This service exclusively searches for literature that cites resources. Please be aware that the total number of searchable documents is limited to those containing RRIDs and does not include all open-access literature.
Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage.
Chikungunya fever is a viral disease transmitted by mosquitoes of the genus Aedes. The infection is usually symptomatic and most common symptoms are fever accompanied by joint pain and swelling. In most cases symptoms subside within a week. However, severe prolonged and disabling joint pain, that may persist for several months, even years, are reported. Although the pathogenesis of Chikungunya infection is not fully understood, the evolution to severe disease seems to be associated with the activation of immune mechanisms and the action of inflammatory mediators. Platelets are recognized as inflammatory cells with fundamental activities in the immune response, maintenance of vascular stability and pathogenicity of several inflammatory and infectious diseases. Although the involvement of platelets in the pathogenesis of viral diseases has gained attention in recent years, their activation in Chikungunya has not been explored. The aim of this study was to analyze platelet activation and the possible role of platelets in the amplification of the inflammatory response during Chikungunya infection. We prospectively included 132 patients attended at the Quinta D'Or hospital and 25 healthy volunteers during the 2016 epidemic in Rio de Janeiro, Brazil. We observed increased expression of CD62P on the surface of platelets, as well as increased plasma levels of CD62P and platelet-derived inflammatory mediators indicating that the Chikungunya infection leads to platelet activation. In addition, platelets from chikungunya patients exhibit increased expression of NLRP3, caspase 4, and cleaved IL-1β, suggestive of platelet-inflammasome engagement during chikungunya infection. In vitro experiments confirmed that the Chikungunya virus directly activates platelets. Moreover, we observed that platelet activation and soluble p-selectin at the onset of symptoms were associated with development of chronic forms of the disease. Collectively, our data suggest platelet involvement in the immune processes and inflammatory amplification triggered by the infection.
Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.
Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.
Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.
Dysregulation of platelet function is causally connected to thrombus formation and cardiovascular diseases. Therefore, assessing platelet reactivity is crucial. However, current platelet function tests come with pitfalls, limiting clinical use. Plasma miRNA signatures have been suggested as novel biomarkers for predicting/diagnosing cardiovascular diseases and monitoring antiplatelet therapy. Here, we provide results from a comprehensive study on the feasibility of using circulatory platelet miRNAs as surrogate markers of platelet activation. We performed small RNA-Seq on different blood cell types to confirm known and identify novel platelet-enriched miRNAs and validated a panel of 16 miRNAs using RT-qPCR. To identify the main carrier of these blood-based platelet miRNAs, we enriched and analyzed distinct microvesicle populations. Platelets were stimulated with GPVI and P2Y12 agonists in vitro to monitor the release of the selected miRNAs following activation. Finally, the miRNA panel was also measured in plasma from mice undergoing the Folts intervention (recurrent thrombus formation in the carotid artery). Applying an unbiased bioinformatics-supported workflow to our NGS data, we were able to confirm a panel of previously established miRNA biomarker candidates and identify three new candidates (i.e., miR-199a-3p, miR-151a-5p, and miR-148b-3p). Basal levels of platelet-derived miRNAs in plasma were mainly complexed with proteins, not extracellular vesicles. We show that changes in miRNA levels due to platelet activation are detectable using RT-qPCR. In addition, we highlight limitations of studying the in vitro release of miRNAs from platelets. In vivo thrombosis resulted in significant elevations of platelet-derived miRNA levels in mice. In conclusion, we provide in-depth evidence that activated platelets release miRNAs, resulting in measurable changes in circulatory miRNA levels, rendering them promising biomarker candidates.
