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/.

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 stemloop 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. SignificanceMicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology.

Heparin-induced thrombocytopenia and thrombosis (HITT) is a severe complication of heparin therapy caused by antibodies to complexes between unfractionated heparin (UFH) and platelet factor 4 (PF4) that form over a narrow molar range of reactants and initiate antibody-induced platelet activation. We observed that UFH and tetrameric PF4 formed ultralarge (> 670 kDa) complexes (ULCs) only over a narrow molar range with an optimal ratio of PF4 to heparin of approximately 1:1. These ULCs were stable and visible by electron microscopy, but they could be dissociated into smaller complexes upon addition of heparin. ULCs formed inefficiently when PF4 was incubated with low-molecular-weight heparin, and none formed with the pentasaccharide fondaparinux sodium. In addition, mutation studies showed that formation of ULCs depended on the presence of PF4 tetramers. The ULCs were more reactive as determined by their capacity to bind to a HITTlike monoclonal antibody and showed greater capacity to promote platelet activation in an antibody-and Fc␥RIIA-dependent manner than were the smaller complexes. The capacity of PF4 to form ULCs composed of multiple PF4 tetramers arrayed in a lattice with several molecules of UFH may play a fundamental role in autoantibody formation, antibody-dependent platelet activation, and the propensity for thrombosis in patients with HITT.

Heparin-induced thrombocytopenia/thrombosis (HIT/HITT) is a severe, life-threatening complication that occurs in 1% to 3% of patients exposed to heparin. Interactions between heparin, human platelet factor 4 (hPF4), antibodies to the hPF4/heparin complex, and the platelet Fc receptor (FcR) for immunoglobulin G, Fc␥RIIA, are the proposed primary determinants of the disease on the basis of in vitro studies. The goal of this study was to create a mouse model that recapitulates the disease process in humans in order to understand the factors that predispose some patients to develop thrombocytopenia and thrombosis and to investigate new therapeutic approaches. Mice that express both human platelet Fc␥RIIA and hPF4 were generated. The Fc␥RIIA/hPF4 mice and controls, transgenic for either Fc␥RIIA or hPF4, were injected with KKO, a mouse monoclonal antibody specific for hPF4/heparin complexes, and then received heparin (20 U/d). Nadir platelet counts for KKO/heparin-treated Fc␥RIIA/hPF4 mice were 80% below baseline values, significantly different (P < .001) from similarly treated controls. Fc␥RIIA/hPF4 mice injected with KKO IntroductionHeparin is one of the most widely used anticoagulants during invasive vascular procedures and to treat thromboembolic diseases. Among patients who receive therapeutic courses of heparin, 1% to 3% will develop an antibody-mediated thrombocytopenia, 1-3 and 30% to 70% of these patients will develop potentially lifethreatening thrombosis. There is abundant evidence that more than 95% of patients with heparin-induced thrombocytopenia (HIT) alone and those with thrombocytopenia and thrombosis (HITT) develop antibodies that recognize complexes between platelet factor 4 (PF4) and heparin. 4-7 PF4, a major component of platelet ␣-granules, is released when platelets are activated. 8,9 It is currently believed that complexes between PF4 and heparin form on the surface of activated platelets, where they are positioned to be recognized by anti-PF4/heparin antibodies. [10][11][12] Antibodies to the PF4/heparin complex have been shown to activate human platelets in vitro via the platelet Fc receptor (FcR) for immunoglobuin (Ig)-G, Fc␥RIIA. [13][14][15][16] The binding of HIT antibodies to activated platelets probably promotes microparticle release as well as platelet-platelet and platelet-vessel wall interactions, predisposing to thrombosis. 15,17 However, the mechanism by which HIT antibodies activate platelets and promote thrombosis is uncertain. It has been proposed that HIT antibodies bind to cell-surface-associated PF4/heparin complexes via the Fab end of the molecule, providing an opportunity to transduce platelet-activating signals through the interaction between the Fc portion of the bound IgG and Fc␥RIIA. 12,13 Fc␥RIIA, the sole Fc␥ receptor expressed on platelets, 18 has been shown to transduce signals without the requirement for another transmembrane partner. 19 A monoclonal antibody to Fc␥RIIA blocks platelet aggregation and secretion induced by HIT antibodies. 12,13 Although increased...

• Unique dataset of human platelet mRNA, miRNA, and physiology reveals mRNAs and miRNAs that differ by age and gender.• Interactive public web tool (www.plateletomics.com) provides biologic insights into platelet function and gene expression.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. (Blood. 2014;123(16):e37-e45)

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.

In our population of VLBW infants, sepsis is frequently associated with thrombocytopenia and an elevation in MPV. However, fungal and Gram-negative pathogens are associated with a lower platelet count and more prolonged thrombocytopenia compared with Gram-positive pathogens. We conclude that common pathogens causing sepsis have different effects on platelet kinetics.

• The procoagulant nature of HIT can be simulated in a microfluidic model using human blood and its components.• PF4/glycosaminoglycans/ immunoglobulin G complexes activate monocytes through FcgRIIA to generate TF and thrombin, leading to coated platelets in HIT.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 fragment crystallizable (Fc)gRIIA 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 FcgRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcgRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V 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 FcgRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk. (Blood. 2016;127(4):464-472) IntroductionHeparin-induced thrombocytopenia (HIT) is an iatrogenic, immunemediated disorder characterized by antibodies that recognize complexes between the platelet chemokine platelet factor 4 (PF4, CXCL4) and heparin or cell surface glycosaminoglycans (GAGs). 1,2 It is estimated that up to 50% of patients with HIT develop thrombosis that might be limb-and/or life-threatening. [3][4][5] Even with early recognition, cessation of heparin, and institution of alternative forms of anticoagulation, recurrent thromboembolic complications may occur and 10% to 20% of patients go on to amputation and/or death. 6 Thus, there is a need for a better understanding of the pathogenesis of HIT and to determine how this information can be used to mitigate the risk of thrombosis.Thrombocytopenia and thrombosis in HIT have been attributed to binding of PF4/heparin/immunoglobulin G (IgG) immune-complexes to the platelets through the IgG fragment crystallizable (Fc) region, which activates platelets through their immunoreceptor tyrosine-based activation motif (ITAM) receptor, FcgRIIA. 7,8 However, monocytes, endothelial cells, and other cell types might also be activated by these immune complexes and contribute to the underlying pathology, 9 but their contribution to the process is less well characterized. Indeed, recent evidence suggests that thrombosis in HIT is initiated by binding of pathogenic antibodies to antigenic complexes of PF4 and GAGs expressed by the endothelium as well as circulating cells, includi...