequenced individuals. Comparison of the levels of mRNA expression across the 10 individuals showed an exceptional level of consistency with pair-wise Pearson correlation values [≥]0.98. The miRNA expression profiles across the 10 individuals showed a similar consistency with pair-wise Pearson correlation values [≥]0.98. Surprisingly, we found that these mRNAs and miRNAs accounted for a little over 1/2 of all of the uniquely mapped sequence reads suggesting the abundant presence of additional non-protein coding RNA (ncRNA) transcripts. Using the annotated entries of the latest release of the ENSEMBL database, we investigated the genetic make-up of these other transcripts. We found that [~]25% of each individual's uniquely mapped reads corresponded to non-protein coding transcripts from mRNA-coding loci. These reads accounted for more than 10,000 distinct such transcripts. In addition, each of the individuals in our cohort expressed an average of [~]1,500 pseudogenes and [~]200 long intergenic non-coding RNAs (lincRNAs). The short RNA profiles of the ten individuals revealed an abundance of diverse categories of ncRNAs including the signal recognition particle RNA (srpRNA), small nuclear RNA (snRNA) and small cytoplasmic RNAs (scRNA). These ncRNAs are involved in the processing of pre-mRNAs and their presence and prevalence in the anucleate platetet suggests the existence of a complex network of mRNA processing that persists after the megakaryocyte fragmentation. We also investigated the RNA-omes of the ten individuals for evidence of transcription of the pyknon category of ncRNAs. Pyknons are of particular interest because each has numerous intergenic and intronic copies whereas nearly all known human protein-coding genes contain one or more pyknons in their mRNA. Recent experimental work has shown that intergenic instances of the pyknons are transcribed in a tissue- and cell-state specific manner. An average of [~]100,000 pyknons are transcribed in each of the 10 sequenced individuals suggesting the possibility of a far-reaching network of interactions that link exonic space to distant non-exonic regions and are active in platelets. Lastly, we found that a large variety of distinct repeat element categories are expressed in the RNA-omes (both short and long) of these individuals. Among the most abundantly represented categories of repeat elements were DNA transposons, long terminal repeat (LTR) retrotransposons, and non-LTR retrotransposons such as long interspersed elements (LINEs) and short interspersed elements (SINEs). In summary, our RNA-seq analyses have revealed a spectrum of platelet transcripts that transcends protein-coding genes and miRNAs. Indeed, the transcripts that have their source in genomic features not previously discussed or analyzed in the platelet context represent a very significant portion of all platelet transcripts. This in turn suggests an unanticipated richness, and presumably commensurate complexity, for the pl telet transcriptome. While the role of these novel non-protein coding RNAs is currently unknown it is expected that at least some of them may be of functional significance which will in turn permit a better understanding of the molecular mechanisms that regulate platelet physiology and may contribute to processes beyond thrombosis and hemostasis. DisclosuresNo relevant conflicts of interest to declare.
    No preview · Article · Jan 2012 · Blood

Publication Stats

2k Citations
448.99 Total Impact Points

Institutions

  • 1998-2016
    • Thomas Jefferson University
      • • Department of Medicine
      • • Department of Pediatrics
      Filadelfia, Pennsylvania, United States
  • 1999-2005
    • Jefferson College
      Хиллсборо, Missouri, United States
  • 1997-1999
    • University of Pennsylvania
      • School of Engineering and Applied Science
      Filadelfia, Pennsylvania, United States
  • 1992-1997
    • The Children's Hospital of Philadelphia
      • • Division of Hematology
      • • Department of Pediatrics
      Filadelfia, Pennsylvania, United States