Search

Venous thromboembolism (VTE) is the third leading cardiovascular cause of death and is conventionally treated with anticoagulants that directly antagonize coagulation. However, recent data have demonstrated that also platelets play a crucial role in VTE pathophysiology. In the current review, we outline how platelets are involved during all stages of experimental venous thrombosis. Platelets mediate initiation of the disease by attaching to the vessel wall upon which they mediate leukocyte recruitment. This process is referred to as immunothrombosis, and within this novel concept inflammatory cells such as leukocytes and platelets directly drive the progression of VTE. In addition to their involvement in immunothrombosis, activated platelets can directly drive venous thrombosis by supporting coagulation and secreting procoagulant factors. Furthermore, fibrinolysis and vessel resolution are (partly) mediated by platelets. Finally, we summarize how conventional antiplatelet therapy can prevent experimental venous thrombosis and impacts (recurrent) VTE in humans.

Structural and biochemical changes in stored platelets are influenced by collection and processing methods. Lesions may appear during platelet concentrate storage, some of which may be involved in adverse transfusion reactions. The preparation and storage of platelet concentrates (PC) may modify and even damage the lipid mediator content. The aim of this study was to investigate the lipidomic profile identified in the supernatants of PCs according to processing and storage conditions, both after leukocyte filtration and contained in platelet additive solution (PAS), comparing single donor apheresis (SDA) products with pooled buffy coat (BC) products.We investigated the accumulation of various lipid mediators including lysophospholipids (LP) and eicosanoids in SDA and BC products stored for 0-5 days. All products were processed following French Blood Establishment (EFS) procedures in accordance with EDQM/GTS European Standards. Both SDA and BC were leukocyte reduced and conserved in 35% autologous donor plasma and 65% platelet additive solution. Lipidomic analysis was performed on PC supernatants using LS/MS spectrometry.Our data demonstrate that lysophosphatidylcholine (LPC) levels were higher in BCs compared to SDAs, with no difference in lysophosphatidic acid (LPA) expression between the two preparation methods. Results for other eicosanoids showed greater similarity; indeed, no clear pattern emerged from analysis of eicosanoids in terms of storage time and process. In general, we observed longitudinal lipid mediator modulation for both SDAs and BCs, particularly at later time points.The expression of LPC and some eicosanoids in BCs could be used as novel biomarkers of PC quality. Future studies are needed to explore their impact on adverse transfusion reactions.

Structural and biochemical changes in stored platelets are influenced by collection and processing methods. During platelet concentrate storage, lesions can appear of which certain can be involved in adverse reaction after transfusion. The aim of this study was to investigate the lipidome in the supernatant of platelet concentrates (PCs) dependent on processing and storage conditions comparing apheresis (SDA) versus buffy coat (BC) platelet concentrate (PC) methods. We investigated various lipid mediator accumulation (Lysophospholipids and Eicosanoids), in either platelet concentrates (SDA-PCs and BC-PCs) stored from 0 to 5 days. All components were produced following French Blood Establishment protocols according to European (EDQM-GTS) Standards; lipids were measured with LS/MS spectrometry. Our data showed no difference between SDA-PC and BC-PC regarding lysophosphatidic acid; however, lysophosphatidylcholine levels were higher in BC-PC than in SDA-PC. Other eicosanoids were affected by either collection or storage conditions of PC. Along the storage, some lipid mediator levels are modulated in SDA-PC and BC-PC. It could be defined as standard platelet components ready for transfusion. To what extent differences, even minimal, matter in clinical outcome remains to be evaluated.

