In the late 1960s, numerous investigators independently demonstrated that platelets are capable of synthesizing proteins. Studies continued at a steady pace over the next 30 years and into the 21st ...century. Collectively, these investigations confirmed that platelets synthesize proteins and that the pattern of protein synthesis changes in response to cellular activation. More recent studies have characterized the mechanisms by which platelets synthesize proteins and have shown that protein synthesis alters the phenotype and functions of platelets. Here, we chronologically review our increased understanding of protein synthetic responses in platelets and discuss how the field may evolve over the next decade.
Summary
Platelets are generated from nucleated precursors referred to as megakaryocytes. The formation of platelets is one of the most elegant and unique developmental processes in eukaryotes. ...Because they enter the circulation without nuclei, platelets are often considered simple, non‐complex cells that have limited functions beyond halting blood flow. However, emerging evidence over the past decade demonstrates that platelets are more sophisticated than previously considered. Platelets carry a rich repertoire of messenger RNAs (mRNAs), microRNAs (miRNAs), and proteins that contribute to primary (adhesion, aggregation, secretion) and alternative (immune regulation, RNA transfer, translation) functions. It is also becoming increasingly clear that the ‘genetic code’ of platelets changes with race, genetic disorders, or disease. Changes in the ‘genetic code’ can occur at multiple points including megakaryocyte development, platelet formation, or in circulating platelets. This review focuses on regulation of the ‘genetic code’ in megakaryocytes and platelets and its potential contribution to health and disease.
Summary
Background
Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self‐limiting febrile illness through to a ...life‐threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV‐induced platelet activation and thrombocytopenia are incompletely understood.
Patients and methods
Freshly isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alteration and activation of cell death pathways. In parallel, we examined direct DENV‐induced activation and apoptosis of platelets obtained from healthy subjects.
Results
We found that platelets from DENV‐infected patients exhibited increased activation by comparison to control subjects. Moreover, platelets from DENV‐infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase‐9 and ‐3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV‐infected patients. Platelet activation, mitochondrial dysfunction and caspase‐dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV‐induced platelet activation was shown to occur through mechanisms largely dependent on DC‐SIGN.
Conclusions
Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, which may contribute to the development of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC‐SIGN as a critical receptor in DENV‐dependent platelet activation.
Platelets have intricate signaling mechanisms and participate in a breadth of cellular interactions. This diversity is frequently unrecognized. In addition to being the chief cellular effectors of ...haemostasis, platelets are innate inflammatory cells that have previously unrecognized molecular pathways and synthetic capacities, which can link innate and adaptive responses in the immune continuum. Characterization of these features and parallel
in vivo observations identify new sentinel, surveillance and information-transfer functions. Recent observations indicate that platelets have key roles in adaptive responses to microbial and antigen challenge, in addition to their well known acute defensive activities in tissue injury, and suggest that these mechanisms can be dysregulated in disease. Ongoing characterization of the platelet transcriptome, secretome and proteome also suggest that additional functions of platelets relevant to innate and adaptive immunity remain to be discovered. This Review is the third in a series on
interactions between haemostasis and inflammation.
Platelet functions beyond hemostasis SMYTH, S. S.; MCEVER, R. P.; WEYRICH, A. S. ...
Journal of thrombosis and haemostasis,
November 2009, 2009-Nov, 2009-11-00, 20091101, Volume:
7, Issue:
11
Journal Article
Peer reviewed
Open access
Although their central role is in the prevention of bleeding, platelets probably contribute to diverse processes that extend beyond hemostasis and thrombosis. For example, platelets can recruit ...leukocytes and progenitor cells to sites of vascular injury and inflammation; they release proinflammatory and anti‐inflammatory and angiogenic factors and microparticles into the circulation; and they spur thrombin generation. Data from animal models suggest that these functions may contribute to atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury, and transplant rejection. This article represents an integrated summary of presentations given at the Fourth Annual Platelet Colloquium in January 2009. The process of and factors mediating platelet–platelet and platelet–leukocyte interactions in inflammatory and immune responses are discussed, with the roles of P‐selectin, chemokines and Src family kinases being highlighted. Also discussed are specific disorders characterized by local or systemic platelet activation, including coronary artery restenosis after percutaneous intervention, alloantibody‐mediated transplant rejection, wound healing, and heparin‐induced thrombocytopenia.
Summary
Essentials
Phosphoinositide 3‐kinase and MAPK pathways crosstalk via PDK1.
