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Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen β-chain were the most differentially expressed proteins between severity groups. Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis. [ABSTRACT FROM AUTHOR]

We aimed to investigate the effects of integrin αIIbβ3 inhibitor tirofiban on hallmarks of platelet activation, degranulation, and aggregation during its use to analyze activated but non-complexed platelets via flow cytometry. To do so, we used washed platelets from healthy human donors. We combined aggregometry, an assay of platelet functionality, with flow cytometry and ELISA to detect and correlate, respectively, platelet aggregation, activation, and granule release. While tirofiban effectively inhibited agonist-induced platelet aggregation (thrombin receptor-activating peptide 6 (TRAP), convulxin (CVX), U46619 and IV.3), the surface expression of P-selectin and CD63 and granule release of RANTES were significantly increased, indicating that tirofiban enhances degranulation, uncoupled from aggregation. The results show that tirofiban alters the activation phenotype of platelets, something that should be considered when using tirofiban to enable flow cytometric analysis of activated but unaggregated platelet suspensions.

Platelets, originally described for their role in blood coagulation, are now also recognized as key players in modulating inflammation, tissue regeneration, angiogenesis, and carcinogenesis. Recent evidence suggests that platelets also influence insulin secretion from pancreatic β cells. The multifaceted functions of platelets are mediated by the factors stored in their alpha granules (AGs) and dense granules (DGs). AGs primarily contain proteins, while DGs are rich in small molecules, and both types of granules are released during blood coagulation. Specific components stored in AGs and DGs are implicated in various inflammatory, regenerative, and tumorigenic processes. However, the relative contributions of AGs and DGs to the regulation of pancreatic β cell function have not been previously explored. In this study, we utilized mouse models deficient in AG content (neurobeachin-like 2 (Nbeal2) -deficient mice) and models with defective DG release (Unc13d -deficiency in bone marrow-derived cells) to investigate the impact of platelet granules on insulin secretion from pancreatic β cells. Our findings indicate that AG deficiency has little to no effect on pancreatic β cell function and glucose homeostasis. Conversely, mice with defective DG release exhibited glucose intolerance and reduced insulin secretion. Furthermore, Unc13d -deficiency in hematopoietic stem cells led to a reduction in adipose tissue gain in obese mice. Obtained data suggest that DGs, but not AGs, mediate the influence of platelets on pancreatic β cells, thereby modulating glucose metabolism. • Dense granules mediate the influence of platelets on insulin secretion. • Deficiency of alpha granules in platelets has no effect on glucose homeostasis. • Unc13D deficiency in hemopoietic cells leads to reduced adiposity in mice. [ABSTRACT FROM AUTHOR]