Blood transfusion is a life-saving procedure in which whole blood, or blood components are provided to a patient. Platelet concentrates (PC) may sometimes induce adverse reactions, that are occasionally severe. PC comprise active biomolecules such as cytokines and lipid mediators. Platelet storage lesions are structural and biochemical changes in PC and vary in collection and processing conditions. We investigated lipid mediators as bioactive molecules along storage conditions and in adverse reactions after transfusion. The PC left over were analysed after transfusion, classified with/out severe reaction. Our data presented a decrease of lysophosphatidylcholine species to produce lysophosphatidic acid species. This balance could lead and be correlated with complication symptoms, as other lipids. Lysophosphatidic acid increased with primarily platelet-inhibitor-lipids when single donor apheresis platelet upon storage. Anti-inflammatory and platelet-induced-inhibition lipids were weakly expressed in case of severe adverse reaction. Thus, a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid could be reliable predictors of serious adverse transfusion reactions transfusion. These data highlighted the importance of PC manufacturing and storage processing, not only to maximize clinical efficacy but also to minimize adverse reactions.

Background The stented coronary artery remains at high-risk of complications, particularly in the form of stent thrombosis and in-stent restenosis. Improving our ability to identify patients at high-risk for these complications may provide opportunities for intervention. PAI-1 has been implicated in the pathophysiology of stent complications in preclinical studies, suggesting it may be a clinically valuable biomarker to predict adverse events following percutaneous coronary intervention. Methods Plasma PAI-1 levels were measured in 910 subjects immediately after coronary angiography between 2015 and 2019. The primary outcome was the incidence of unplanned revascularization (UR) at 12 months. The secondary outcome was the incidence of major adverse cardiac events (MACE). Results UR and MACE occurred in 49 and 103 patients in 12 months. Reduced plasma PAI-1 levels were associated with UR (4386.1 pg/mL [IQR, 2778.7–6664.6], n = 49, vs. 5247.6 pg/mL [IQR, 3414.1–7836.1], n = 861; p = 0.04). Tertile PAI-1 levels were predictive of UR after adjustment for known clinical risk factors associated with adverse outcomes. In post-hoc landmark analysis, UR was enhanced with low plasma PAI-1 levels for late complications (beyond 30 days). Finally, an updated systematic review and meta-analysis did not reveal an association between plasma PAI-1 and MACE. Conclusion PAI-1 levels are not independently associated with UR nor MACE in patients undergoing angiography but associated with UR following adjustment with known clinical factors. In our landmark analysis, low PAI-1 levels were associated with UR for late stent complications. As such, future studies should focus on the mediatory role of PAI-1 in the pathogenesis of stent complications.

Patients with established coronary artery disease remain at elevated risk of major adverse cardiac events. The goal of this study was to evaluate the utility of plasminogen activator inhibitor-1-positive platelet-derived extracellular vesicles as a biomarker for major adverse cardiac events and to explore potential underlying mechanisms. Our study suggests these extracellular vesicles as a potential biomarker to identify and a therapeutic target to ameliorate neointimal formation of high-risk patients.