Patients with glioblastoma multiforme (GBM) suffer from an increased incidence of vascular thrombotic events. However, key influencing factors of the primary hemostasis have not been characterized in GBM patients to date. Thus, the present study determines the activation level of circulating platelets in GBM patients, in-vitro reactivity to agonist-induced platelet stimulation and the formation of circulating platelet-leucocyte conjugates as well as the plasma levels of the proinflammatory lipid mediator sphingosine-1-phosphate (S1P). The endogenous thrombin potential (ETP) was determined as global marker for hemostasis. The 21 GBM patients and 21 gender and age matched healthy individuals enrolled in this study did not differ in mean total platelet count. Basal surface expression of platelet CD63 determined by flow cytometry was significantly increased in GBM patients compared to controls as was observed for the concentration of soluble P-selectin in the plasma of GBM patients. While the ETP was not affected, the immunomodulatory lipid S1P was significantly decreased in peripheral blood in GBM. Interestingly, monocyte expression of PSGL-1 (CD162) was decreased in GBM patient blood, possibly explaining the rather decreased formation of platelet-monocyte conjugates. Our study reveals an increased CD63 expression and P-selectin expression/ secretion of circulating platelets in GBM patients. In parallel a down-modulated PSGL-1 expression in circulating monocytes and a trend towards a decreased formation of heterotypic platelet-monocyte conjugates in GBM patients was seen. Whether this and the observed decreased plasma level of the immunomodulatory S1P reflects a systemic anti-inflammatory status needs to be addressed in future studies.
Platelets are anucleate blood cells with reported roles in hemostasis and immune responses, which possess a functional receptor for bacterial lipopolysaccharides (LPSs), the well-known inducers of inflammation. However, LPSs effects on platelets are contradictory. Here we aim to investigate mechanisms of platelet functioning in the presence of LPS and to find the cause of the discrepancy in the previously published data. Cell activity was analyzed by flow cytometry, western blotting, and aggregometry. Thrombus growth was assessed by fluorescent microscopy. LPS' activity was checked by their capability to induce PMN activation. However, LPSs did not substantially affect either thrombus growth in flow chambers, irreversible platelet aggregation, or platelet responses to strong activation. Platelet aggregation in response to 1 μM of ADP was significantly inhibited by LPSs. Flow cytometry analysis revealed that platelet activation responses to weak stimulation were also diminished by LPSs, while VASP phosphorylation was weakly increased. Additionally, LPSs were capable of inhibition of ADP-induced P2-receptor desensitization. Incubation of platelets with a pan-PDE inhibitor IBMX significantly enhanced the LPSs-induced platelet inhibition, implying cAMP/cGMP dependent mechanism. The discrepancy in the previously published data could be explained by LPS-induced weak inhibition of platelet activation and the prevention of platelet desensitization.
Plateletpheresis is generally a safe procedure for platelet donation. Studies on the effects of haemostatic parameters and possible association between automated plateletpheresis and hypercoagulable state are limited. Hence, this study aimed to investigate the effects of plateletpheresis on regular donors using haemostatic parameters, i.e. natural anticoagulant proteins, platelet indices, and platelet activation markers.
Blood platelet dysfunctions are strongly involved in the development of the micro- and macrovascular complications in diabetes mellitus (DM). However, the molecular causes of abnormal platelet activation in DM remain unclear. Experimental data suggests that platelet mitochondria can regulate the prothrombotic phenotype of platelets, and changes in these organelles may influence platelet activation and modify platelet responses to stimulation. The present study evaluates the impact of DM on mitochondrial respiratory parameters and blood platelet activation/reactivity in a rat model of experimental diabetes following 1, 2.5 and 5 months of streptozotocin (STZ)-induced diabetes. Moreover, a mild inhibition of the mitochondrial respiratory chain with the use of metformin under in vitro and in vivo conditions was tested as a method to reduce platelet activation and reactivity. The platelets were studied with a combination of flow cytometry and advanced respirometry. Our results indicate that prolonged exposure of blood platelets to high concentrations of glucose, as in diabetes, can result in elevated blood platelet mitochondrial respiration; this may be an effect of cell adaptation to the high availability of energy substrates. However, as these alterations occur later than the changes in platelet activation/reactivity, they may not constitute the major reason for abnormal platelet functioning in DM. Moreover, metformin was not able to inhibit platelet activation and reactivity under in vitro conditions despite causing a decrease in mitochondrial respiration. This indicates that the beneficial effect of metformin on the coagulation system observed in vivo can be related to other mechanisms than via the inhibition of platelet activation.
