RESUMO
BACKGROUND: The small noncoding RNAs (sncRNAs) in megakaryocytes (MKs) and platelets are not well characterized. Neither is the impact of SARS-CoV-2 infection on the sncRNAs of platelets. OBJECTIVES: To investigate the sorting of MK sncRNAs into platelets, and the differences in the platelet sncRNAomes of healthy donors (HDs) and COVID-19 patients. METHODS: We comprehensively profiled sncRNAs from MKs cultured from cord blood-derived CD34+ cells, platelets from HDs, and platelets from patients with moderate and severe SARS-CoV-2 infection. We also comprehensively profiled Argonaute (AGO)-bound sncRNAs from the cultured MKs. RESULTS: We characterized the sncRNAs in MKs and platelets and can account for â¼95% of all sequenced reads. We found that MKs primarily comprise microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), rRNA-derived fragments (rRFs), and Y RNA-derived fragments (yRFs) in comparable abundances. The platelets of HDs showed a skewed distribution by comparison: 56.7% of all sncRNAs are yRFs, 34.4% are isomiRs, and <2.0% are tRFs and rRFs. Most isomiRs in MKs and platelets are either noncanonical, nontemplated, or both. When comparing MKs and platelets from HDs, we found numerous isomiRs, tRFs, rRFs, and yRFs showing opposite enrichments or depletions, including molecules from the same parental miRNA arm, tRNA, rRNA, or Y RNA. The sncRNAome of platelets from patients with COVID-19 is skewed compared to that of HDs with only 19.8% of all sncRNAs now being yRFs, isomiRs increasing to 63.6%, and tRFs and rRFs more than tripling their presence to 6.1%. CONCLUSION: The sncRNAomes of MKs and platelets are very rich and more complex than it has been believed. The evidence suggests complex mechanisms that sort MK sncRNAs into platelets. SARS-CoV-2 infection acutely alters the contents of platelets by changing the relative proportions of their sncRNAs.
Assuntos
COVID-19 , MicroRNAs , Pequeno RNA não Traduzido , Humanos , Megacariócitos , SARS-CoV-2/genética , Plaquetas , MicroRNAs/genética , RNA de Transferência/genéticaRESUMO
Protease-activated receptor 4 (PAR4) (gene F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not known whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice in which F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared with hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7,620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of nonstandard antiplatelet therapies for patients expressing PAR4 Thr120.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Animais , Camundongos , Receptores de Trombina/genética , Agregação Plaquetária/genética , Plaquetas/fisiologia , Inibidores da Agregação Plaquetária/farmacologia , Acidente Vascular Cerebral/genética , Receptor PAR-1RESUMO
Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.
Assuntos
Endocitose , Fibrinogênio , Proteínas de Membrana , Sepse , Animais , Camundongos , Clatrina , Fibrinogênio/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sepse/genéticaRESUMO
The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbß3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.
Assuntos
RNA Mensageiro , Humanos , Animais , CamundongosRESUMO
Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5'-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.
Assuntos
Plaquetas , Megacariócitos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fatores de Troca do Nucleotídeo Guanina , Humanos , Fenótipo , TrombopoeseRESUMO
BACKGROUND: Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. OBJECTIVES: As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. PATIENTS/METHODS: Expression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex-matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. RESULTS: The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. CONCLUSIONS: During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.