Background: A significant burden of atherosclerotic disease is driven by inflammation. Recently, microRNAs (miRNAs) have emerged as important factors driving and protecting from atherosclerosis. miR-223 regulates cholesterol metabolism and inflammation via targeting both cholesterol biosynthesis pathway and NF k B signaling pathways; however, its role in atherosclerosis has not been investigated. We hypothesize that miR-223 globally regulates core inflammatory pathways in macrophages in response to inflammatory and atherogenic stimuli thus limiting the progression of atherosclerosis. Methods and Results: Loss of miR-223 in macrophages decreases Abca1 gene and protein expression as well as cholesterol efflux to apoA1 (Apolipoprotein A1) and enhances proinflammatory gene expression. In contrast, overexpression of miR-223 promotes the efflux of cholesterol and macrophage polarization toward an anti-inflammatory phenotype. These beneficial effects of miR-223 are dependent on its target gene, the transcription factor Sp3 . Consistent with the antiatherogenic effects of miR-223 in vitro, mice receiving miR223 −/− bone marrow exhibit increased plaque size, lipid content, and circulating inflammatory cytokines (ie, IL-1β). Deficiency of miR-223 in bone marrow–derived cells also results in an increase in circulating pro-atherogenic cells (total monocytes and neutrophils) compared with control mice. Furthermore, the expression of miR-223 target gene ( Sp3 ) and pro-inflammatory marker ( Il-6 ) are enhanced whereas the expression of Abca1 and anti-inflammatory marker ( Retnla ) are reduced in aortic arches from mice lacking miR-223 in bone marrow–derived cells. In mice fed a high-cholesterol diet and in humans with unstable carotid atherosclerosis, the expression of miR-223 is increased. To further understand the molecular mechanisms underlying the effect of miR-223 on atherosclerosis in vivo, we characterized global RNA translation profile of macrophages isolated from mice receiving wild-type or miR223 −/− bone marrow. Using ribosome profiling, we reveal a notable upregulation of inflammatory signaling and lipid metabolism at the translation level but less significant at the transcription level. Analysis of upregulated genes at the translation level reveal an enrichment of miR-223-binding sites, confirming that miR-223 exerts significant changes in target genes in atherogenic macrophages via altering their translation. Conclusions: Our study demonstrates that miR-223 can protect against atherosclerosis by acting as a global regulator of RNA translation of cholesterol efflux and inflammation pathways.

Platelets are anucleate cytoplasmic fragments derived from the fragmentation of medullary megakaryocytes. Activated platelets adhere to the damaged endothelium by means of glycoproteins on their surface, forming the platelet plug. Activated platelets can also secrete the contents of their granules, notably the growth factors contained in the α-granules, which are involved in platelet aggregation and maintain endothelial activation, but also contribute to vascular repair and angiogenesis. Platelets also have a major inflammatory and immune function in antibacterial defence, essentially through their Toll-like Receptors (TLRs) and Sialic acid-binding immunoglobulin-type lectin (SIGLEC). Platelet activation also contributes to the extensive release of anti- or pro-inflammatory mediators such as IL-1β, RANTES (Regulated on Activation, Normal T Expressed and Secreted) or CD154, also known as the CD40-ligand. Platelets are involved in the direct activation of immune cells, polynuclear neutrophils (PNNs) and dendritic cells via the CD40L/CD40 complex. As a general rule, all of the studies presented in this review show that platelets are capable of covering most of the stages of inflammation, primarily through the CD40L/CD40 interaction, thus confirming their own role in this pathophysiological condition.

COVID-19 convalescent plasma (CCP) contains neutralising anti-SARS-CoV-2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients.CCP (n = 766) was compared to non-convalescent control plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralising auto-Abs to type I IFNs and reported adverse events in the recipients.CCP exhibited a statistically significant increase in IL-6 and TNF-alpha levels (0.531 ± 0.04 vs 0.271 ± 0.04; (95% confidence interval [CI], 0.07371-0.4446; p = 0.0061) and 0.900 ± 0.07 vs 0.283 ± 0.07 pg/mL; (95% [CI], 0.3097-0.9202; p = 0.0000829) and lower IL-10 (0.731 ± 0.07 vs 1.22 ± 0.19 pg/mL; (95% [CI], -0.8180 to -0.1633; p = 0.0034) levels than control plasma. Neutralising auto-Abs against type I IFNs were detected in 14/766 (1.8%) CCPs and were not associated with reported adverse events when transfused. Inflammatory markers and bioactivity in CCP with or without auto-Abs, or in CCP whether or not linked to adverse events in transfused patients, did not differ to a statistically significant extent.Overall, CCP exhibited moderately increased inflammatory markers compared to the control plasma with no discernible differences in ex-vivo bioactivity. Auto-Abs to type I IFNs detected in a small fraction of CCP were not associated with reported adverse events or differences in inflammatory markers. Additional studies, including careful clinical evaluation of patients treated with CCP, are required in order to further define the clinical relevance of these findings.French National Blood Service-EFS, the Association "Les Amis de Rémi" Savigneux, France, the "Fondation pour la Recherche Médicale (Medical Research Foundation)-REACTing 2020".

