ALBERT

Description: 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 immunoprecipitation 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 unreported 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.

Description: There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood–brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Description: Abstract 153 Outside-in signal transduction is one of several amplification pathways that platelets employ following their adhesion to components of the extracellular matrix or to multivalent ligands like fibrinogen and von Willebrand factor. Previous studies have shown that outside-in signal transduction initiated by the binding of fibrinogen to its receptor, the integrin αIIbβ3, results in the activation of β3-associated Src family kinases (J Cell Biol 157:265, 2002; J Cell Sci 121:1641, 2008) that phosphorylate key tyrosine residues within the cytoplasmic domain of the transmembrane immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, Fcγ;RIIa (Blood 112:2780, 2008). “Activation” of FcγRIIa sets off a cascade of events that facilitate assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2. PLCγ2, through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. Curiously, mice lack the gene encoding FcγRIIa, and murine platelets are notoriously poor at sending αIIbβ3-mediated outside-in signals into the cell. The purpose of the current investigation, therefore, was to determine whether enforced expression of FcγRIIa in mouse platelets might serve to amplify murine platelet activation downstream of ligand binding to αIIbβ3. We found that the spreading of platelets derived from FcγRIIa transgenic mice was more robust and extensive compared to that of their wild-type counterparts. ADP- and collagen related peptide-induced granule secretion, as measured by P-selectin exposure, was also enhanced, as was the degree of integrin of αIIbβ3 activation, as reported by the binding of the activation-dependent monoclonal antibody, JON/A. Taken together, these data provide further support for importance of FcγRIIa in transmitting αIIbβ3-mediated amplification signals into the cell, and help to explain the often-observed defect in αIIbβ3-mediated signal transduction in murine platelets. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy.

Description: 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 platelet 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. Disclosures: No relevant conflicts of interest to declare.

Description: Platelet FcγRIIa is an important component of heparin-induced thrombocytopenia and other immune-mediated thrombocytopenia and thrombosis syndromes. Platelet FcγRIIa, an ITAM receptor, signals not only to cause aggregation and secretion, but also to PS exposure in the platelet procoagulant response when platelets are co-stimulated via the GCPR PARs. The molecular mechanisms downstream of FcγRIIa that lead to PS exposure are incompletely understood. Recently, we (WB; Ahmad et al., JTH 2011; 9:2077) demonstrated that downstream of PAR and GPVI/FcRγ, another ITAM receptor, there are CalDAG-GEF I (CDGI)-dependent and CDGI-independent, ADP/P2Y12-dependent parallel signaling pathways to PS exposure. In this study, we investigated the molecular signaling requirements for PS exposure downstream of FcγRIIa + PAR dual stimulation. We studied the exposure of PS in FcγRIIa transgenic (tg) mouse platelets following dual stimulation through FcγRIIa via anti-mouse CD9 and through PAR4 via PAR4 activating peptide (PAR4AP; AYPGKF). Washed platelets from FcγRIIa-tg mice and FcγRIIa-tg/CDG1-/- mice were stimulated with varying concentrations of anti-mouse CD9 and PAR4-AP (200uM) under static conditions and immediately measured for PS exposure by labeled Annexin-V in flow cytometry. We observed that at 0.5ug/ml of anti-mouse CD9 that the PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 70% of the FcγRIIa-tg platelets. However, when the platelets are pre-incubated with P2Y12 inhibitor MesAMP at 100uM, the PS exposure of the FcγRIIa-tg platelets is decreased by approximately 50% (n=3). For the FcγRIIa-tg/CDG1-/- platelets in the presence of MesAMP, PS exposure is completely abolished (n=3). This indicates that CDG1 contributes part of the signal that leads to PS exposure, while ADP/P2Y12 contributes the other CDGI-independent part of PS exposure downstream of FcγRIIa and GPCR dual stimulation. At a lower concentration of anti-mouse CD9 (0.25ug/ml; near threshold), the level of PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 80% of the FcgRIIa-tg platelets. In addition, at 0.25 ug/ml of anti-CD9, FcγRIIa-tg/CDG1-/- platelets pre-incubated with the P2Y12 inhibitor revealed no stimulated PS exposure. This observation indicates that ADP/P2Y12 plays a more significant role in PS exposure as the concentration of FcγRIIa stimulant nears threshold. Eradication of procoagulant PS exposure may require targeting of both the CDGI-dependent and CDGI-independent pathways for optimum therapeutic benefit in HIT and other immune-mediated thrombocytopenia and thrombosis disorders. CDGI inhibitors useful in human platelets will allow translation of these findings. Disclosures: No relevant conflicts of interest to declare.

Description: FcγRIIa is widely expressed on hematopoietic cells. There are two known allelic polymorphic forms of FcγRIIa, FcγRIIa-R131 and FcγRIIa-H131, which differ in the amino acid at position 131 in the second Ig-like domain. In contrast to FcγRIIa-R131, FcγRIIa-H131binds hIgG2 but not mIgG1, and this differential binding has clinical implications for host defense, autoimmune disease, immunohematologic disease, and response to therapeutic monoclonal antibodies. We identified a novel FcγRIIA genotype in a healthy individual homozygous for FcγRIIA R/R131 in whom a C to A substitution at codon 127 changes glutamine (Q) to lysine (K) in one of the two FcγRIIA genes. This individual's homozygosity for FcγRIIA-R/R131 leads to the prediction that the receptors on her cells would not bind hIgG2. Monocyte and neutrophil phagocytosis of hIgG2-opsonized erythrocytes was significantly higher (P 〈 .05) for cells from this K/Q127, R/R131 individual than for Q/Q127, R/R131 donors. Platelet aggregation stimulated by an mIgG1 anti-CD9 antibody in this individual was significantly different (P 〈 .05) from Q/Q127, H/R131 and Q/Q127, H/H131 donors and similar to Q/Q127, R/R131. Our data show that the K127/R131 receptors have a unique phenotype, binding both hIgG2 and mIgG1. Further functionally significant mutations in human Fcγ receptors and possible novel mechanisms for inherited differences in disease susceptibility should be sought with unbiased screening methods.

Description: Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.