Assuntos
Plaquetas/enzimologia , Glucuronidase/metabolismo , Sepse , Células Endoteliais , Glucuronidase/genética , Humanos , ProteômicaRESUMO
Patients with heart failure (HF) and left ventricular assist devices (LVAD) have dysregulated thrombo-inflammatory responses, mediated in part by platelets. While studies of platelet activation have been undertaken in HF, changes in the platelet transcriptome in HF patients following mechanical unloading with an LVAD have not been investigated. We prospectively enrolled and longitudinally followed advanced HF patients (n = 32) for a mean of 57 months post-LVAD implantation. For comparison, healthy donors were also enrolled (n = 20). Platelets were hyperactive in HF, as evidenced by significantly increased formation of circulating platelet-monocyte aggregate formation. Platelet transcriptome interrogation by next-generation RNA-sequencing identified that the expression of numerous genes (n = 588) was significantly (FDR < 0.05) altered in HF patients prior to LVAD implantation. Differentially expressed genes were predicted to have roles in angiogenesis, immune and inflammatory responses, apoptosis, and cardiac muscle contraction. 90 days following LVAD implantation, the majority (80%) of differentially expressed genes in HF patients normalized, as compared to the platelet transcriptomes of healthy donors. In conclusion, advanced HF is associated with marked alterations in the platelet transcriptome. While LVAD implantation to off load the failing heart results in resolution in the majority of differentially expressed genes, a subset of the platelet transcriptome remains persistently altered.
Assuntos
Plaquetas/metabolismo , Insuficiência Cardíaca/sangue , Adolescente , Adulto , Idoso , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Transcriptoma , Adulto JovemRESUMO
RATIONALE: Longitudinal studies are required to distinguish within versus between-individual variation and repeatability of gene expression. They are uniquely positioned to decipher genetic signal from environmental noise, with potential application to gene variant and expression studies. However, longitudinal analyses of gene expression in healthy individuals-especially with regards to alternative splicing-are lacking for most primary cell types, including platelets. OBJECTIVE: To assess repeatability of gene expression and splicing in platelets and use repeatability to identify novel platelet expression quantitative trait loci (QTLs) and splice QTLs. METHODS AND RESULTS: We sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examined within and between individual variation and repeatability of platelet RNA expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression is generally stable between and within-individuals over time-with the exception of a subset of genes enriched for the inflammation gene ontology. We show an enrichment among repeatable genes for associations with heritable traits, including known and novel platelet expression QTLs. Several exon skipping events were also highly repeatable, suggesting heritable patterns of splicing in platelets. One of the most repeatable was exon 14 skipping of SELP. Accordingly, we identify rs6128 as a platelet splice QTL and define an rs6128-dependent association between SELP exon 14 skipping and race. In vitro experiments demonstrate that this single nucleotide variant directly affects exon 14 skipping and changes the ratio of transmembrane versus soluble P-selectin protein production. CONCLUSIONS: We conclude that the platelet transcriptome is generally stable over 4 years. We demonstrate the use of repeatability of gene expression and splicing to identify novel platelet expression QTLs and splice QTLs. rs6128 is a platelet splice QTL that alters SELP exon 14 skipping and soluble versus transmembrane P-selectin protein production.
Assuntos
Processamento Alternativo , Plaquetas/metabolismo , Selectina-P/genética , Locos de Características Quantitativas/genética , RNA-Seq/métodos , Transcriptoma/genética , Éxons/genética , Ontologia Genética , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.
Assuntos
Envelhecimento , Plaquetas/imunologia , Inflamação/imunologia , Mitocôndrias/imunologia , Trombose/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Plaquetas/patologia , Inflamação/patologia , Megacariócitos/imunologia , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Ativação Plaquetária , Trombose/patologiaRESUMO
PURPOSE OF REVIEW: Platelets are specialized effector cells that rapidly respond to sites of vascular injury. However, emerging data demonstrate that platelets possess diverse functions that also mediate inflammatory responses and neurological diseases. These functions are relevant to disease processes prevalent among older adults and likely influence susceptibility to thrombotic and inflammatory disorders. RECENT FINDINGS: Platelet counts decrease in aged individuals whereas platelet reactivity increases. The platelet transcriptome is altered in aged individuals resulting in altered platelet function and exaggerated inflammation. Platelet signaling to monocytes in aging results in significantly more cytokines because of increased platelet-derived granzyme A. Platelet activation in aging appears to be driven, in part, because of increased reactive oxygen species and activation of the mammalian target of rapamycin pathway. Increased platelet hyperactivity in diseases is associated with aging, such cardiovascular disease and sepsis, exaggerate inflammation and thrombosis. Noncanonical functions of platelets influence the development of neurological diseases including Alzheimer's disease. SUMMARY: Although there have been advances dissecting the molecular mechanisms regarding aging-related changes in platelets, many knowledge gaps still remain. Studies filling these gaps are likely to identify new mechanisms driving aging-related changes in platelet gene expression and function, and contributing to injurious thrombo-inflammation in older adults.
Assuntos
Envelhecimento/metabolismo , Plaquetas/metabolismo , Envelhecimento/patologia , Plaquetas/patologia , HumanosRESUMO
Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.
Assuntos
Imunidade Inata/imunologia , Megacariócitos/imunologia , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/imunologia , Antivirais/imunologia , Dengue/imunologia , Vacinas contra Dengue/imunologia , HumanosRESUMO
BACKGROUND: Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. OBJECTIVES: The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. METHODS: The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. RESULTS: Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. CONCLUSIONS: The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103).
Assuntos
Plaquetas/fisiologia , Calgranulina B/imunologia , Monócitos/fisiologia , Doença Arterial Periférica , Adulto , Reprogramação Celular/imunologia , Feminino , Hemostasia , Humanos , Macrófagos/fisiologia , Masculino , Pessoa de Meia-Idade , Selectina-P/imunologia , Doença Arterial Periférica/sangue , Doença Arterial Periférica/fisiopatologia , Ativação Plaquetária/imunologiaRESUMO
Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.
Assuntos
Aterosclerose/imunologia , Calgranulina A/fisiologia , Calgranulina B/fisiologia , Diabetes Mellitus Experimental/imunologia , Neutrófilos/metabolismo , Trombocitose/imunologia , Animais , Aterosclerose/metabolismo , Plaquetas/fisiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Trombocitose/metabolismoRESUMO
OBJECTIVE: Low to moderate inorganic arsenic (iAs) exposure is independently associated with cardiovascular disease (CVD), particularly for patients with diabetes mellitus (DM). The mechanism of increased CVD risk from iAs exposure in DM has not been adequately characterized. We evaluated whether increasing concentrations of glucose enhance the effects of iAs on platelet and megakaryocyte activity, key steps in atherothrombosis. METHODS: Healthy donor whole blood was prepared in a standard fashion and incubated with sodium arsenite in a range from 0 to 10 µM. iAs-induced platelet activation was assessed by platelet receptor CD62P (P-selectin) expression and monocyte-platelet and leukocyte-platelet aggregation (MPA and LPA, respectively) in the presence of increasing sodium arsenite and glucose concentrations. Megakaryocyte (Meg-01) cell adhesion and gene expression was assessed after incubation with or without iAs and increasing concentrations of D-glucose. RESULTS: Platelet activity markers increased significantly with 10 vs. 0 µM iAs (P < 0.05 for all) and with higher D-glucose concentrations. Platelet activity increased significantly following co incubation of 1 and 5 µM iAs concentrations with hyperglycemic D-glucose (P < 0.01 for both) but not after incubation with euglycemic D-glucose. Megakaryocyte adhesion was more pronounced after co incubation with iAs and hyperglycemic than euglycemic D-glucose, while gene expression increased significantly to iAs only after co incubation with hyperglycemic D-glucose. CONCLUSION: We demonstrate that glucose concentrations common in DM potentiate the effect of inorganic arsenic exposure on markers of platelet and megakaryocyte activity. Our results support recent observational cohort data that DM enhances the vasculotoxic effects of arsenic exposure, and suggest that activation of the platelet-megakaryocyte hemostatic axis is a pathway through which inorganic arsenic confers atherothrombotic risk, particularly for patients with DM.