Introduction and objective:Target lesion failure continues to limit the efficacy of percutaneous coronary intervention despite advancements in stent design and medical therapy. Identification of biomarkers to risk stratify patients after percutaneous coronary intervention has the potential to focus therapies on cohorts with increased benefits. Plasminogen activator inhibitor-1 has been identified as a candidate biomarker. Herein, we evaluate biological variables which impact plasminogen activator inhibitor-1 levels and analytical characteristics which impact its utility as a biomarker in humans.Methods:Plasma plasminogen activator inhibitor-1 was measured in 689 patients undergoing coronary angiography. Plasminogen activator inhibitor-1 levels were measured. Clinical and procedural characteristics were collected in a prospective registry.Results:Plasma plasminogen activator inhibitor-1 analytical ( CVa= 4.1%), intra-individual ( CVi= 44.0%) and inter-individual ( CVg= 118.6%) variations with reference change value of 122.3% were calculated. Plasminogen activator inhibitor-1 levels were elevated in patients with cardiovascular risk factors, including type 2 diabetes, pre-diabetes, smokers, obesity, hypertension, and daytime variation in procedure and blood draw.Conclusion:Variation in plasma plasminogen activator inhibitor-1 levels is influenced by multiple biological and procedural characteristics. The performance of plasma plasminogen activator inhibitor-1 is consistent with biomarkers in clinical use (N-terminal pro-B-type natriuretic peptide and C-reactive protein) and its applicability is promising.

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) – for comparative purposes in individuals with another chronic neurodegenerative condition – and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD.

International audience; Extracellular vesicles (EVs) are small vesicles released by the majority of cells in response to cell activation or death stimuli. They are grouped as small EVs or exosomes, large EVs such as microvesicles (MVs) and apoptotic bodies, resulting from distinct mechanisms of generation. EVs are released into the extracellular space, in most human biological fluids and tissues, including atherosclerotic plaques. They transport complex cargo of bioactive molecules, including proteins, lipids and genetic material and are therefore involved in pathophysiological pathways of cell-cell communication. Indeed, EVs are involved in several processes such as inflammation, coagulation, vascular dysfunction, angiogenesis and senescence, contributing to the initiation and progression of atherothrombotic diseases. Consequently, they behave as a determinant of atherosclerotic plaque vulnerability leading to major cardiovascular disorders. Over the last decade, the field of EVs research has grown, highlighting their involvement in atherosclerosis. However, limitations in both detection methodologies and standardisation have hindered implementation of EVs in the clinical settings. This review summarizes the effect of EVs in atherosclerosis development, progression and severity, with specific attention devoted to their ambivalent roles in senescence and hemostasis. This review will also highlight the role of MVs as multifaceted messengers, able to promote or to attenuate atherosclerosis progression. Finally, we will discuss the main technical challenges and prerequisites of standardization for driving EVs to the clinics and delineate their relevance as emergent biomarkers and innovative therapeutic approaches in atherosclerosis.