PDK1 is required for adenosine diphosphate‐induced platelet activation and thromboxane generation.
PDK1 regulates ...RAF proto‐oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway.
Genetic ablation of PDK1 protects against platelet‐dependent thrombosis in vivo.
Summary
Background
Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide‐dependent protein kinase 1 (PDK1) regulates protease‐activated receptor 4‐induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown.
Objective
To establish the mechanism of PDK1 in ADP‐induced platelet activation and thrombosis.
Methods
We assessed the role of PDK1 on 2MeSADP‐induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX‐795, which inhibits PDK1, or by using platelet‐specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model.
Results
PDK1 inhibition with BX‐795 reduced 2‐methylthio‐ADP (2MeSADP)‐induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1−/− mice. PDK1 was also necessary for the phosphorylation of mitogen‐activated protein kinase kinase 1/2 (MEK1/2), extracellular signal‐regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen‐activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf‐1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine‐induced pulmonary embolism.
Conclusion
PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.
Essentials
Platelets express retinoic acid receptor (RAR)α protein, specifically binding target mRNAs.
mRNAs under RARα control include MAP1LC3B2, SLAIN2, and ANGPT1.
All‐trans retinoic acid (atRA) ...releases RARα from its target mRNA.
RARα expressed in human platelets exerts translational control via direct mRNA binding.
Summary
Background
Translational control mechanisms in platelets are incompletely defined. Here, we determined whether the nuclear transcription factor RARα controls protein translational events in human platelets.
Methods
Isolated human platelets were treated with the pan‐RAR agonist all‐trans‐retinoic acid (atRA). Global and targeted translational events were examined.
Results
Stimulation of platelets with atRA significantly increased global protein expression. RARα protein bound to a subset of platelet mRNAs, as measured by next‐generation RNA‐sequencing. In‐depth analyses of 5′ and 3′‐untranslated regions of the RARα‐bound mRNAs revealed consensus RARα binding sites in microtubule‐associated protein 1 light chain 3 beta 2 (MAP1LC3B2), SLAIN motif‐containing protein 2 (SLAIN2) and angiopoietin‐1 (ANGPT1) transcripts. When platelets were treated with atRA, binding interactions between RARα protein and mRNA for MAP1LC3B2, SLAIN2 and ANGPT1 were significantly decreased. Consistent with the release of bound RARα protein from MAP1LCB2mRNA, we observed an increase in the synthesis of MAP1LC3B2 protein.
Conclusions
These findings provide the first evidence that RARα, a nuclear transcriptional factor, regulates synthetic events in anucleate human platelets. They also reveal an additional non‐genomic role for RARα in platelets that may have implications for the vitamin A‐dependent signaling in humans.
Background: Activated platelets have previously‐unrecognized mechanisms of post‐transcriptional gene expression that may influence hemostasis and inflammation. A novel pathway involves splicing of ...pre‐mRNAs in resting platelets to mature, translatable mRNAs in response to cellular activation. Objectives: We asked if bacterial products and host agonists present in the septic milieu induce tissue factor pre‐mRNA splicing in platelets from healthy subjects. In parallel, we asked if spliced tissue factor (TF) mRNA is present in platelets from septic patients in a proof‐of‐principle analysis. Patients/methods: TF pre‐mRNA and mRNA expression patterns were characterized in platelets from septic patients and in platelets isolated from healthy subjects activated with bacteria, toxins and inflammatory agonists. Procoagulant activity was also measured. Results and conclusions: Live bacteria, staphylococcal α‐toxin and lipopolysaccharide (LPS) induced TF pre‐mRNA splicing in platelets isolated from healthy subjects. Toxin‐stimulated platelets accelerated plasma clotting, a response that was blocked by a previously‐characterized splicing inhibitor and by an anti‐tissue factor antibody. Platelets from septic patients expressed spliced TF mRNA, whereas it was absent from unselected and age‐matched control subjects. Tissue factor‐dependent procoagulant activity was elevated in platelets from a subset of septic patients. Thus, bacterial and host factors induce splicing of TF pre‐mRNA, expression of TF mRNA and tissue factor‐dependent clotting activity in human platelets. TF mRNA is present in platelets from some septic patients, indicating that it may be a marker of altered platelet phenotype and function in sepsis and that splicing pathways are induced in this syndrome.
Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes ...pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1β pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.
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