We aimed to investigate the effects of integrin αIIbβ3 inhibitor tirofiban on hallmarks of platelet activation, degranulation, and aggregation during its use to analyze activated but non-complexed platelets via flow cytometry. To do so, we used washed platelets from healthy human donors. We combined aggregometry, an assay of platelet functionality, with flow cytometry and ELISA to detect and correlate, respectively, platelet aggregation, activation, and granule release. While tirofiban effectively inhibited agonist-induced platelet aggregation (thrombin receptor-activating peptide 6 (TRAP), convulxin (CVX), U46619 and IV.3), the surface expression of P-selectin and CD63 and granule release of RANTES were significantly increased, indicating that tirofiban enhances degranulation, uncoupled from aggregation. The results show that tirofiban alters the activation phenotype of platelets, something that should be considered when using tirofiban to enable flow cytometric analysis of activated but unaggregated platelet suspensions. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) a leading cause of pediatric and adult morbidity and mortality worldwide. It can cause complications in multiple organs, thus increasing hospital stays and costs. However, RSV-based studies have primarily focused on effects in the lungs and blood, thereby potentially neglecting critical genes and pathways. Hence, studying RSV infection via a novel multi-organ approach is important. In this study, lung, intestine, brain, and spleen tissues from six BALB/c mice (6–8 weeks old; three in control group and three in RSV-infected group) were subjected to RNA sequencing. Differentially expressed genes (DEGs) in each organ were obtained and functional enrichment analysis was performed. We first used CIBERSORT to evaluate the immune-infiltration landscape. Subsequently, common DEGs (co-DEGs) among the four organs were analyzed to identify key genes and pathways. After quantitative reverse transcription-polymerase chain reaction, western blotting, and external validation analysis of key hub genes, their correlation with immune cells and potential functions were explored. We found that the host response to RSV infection varied among the four organs regarding gene expression profiles and immune cell infiltration. Analysis of the 16 co-DEGs indicated enrichment in the platelet and neutrophil degranulation pathways. Importantly, the key gene hemopexin (Hpx) was strongly correlated with the immune cell fraction in the lungs and may participate in the regulation of platelet activation and immune response. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) a leading cause of pediatric and adult morbidity and mortality worldwide. It can cause complications in multiple organs, thus increasing hospital stays and costs. However, RSV-based studies have primarily focused on effects in the lungs and blood, thereby potentially neglecting critical genes and pathways. Hence, studying RSV infection via a novel multi-organ approach is important. In this study, lung, intestine, brain, and spleen tissues from six BALB/c mice (6–8 weeks old; three in control group and three in RSV-infected group) were subjected to RNA sequencing. Differentially expressed genes (DEGs) in each organ were obtained and functional enrichment analysis was performed. We first used CIBERSORT to evaluate the immune-infiltration landscape. Subsequently, common DEGs (co-DEGs) among the four organs were analyzed to identify key genes and pathways. After quantitative reverse transcription-polymerase chain reaction, western blotting, and external validation analysis of key hub genes, their correlation with immune cells and potential functions were explored. We found that the host response to RSV infection varied among the four organs regarding gene expression profiles and immune cell infiltration. Analysis of the 16 co-DEGs indicated enrichment in the platelet and neutrophil degranulation pathways. Importantly, the key gene hemopexin (Hpx) was strongly correlated with the immune cell fraction in the lungs and may participate in the regulation of platelet activation and immune response.

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed “reperfusion injury.” Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed "reperfusion injury." Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury. [ABSTRACT FROM AUTHOR]

Background/Aims: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by αIIbβ3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. Conclusions: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

Background/Aims: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by αIIbβ3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. Conclusions: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation. © 2016 The Author(s) Published by S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We report here on the influence of the platelet antagonist clopidogrel (Plavix) on bovine platelet function. We first evaluated the capacity of clopidogrel to inhibit adenosine diphosphate (ADP)-stimulated platelet function in the bovine species, using an ex vivo approach with blood from treated animals. Platelets isolated from treated calves displayed rapid and consistent reduction in function (aggregation, thromboxane production) upon ADP, but not platelet activating factor (PAF), stimulation. We then examined the possibility that clopidogrel could influence Mannheimia haemolytica pneumonia pathobiology using an experimental challenge model. We were unable to detect significant differences between clopidogrel treated and untreated animals when challenged with intra-tracheal inoculation of M. haemolytica. There was a trend towards inhibition of platelet degranulation in the affected regions of lungs from clopidogrel treated calves, and pre-treated challenged animals had similar amounts of fibrin deposition and enhanced fibrous tissue formation in their lungs when compared with control counterparts. [ABSTRACT FROM AUTHOR]

During haemodialysis (HD), platelets (PLTs) are activated and release granule contents. As HD treatment occurs three times a week, it has been demonstrated that PLTs are exhausted due to the repetitive character of the treatment. To identify PLT depletion mor-phologically, PLT evaluation was performed by light microscopy and electron microscopy (EM) in a chronic HD subject and a healthy reference subject. Blood samples were taken before the start of HD treatment for measure-ment of PLT count, PLT volume and size parameters. Blood smears were screened by light microscopy for qualitative evaluation of PLT granule containing cytoplasm, as indicat-ed by its staining density. Morphological PLT parameters of surface area and size of dense bodies were assessed by EM. Data were com-pared with results of a group of 20 chronic HD subjects and a group of 20 healthy reference subjects. With respect to the percentage of PLTs with appropriate staining density (>75%), light microscopic evaluation showed that this value (9%) was within the range of a group of chronic HD subjects, but considerably below the reference range (70%). EM evalua-tion revealed an average PLT surface area and dense bodies area of respectively 42% and 31%, if the healthy reference subject was set on 100%. PLTs from a chronic HD subject are con-siderably smaller and substantially less granu-lar than PLTs from a healthy reference subject. These findings support the hypothesis of PLT depletion in chronic HD subjects due to fre-quent PLT activation and/or increased urea concentrations. [ABSTRACT FROM AUTHOR]