Objective: To evaluate the utility of plasminogen activator inhibitor-1 positive platelet-derived extracellular vesicles (PAI-1 + PEV) as a biomarker for major adverse cardiac events (MACE) following angiography. Background: The stented coronary artery is at high-risk for complications, predominantly in the form of stent thrombosis and in-stent restenosis. Clinical risk scores have been attempted but no current models nor biomarkers yet accurately identifies the high-risk cohort following revascularization. Methods: PAI-1 + PEV was measured by flow cytometry in 172 patients undergoing coronary angiography. Biological characteristics and utility of PAI-1 + PEV as a biomarker were evaluated. The primary outcome was the incidence of MACE (composite of death, myocardial infarction, cerebrovascular accident, and unplanned revascularization) at 12 months. Results: During a median follow-up period of 377 days (IQR, 269.5 to 442.5 days), 38 patients (20.9%) experienced MACE. In this study, the existence of PAI-1 + PEV complex was validated by flow cytometry (Figure 1A). Furthermore, low log-transformed PAI-1 + PEV levels were associated with MACE (4.17 0.40 vs. 4.33 0.59 logPAI-1 + PEV, p=0.02). After adjustment for known clinical risk factors, low PAI-1 + PEV levels were independently associated with MACE with a hazard ratio of 7.79 (95% CI, 1.87 to 32.4, p=0.005). Finally, low plasma PAI-1 + PEV levels was predictive of MACE in both the discovery and validation cohort (Figure 1B-C). Conclusion: Our results demonstrate the existence of a PAI-1 + PEV and its potential utility as a biomarker to predict MACE. Low plasma PAI-1 + PEV levels was predictive of MACE in both the discovery and validation cohort.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.

The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even when LDL is low. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for cardiovascular disease. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote the efflux of excess cholesterol from lipid-laden macrophages in the vessel wall to the liver. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient, and safe delivery of miRNAs in vivo. We sought to develop chitosan nanoparticles (chNPs) that can deliver functional miRNA mimics to macrophages and to determine if these nanoparticles can alter cholesterol efflux and reverse cholesterol transport in vivo. We developed chNPs with a size range of 150-200 nm via the ionic gelation method using tripolyphosphate (TPP) as a cross-linker. In this method, negatively charged miRNAs were encapsulated in the nanoparticles by ionic interactions with polymeric components. We then optimized the efficiency of intracellular delivery of different formulations of chitosan/TPP/miRNA to mouse macrophages. Using a well-defined miRNA with roles in macrophage cholesterol metabolism, we tested whether chNPs could deliver functional miRNAs to macrophages. We find chNPs can transfer exogenous miR-33 to naive macrophages and reduce the expression of ABCA1, a potent miR-33 target gene, both in vitro and in vivo, confirming that miRNAs delivered via nanoparticles can escape the endosomal system and function in the RISC complex. Because miR-33 and ABCA1 play a key role in regulating the efflux of cholesterol from macrophages, we also confirmed that macrophages treated with miR-33-loaded chNPs exhibited reduced cholesterol efflux to apolipoprotein A1, further confirming functional delivery of the miRNA. In vivo, mice treated with miR33-chNPs showed decreased reverse cholesterol transport (RCT) to the plasma, liver, and feces. In contrast, when efflux-promoting miRNAs were delivered via chNPs, ABCA1 expression and cholesterol efflux into the RCT pathway were improved. Over all, miRNAs can be efficiently delivered to macrophages via nanoparticles, where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA nanoparticles can be used in vivo to target atherosclerotic lesions.

Stent thrombosis remains an infrequent but significant complication following percutaneous coronary intervention. Preclinical models to rapidly screen and validate therapeutic compounds for efficacy are lacking. Herein, we describe a reproducible, high throughput and cost-effective method to evaluate candidate therapeutics and devices for either treatment or propensity to develop stent thrombosis in an

Phospholipase A

Supplemental material, Supplemental_Tables_for_PAI-1_RTR for Performance of plasminogen activator inhibitor-1 as a biomarker in patients undergoing coronary angiography: Analytical and biological considerations by Richard G Jung, Trevor Simard, Pietro Di Santo, Alisha Labinaz, Robert Moreland, Anne-Claire Duchez, Kamran Majeed, Pouya Motazedian, Rebecca Rochman, Young Jung and Benjamin Hibbert in Diabetes & Vascular Disease Research

Stent thrombosis remains an infrequent but significant complication following percutaneous coronary intervention. Preclinical models to rapidly screen and validate therapeutic compounds for efficacy are lacking. Herein, we describe a reproducible, high throughput and cost-effective method to evaluate candidate therapeutics and devices for either treatment or propensity to develop stent thrombosis in an in vitro bench-top model. Increasing degree of stent malapposition (0.00 mm, 0.10 mm, 0.25 mm and 0.50 mm) was associated with increasing thrombosis and luminal area occlusion (4.1 ± 0.5%, 6.3 ± 0.5%, 19.7 ± 4.5%, and 92.6 ± 7.4%, p < 0.0001, respectively). Differences in stent design in the form of bare-metal, drug-eluting, and bioresorbable vascular scaffolds demonstrated differences in stent thrombus burden (14.7 ± 3.8% vs. 20.5 ± 3.1% vs. 86.8 ± 5.3%, p < 0.01, respectively). Finally, thrombus burden was significantly reduced when healthy blood samples were incubated with Heparin, ASA/Ticagrelor (DAPT), and Heparin+DAPT compared to control (DMSO) at 4.1 ± 0.6%, 6.9 ± 1.7%, 4.5 ± 1.2%, and 12.1 ± 1.8%, respectively (p < 0.01). The reported model produces high throughput reproducible thrombosis results across a spectrum of antithrombotic agents, stent design, and degrees of apposition. Importantly, performance recapitulates clinical observations of antiplatelet/antithrombotic regimens as well as device and deployment characteristics. Accordingly, this model may serve as a screening tool for candidate therapies in preclinical evaluation.

Background: the frequency and severity of cardiovascular disease (CVD) is higher in patients suffering from Rhumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) than healthy individuals, likely due to the direct impact of systemic inflammation on the atherosclerotic plaque. Several factors can contribute to cardiovascular damage, including pro-inflammatory lipid mediators such as lysophosphatidic acid (LPA) produced by a lysophospholipase enzyme named autotaxin (ATX). Extracellular vesicles (EVs) are also abundant in blood from patient developing atherosclerotic plaque, and recently EVs from arthritic synovial fluid were found to interact with phospholipase and release inflammatory lipids. Objective: we therefore sought to understand how EVs and ATX may cooperate in the context of rheumatoid disease to promote inflammation. Results: we observed that plasma from RA and SLE patients, analysed by ELISA, contained high concentrations of ATX compared to the plasma from healthy people (pvalue =0.0001). The plasma of these patients, analysed by nanoparticle flow cytometry, also contained high concentration of EVs and these EVs are decorated by ATX, on their surface. As the presence of EVs indicated cell activation due to different endogenous stimuli such as LPA, we found in vitro that different species of LPA activate circulating blood cell to produce EVs (observed by flow cytometry and electronic microscopy), suggesting that LPA serves as an activating signal for EV production. In summary, our results show that ATX bind EVs and produces LPA that activates circulating cells in RA and SLE patients. ATX and LPA could serve as biomarkers and therapeutic target in accelerated CVD in patients with rheumatoid diseases.

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

Platelets play a crucial role in the maintenance of hemostasis, as well as in thrombosis. Upon activation, platelets release small membrane-bound microparticles (MPs) containing bioactive proteins and genetic materials from their parental cells that may be transferred to, and exert potent biological effects in, recipient cells of the circulatory system. Platelets have been shown to contain an abundant and diverse array of microRNAs, and platelet-derived MPs are the most abundant microvesicles in the circulation. Here we demonstrate that human platelets activated with thrombin preferentially release their miR-223 content in MPs. These MPs can be internalized by human umbilical vein endothelial cells (HUVEC), leading to the accumulation of platelet-derived miR-223. Platelet MPs contain functional Argonaute 2 (Ago2)•miR-223 complexes that are capable of regulating expression of a reporter gene in recipient HUVEC. Moreover, we demonstrate a role for platelet MP-derived miR-223 in the regulation of 2 endogenous endothelial genes, both at the messenger RNA and protein levels. Our results support a scenario by which platelet MPs may act as intercellular carriers of functional Ago2•microRNA complexes that may exert heterotypic regulation of gene expression in endothelial cells, and possibly other recipient cells of the circulatory system.

Extracellular vesicles (EV) are small membrane vesicles produced by cells upon activation and apoptosis. EVs are heterogeneous according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. Whereas it is apparent that EVs are implicated in intercellular communication, they can also be used as biomarkers. Continuous improvements in pre-analytical parameters and flow cytometry permit more efficient assessment of EVs; however, methods to more objectively distinguish EVs from cells and background, and to interpret multiple single-EV parameters are lacking. We used spanning-tree progression analysis of density-normalized events (SPADE) as a computational approach for the organization of EV subpopulations released by platelets and erythrocytes. SPADE distinguished EVs, and logically organized EVs detected by high-sensitivity flow cytofluorometry based on size estimation, granularity, mitochondrial content, and phosphatidylserine and protein receptor surface expression. Plasma EVs were organized by hierarchy, permitting appreciation of their heterogeneity. Furthermore, SPADE was used to analyze EVs present in the synovial fluid of patients with inflammatory arthritis. Its algorithm efficiently revealed subtypes of arthritic patients based on EV heterogeneity patterns. Our study reveals that computational algorithms are useful for the analysis of high-dimensional single EV data, thereby facilitating comprehension of EV functions and biomarker development.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naive mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20 S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

SummaryPlatelet microparticles (MPs) represent the most abundant MPs subtype in the circulation, and can mediate intercellular communication through delivery of bioactives molecules, such as cytokines, proteins, lipids and RNAs. Here, we show that platelet MPs can be internalised by primary human macrophages and deliver functional miR-126–3p. The increase in macrophage miR-126–3p levels was not prevented by actinomycin D, suggesting that it was not due to de novo gene transcription. Platelet MPs dose-dependently downregulated expression of four predicted mRNA targets of miR-126–3p, two of which were confirmed also at the protein level. The mRNA downregulatory effects of platelet MPs were abrogated by expression of a neutralising miR-126–3p sponge, implying the involvement of miR-126–3p. Transcriptome-wide, microarray analyses revealed that as many as 66 microRNAs and 653 additional RNAs were significantly and differentially expressed in macrophages upon exposure to platelet MPs. More specifically, platelet MPs induced an upregulation of 34 microRNAs and a concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1 and TNF. These changes were associated with reduced CCL4, CSF1 and TNF cytokine/chemokine release by macrophages, and accompanied by a marked increase in their phagocytic capacity. These findings demonstrate that platelet MPs can modify the transcriptome of macrophages, and reprogram their function towards a phagocytic phenotype.Supplementary Material to this article is available online at www.thrombosis-online.com.

Platelet microparticles are small extracellular vesicles abundant in blood. The present review will introduce the mechanisms underlying the generation of microparticles, and will describe the diverse microparticle subtypes identified to date. The most appropriate methodologies used to distinguish microparticle subtypes will be also presented.Both the megakaryocytes and platelets can generate microparticles. Circulating microparticles originating from megakaryocytes are distinguished from those derived from activated platelets by the presence of CD62P, LAMP-1, and immunoreceptor-based activation motif receptors. Close examination of platelet activation has shed light on a novel mechanism leading to microparticle production. Under physiologic flow, microparticles bud off from long membrane strands formed by activated platelets. Furthermore, mounting evidence supports the notion of microparticle heterogeneity. Platelet microparticles are commonly characterized by the expression of surface platelet antigens and phosphatidylserine. In fact, only a fraction of platelet microparticles harbor phosphatidylserine, and a distinct subset contains respiratory-competent mitochondria. During disease, the microparticle surface may undergo posttranslational modifications such as citrullination, further supporting the concept of microparticle diversity.An appreciation of the microparticle heterogeneity will support their development as potential biomarkers and may reveal functions unique to each microparticle subtype in health and disease.