HNG, a highly potent mutant of the anti-Alzheimer peptide-humanin, has been shown to protect against ischaemia-reperfusion (I/R) injury. However, the underlying mechanism related to platelet activation remains unknown. We proposed that HNG has an effect on platelet function and thrombus formation. In this study, platelet aggregation, granule secretion, clot retraction, integrin activation and adhesion under flow conditions were evaluated. In mice receiving HNG or saline, cremaster arterial thrombus formation induced by laser injury, tail bleeding time and blood loss were recorded. Platelet microtubule depolymerization was evaluated using immunofluorescence staining. Results showed that HNG inhibited platelet aggregation, P-selectin expression, ATP release, and αIIb β3 activation and adhesion under flow conditions. Mice receiving HNG had attenuated cremaster arterial thrombus formation, although the bleeding time was not prolonged. Moreover, HNG significantly inhibited microtubule depolymerization, enhanced tubulin acetylation in platelets stimulated by fibrinogen or microtubule depolymerization reagent, nocodazole, and inhibited AKT and ERK phosphorylation downstream of HDAC6 by collagen stimulation. Therefore, our results identified a novel role of HNG in platelet function and thrombus formation potentially through stabilizing platelet microtubules via tubulin acetylation. These findings suggest a potential benefit of HNG in the management of cardiovascular diseases.
CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40, leading to many effects depending on target cell type. Platelets express CD40L and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanisms. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling. Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated platelet activation. Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin αIIbβ3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression platelet receptors. Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.
The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbβ3 integrin clustering following thrombin stimulation. This αIIbβ3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets.
In resting platelets, the integrin αIIbβ3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation.
The complex process of platelet formation originates with the hematopoietic stem cell, which differentiates through the myeloid lineage, matures, and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRα+ perivascular cells, derived from human umbilical cord tissue, which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression, yielding platelets that are functional with low basal activation levels, a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo.
Inflammation and thrombosis are two responses that are linked through a number of mechanisms, one of them being the complement system. Various proteins of the complement system interact specifically with platelets, which, in turn, activates them and promotes thrombosis. In this paper, we show that the converse is also true: activated platelets can activate the complement system. As assessed by flow cytometry and immunoblotting, C3 deposition increased on the platelet surface upon cell activation with different agonists. Activation of the complement system proceeded to its final stages, which was marked by the increased generation of the anaphylotoxin C3a and the C5b-9 complex. We identified P-selectin as a C3b-binding protein, and confirmed by surface plasmon resonance binding that these two proteins interact specifically with a dissociation constant of 1 microM. Using heterologous cells expressing P-selectin, we found that P-selectin alone is sufficient to activate the complement system, marked by increases in C3b deposition, C3a generation, and C5b-9 formation. In summary, we have found that platelets are capable of activating the complement system, and have identified P-selectin as a receptor for C3b capable of initiating complement activation. These findings point out an additional mechanism by which inflammation may localize to sites of vascular injury and thrombosis.
There is evidence for the effects of platelet inhibition on innate immune activation. Circulating microRNAs (miRNAs) have been implicated as markers of platelet and leukocyte activation. In the present study, we assessed the effects of P2Y12 inhibitors on platelet and leukocyte miRNAs during endotoxemia. Healthy volunteers were randomly assigned to receive oral ticagrelor (n = 10), clopidogrel (n = 8) or no drug (n = 8) for one week, followed by an intravenous bolus of 2 ng/kg endotoxin. Serum was collected at baseline, after one week of antiplatelet treatment and 6 and 24 h after endotoxin administration. MiRNAs were screened using LNA-based qPCR, followed by TaqMan-qPCR validation of candidates. Clinical validation was performed in 41 sepsis patients. Platelet-enriched miR-197, miR-223 and miR-223* were decreased in volunteers following antiplatelet therapy. Endotoxin increased platelet miRNAs, whilst the opposite effect was seen for leukocyte-enriched miR-150. Neither of these endotoxin-mediated effects were altered by P2Y12 inhibitors. Sepsis patients with fatal outcomes (n = 12) had reduced miR-150 levels compared with survivors (n = 29). In conclusion, we show that miR-150 is downregulated in experimental endotoxemia and can predict survival in sepsis but is unaffected by P2Y12 inhibition. While P2Y12 inhibition reduces platelet-associated miRNAs in healthy volunteers, it fails to attenuate the response of platelet miRNAs to endotoxemia.
Welcome to the FDI Lab - SciCrunch.org Resources search. From here you can search through a compilation of resources used by FDI Lab - SciCrunch.org and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that FDI Lab - SciCrunch.org has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on FDI Lab - SciCrunch.org then you can log in from here to get additional features in FDI Lab - SciCrunch.org such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into FDI Lab - SciCrunch.org you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the facets that you can filter your papers by.
From here we'll present any options for the literature, such as exporting your current results.
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
Year:
Count: