Circulating Platelets as Mediators of Immunity, Inflammation, and Thrombosis.

Platelets, non-nucleated blood components first described over 130 years ago, are recognized as the primary cell regulating hemostasis and thrombosis. The vascular importance of platelets has been attributed to their essential role in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism. Increasing knowledge on the platelets' role in the vasculature has led to many advances in understanding not only how platelets interact with the vessel wall but also how they convey changes in the environment to other circulating cells. In addition to their well-described hemostatic function, platelets are active participants in the immune response to microbial organisms and foreign substances. Although incompletely understood, the immune role of platelets is a delicate balance between its pathogenic response and its regulation of thrombotic and hemostatic functions. Platelets mediate complex vascular homeostasis via specific receptors and granule release, RNA transfer, and mitochondrial secretion that subsequently regulates hemostasis and thrombosis, infection, and innate and adaptive immunity.

New paradigms in thrombosis: novel mediators and biomarkers platelet RNA transfer.

Platelets, anucleated cells with a central role in hemostasis and inflammation, contain messenger RNAs and microRNAs of unknown functionality and clinical relevance. Historically, platelet RNA was viewed as merely a remnant of platelet biogenesis; however, several studies now refute this assumption. Studies have shown that platelets can actively translate RNA to protein and that specific RNA profiles correlate with select human clinical phenotypes. These studies support a more fluid role for platelet RNA in platelet function and disease development. Our lab and others have recently studied the platelet's unique ability to transfer RNA to recipient cells and the effect this transfer has on the recipient cells' functions. This transfer may represent a previously unknown form of vascular cell communication and modulation. Unlike the well-characterized thrombotic properties of platelets, the nature and purpose of platelet RNA transfer has not been determined, partly due to limitations in techniques used to manipulate platelet RNA profiles. Defining the mechanism of RNA transfer and its role in the vascular system will allow for the better understanding of how platelets function in both their traditional thrombotic role and non-traditional functions, potentially having widespread implications in several fields.

Lipopolysaccharide-induced expression of TRAIL promotes dendritic cell differentiation.

Tumour necrosis factor-related apoptosis inducing ligand (TRAIL) is a death-inducing cytokine whose physiological function is not well understood. Here, we show that TRAIL has a role in programming human dendritic cell (DC) differentiation. TRAIL expression was strongly induced in DCs upon stimulation with lipopolysaccharide (LPS) or Polyinosine-polycytidylic acid (poly(I:C)) stimulation. Blockade of TRAIL with neutralizing antibody partially inhibited LPS-induced up-regulation of co-stimulatory molecules and the expression of inflammatory cytokines including interleukin-12 (IL-12) p70. In addition, neutralization of TRAIL in LPS-treated DCs inhibited the DC-driven differentiation of T cells into interferon-gamma (IFN-gamma) -producing effectors. The effects of TRAIL neutralization in poly(I:C)-treated DCs were similar, except that IL-12 production and the differentiation of effector T cells into IFN-gamma producers were not inhibited. Strikingly, TRAIL stimulation alone was sufficient to induce morphological changes resembling DC maturation, up-regulation of co-stimulatory molecules, and enhancement of DC-driven allogeneic T-cell proliferation. However, TRAIL alone did not induce inflammatory cytokine production. We further show that the effects of TRAIL on DC maturation were not the result of the induction of apoptosis, but may involve p38 activation. Hence, our data demonstrate that TRAIL co-operates with other cytokines to facilitate DC functional maturation in response to Toll-like receptor activation.

The role of platelets in mediating a response to human influenza infection.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.

Micro RNAs from DNA Viruses are Found Widely in Plasma in a Large Observational Human Population.

Viral infections associate with disease risk and select families of viruses encode miRNAs that control an efficient viral cycle. The association of viral miRNA expression with disease in a large human population has not been previously explored. We sequenced plasma RNA from 40 participants of the Framingham Heart Study (FHS, Offspring Cohort, Visit 8) and identified 3 viral miRNAs from 3 different human Herpesviridae. These miRNAs were mostly related to viral latency and have not been previously detected in human plasma. Viral miRNA expression was then screened in the plasma of 2763 participants of the remaining cohort utilizing high-throughput RT-qPCR. All 3 viral miRNAs associated with combinations of inflammatory or prothrombotic circulating biomarkers (sTNFRII, IL-6, sICAM1, OPG, P-selectin) but did not associate with hypertension, coronary heart disease or cancer. Using a large observational population, we demonstrate that the presence of select viral miRNAs in the human circulation associate with inflammatory biomarkers and possibly immune response, but fail to associate with overt disease. This study greatly extends smaller singular observations of viral miRNAs in the human circulation and suggests that select viral miRNAs, such as those for latency, may not impact disease manifestation.

The role of RNA uptake in platelet heterogeneity.

The role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer. The purpose of this study is to determine if platelet RNA variance is the result of horizontal cellular transfer between platelets and other vascular cells. Utilising platelet sorting and RNA sequencing, we found that smaller platelets contained a more diverse set of transcripts than larger platelets. Further investigation using fluorescence imaging, gene expression analyses and in vitro and in vivo modelling revealed that platelets take up RNA from other vascular cells in a complex manner, revealing a dynamic role for platelets in modulating vascular homeostasis through bidirectional RNA transfer. The resultant RNA profile heterogeneity suggests unique functional roles for platelets dependent on size and complexity. This study expands our basic understanding of platelet function and heterogeneity and is the first to evaluate endogenous vascular RNA uptake and its relation to platelet processes. Our findings describe a novel endogenous phenomenon that can help elucidate the platelet's role in these non-thrombotic and haemostatic fields, as well as present potential for diagnostic and therapeutic development.

Specific Inflammatory Stimuli Lead to Distinct Platelet Responses in Mice and Humans.

INTRODUCTION: Diverse and multi-factorial processes contribute to the progression of cardiovascular disease. These processes affect cells involved in the development of this disease in varying ways, ultimately leading to atherothrombosis. The goal of our study was to compare the differential effects of specific stimuli--two bacterial infections and a Western diet--on platelet responses in ApoE-/- mice, specifically examining inflammatory function and gene expression. Results from murine studies were verified using platelets from participants of the Framingham Heart Study (FHS; n = 1819 participants). METHODS: Blood and spleen samples were collected at weeks 1 and 9 from ApoE-/- mice infected with Porphyromonas gingivalis or Chlamydia pneumoniae and from mice fed a Western diet for 9 weeks. Transcripts based on data from a Western diet in ApoE-/- mice were measured in platelet samples from FHS using high throughput qRT-PCR. RESULTS: At week 1, both bacterial infections increased circulating platelet-neutrophil aggregates. At week 9, these cells individually localized to the spleen, while Western diet resulted in increased platelet-neutrophil aggregates in the spleen only. Microarray analysis of platelet RNA from infected or Western diet-fed mice at week 1 and 9 showed differential profiles. Genes, such as Serpina1a, Ttr, Fgg, Rpl21, and Alb, were uniquely affected by infection and diet. Results were reinforced in platelets obtained from participants of the FHS. CONCLUSION: Using both human studies and animal models, results demonstrate that variable sources of inflammatory stimuli have the ability to influence the platelet phenotype in distinct ways, indicative of the diverse function of platelets in thrombosis, hemostasis, and immunity.

Preligand assembly domain-mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential therapeutic value against cancers because of its selective cytotoxicity to many transformed, but not normal, cells. The "decoy receptors" TRAIL-R3 (TR3) and TRAIL-R4 (TR4) were believed to negatively regulate TRAIL-induced cytotoxicity by competing for ligand binding with TRAIL-R1 (TR1) and TRAIL-R2 (TR2). Here, we show that inhibition of TRAIL-induced apoptosis by TR4 critically depends on its association with TR2 via the NH(2)-terminal preligand assembly domain overlapping the first partial cysteine-rich domain of both receptors. By contrast, ligand binding by TR4 is dispensable for its apoptosis inhibitory function, thereby excluding the possibility that TR4 was a "decoy" to inhibit apoptosis by binding up TRAIL. In primary CD8(+) T cells, which express only TR2 and TR4 and are resistant to TRAIL-induced apoptosis, stimulation with phorbol myristate acetate abrogated the ligand-independent interaction between TR2 and TR4 and enhanced their sensitivity to TRAIL-induced apoptosis. Hence, whereas most TNF receptors normally form only homotrimeric complexes, the preligand assembly domains in TR2 and TR4 permit mixed complex formation as a means to regulate apoptosis induction. We propose that TR4 is a "regulatory" rather than "decoy" receptor that inhibits apoptosis signaling by TRAIL through this previously uncharacterized ligand-independent mechanism.