Assuntos
Arsênio/farmacologia , Plaquetas/metabolismo , Hiperglicemia/patologia , Megacariócitos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Adesão Celular/efeitos dos fármacos , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Selectina-P/metabolismo , Trombina/farmacologiaRESUMO
Platelet activity plays a major role in hemostasis with increased platelet activity likely contributing to the pathogenesis of atherothrombosis. We sought to identify associations between platelet activity variability and platelet-related genes in healthy controls. Transcriptional profiling of platelets revealed that WD-40 repeat domain 1 (WDR1), an enhancer of actin-depolymerizing factor activity, is downregulated in platelet messenger RNA (mRNA) from subjects with a hyperreactive platelet phenotype. We used the human megakaryoblastic cell line MEG-01 as an in vitro model for human megakaryocytes and platelets. Stimulation of MEG-01 with thrombin reduced levels of WDR1 transcripts and protein. WDR1 knockdown (KD) in MEG-01 cells increased adhesion and spreading in both the basal and activated states, increased F-actin content, and increased the basal intracellular calcium concentration. Platelet-like particles (PLPs) produced by WDR1 KD cells were fewer in number but larger than PLPs produced from unmodified MEG-01 cells, and had significantly increased adhesion in the basal state and upon thrombin activation. In contrast, WDR1 overexpression reversed the WDR1 KD phenotype of megakaryocytes and PLPs. To translate the clinical significance of these findings, WDR1 expression was measured in platelet RNA from subjects with established cardiovascular disease (n = 27) and age- and sex-matched controls (n = 10). The WDR1 mRNA and protein level was significantly lower in subjects with cardiovascular disease. These data suggest that WDR1 plays an important role in suppressing platelet activity, where it alters the actin cytoskeleton dynamics, and downregulation of WDR1 may contribute to the platelet-mediated pathogenesis of cardiovascular disease.
Assuntos
Aterosclerose/metabolismo , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/biossíntese , Adesividade Plaquetária , Adulto , Aterosclerose/genética , Aterosclerose/patologia , Plaquetas/patologia , Linhagem Celular , Citoesqueleto/genética , Citoesqueleto/patologia , Feminino , Humanos , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Proteínas dos Microfilamentos/genéticaRESUMO
Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos B/imunologia , Quimiotaxia de Leucócito/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Adesão Celular/imunologia , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismo , Proteínas rap1 de Ligação ao GTP/imunologia , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.
Assuntos
Apolipoproteína B-100/metabolismo , Insulina/metabolismo , Animais , Autofagia , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Insulina/farmacologia , Lipoproteínas VLDL/metabolismo , Camundongos , Camundongos Knockout , Proteólise/efeitos dos fármacosRESUMO
BACKGROUND: Mechanisms for increased cardiovascular risk in HIV-1-infected adults are incompletely understood, but platelet activation and immune activation leading to a prothrombotic state have been proposed as significant contributors. Aspirin has antiplatelet and immunomodulatory properties. We explored whether 1 week of low-dose aspirin attenuates platelet activation and immune activation in HIV-1-infected and virologically suppressed adults on antiretroviral therapy. METHODS: Platelet activation and immune activation were measured in HIV-1-infected subjects virologically suppressed on antiretroviral therapy and controls before and after 1 week of low-dose aspirin. RESULTS: Compared with control subjects, HIV-1-infected subjects had increased platelet activation, as measured by spontaneous platelet aggregation and aggregation in response to adenosine diphosphate, collagen, and arachidonic acid. After aspirin therapy, percent aggregation decreased similarly in both HIV-1-infected and control subjects to all platelet agonists tested except aggregation in response to arachidonic acid, which remained elevated in the HIV-1-infected group. HIV-1-infected subjects exhibited increased markers of T-cell activation (CD38 and HLA-DR) and monocyte activation (sCD14), which decreased after 1 week of aspirin therapy. Moreover, leukocyte responses to Toll-like receptor stimulation were enhanced after 1 week of aspirin therapy. In vitro studies showed that HIV-1 plasma could activate healthy platelets, which in turn activated monocytes, implicating a direct role for activated platelets in immune activation. CONCLUSIONS: Our data demonstrate that heightened platelet activation and immune activation in treated HIV-1 disease are attenuated by 1 week of aspirin therapy. Aspirin should be further studied for its antithrombotic and immunomodulatory benefits in treated HIV-1 disease.