Platelet-derived Extracellular Vesicles Research Articles

Targeted delivery of dexamethasone in acute pneumonia.

Pneumonia has contributed to significant mortality owing to the irreversible injury to the lungs and severe inflammation of the tissue. Dexamethasone (DEX) is regarded as an effective drug to relieve the level of pneumonia, while the adverse effect of which is non-negligible. Here, we developed a targeted delivery strategy based on platelet-derived extracellular vesicles (PEVs), which are naturally occurring nanoparticles released by platelets, for DEX delivery in acute pneumonia, aiming to reduce the side effects and improve the therapeutic efficacy. Our strategy may shed light on the problems in DEX-based acute pneumonia therapy clinically.

Circulating extracellular vesicles as new inflammation marker in HIV infection.

Extracellular vesicles, released by cell pullulation, are surrounded by a phospholipid bilayer and carry proteins as well and genetic material. It has been shown that extracellular vesicles mediate intercellular communication in several conditions, such as inflammation, immunodeficiency, tumor growth, and viral infections. Here, we analyzed circulating levels of extracellular vesicles in order to clarify their role in chronic inflammation mechanisms characterizing HIV patients. We analyzed and subtyped circulating levels of extracellular vesicles, through a recently developed flow cytometry method. In detail, endothelial-derived extracellular vesicles (CD31+/CD41a-/CD45-, EMVs), extracellular vesicles stemming from leukocytes (CD45+, LMVs) and platelets (CD41a+/CD31+) were identified and enumerated. Moreover, we analyzed the extracellular vesicle protein cargo with proteomic analysis. Circulating levels of total extracellular vesicles, EMVs and LMVs were significantly lower in the HIV+ patients than in healthy subjects, whereas platelet-derived extracellular vesicles resulted higher in patients than in the healthy population. Proteomic analysis showed the upregulation of gammaIFN and IL1α, and down-regulation of OSM, NF-kB, LIF, and RXRA signaling resulted activated in this patients. These data demonstrate, for the first time that HIV infection induces the production of extracellular vesicles containing mediators that possibly feed the chronic inflammation and the viral replication. These two effects are connected as the inflammation itself induces the viral replication. We, therefore, hypothesize that HIV infection inhibits the production of extracellular vesicles that carry anti-inflammatory molecules.

Platelet-derived extracellular vesicles to target plaque inflammation for effective anti-atherosclerotic therapy

Atherosclerosis is a kind of chronic inflammatory diseases characterized by dysfunction of local immune responses. Here we engineer platelet-derived extracellular vesicles (PEVs) to load MCC950, an NLRP3-inflammasome inhibitor, for atherosclerosis-targeted therapy. PEVs which are readily collected from the activated platelets selectively bind multiple cell types associated with the formation of atherosclerotic plaque in vivo. Intravenous administration of MCC950-PEVs could significantly reduce the formation of atherosclerotic plaques, lower the local inflammation and inhibit proliferation of macrophages and T cells at the plaque site compared with free drug administration in ApoE-KO mice. Our strategy suggests the promise of PEVs for targeted drug delivery for treatment of atherosclerosis.

Abstract 15603: Discovery of Plasminogen Activator Inhibitor-1 Platelet-derived Extracellular Vesicles to Predict Major Adverse Cardiac Events

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.

Differential Profile of Systemic Extracellular Vesicles From Sporadic and Familial Alzheimer's Disease Leads to Neuroglial and Endothelial Cell Degeneration.

Evidence suggests that extracellular vesicles (EVs) act as mediators and biomarkers of neurodegenerative diseases. Two distinct forms of Alzheimer disease (AD) are known: a late-onset sporadic form (SAD) and an early-onset familial form (FAD). Recently, neurovascular dysfunction and altered systemic immunological components have been linked to AD neurodegeneration. Therefore, we characterized systemic-EVs from postmortem SAD and FAD patients and evaluated their effects on neuroglial and endothelial cells. We found increase CLN-5 spots with vesicular morphology in the abluminal portion of vessels from SAD patients. Both forms of AD were associated with larger and more numerous systemic EVs. Specifically, SAD patients showed an increase in endothelial- and leukocyte-derived EVs containing mitochondria; in contrast, FAD patients showed an increase in platelet-derived EVs. We detected a differential protein composition for SAD- and FAD-EVs associated with the coagulation cascade, inflammation, and lipid-carbohydrate metabolism. Using mono- and cocultures (endothelium-astrocytes-neurons) and human cortical organoids, we showed that AD-EVs induced cytotoxicity. Both forms of AD featured decreased neuronal branches area and astrocytic hyperreactivity, but SAD-EVs led to greater endothelial detrimental effects than FAD-EVs. In addition, FAD- and SAD-EVs affected calcium dynamics in a cortical organoid model. Our findings indicate that the phenotype of systemic AD-EVs is differentially defined by the etiopathology of the disease (SAD or FAD), which results in a differential alteration of the NVU cells implied in neurodegeneration.

Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function

Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.

Platelet-derived extracellular vesicles regulate cell cycle progression and cell migration in breast cancer cells

Platelets have been extensively implicated in the progression of cancer and platelet-derived extracellular vesicles (PEVs) are gaining growing attention as potential mediators of the platelet-cancer interplay. PEVs are shed from platelet membrane in response to extracellular stimuli and carry important biological signals for intercellular communication. In this study we demonstrate that PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. PEVs were massively internalized by the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not by the MCF-7 cell line. In SKBR3 cells, PEVs decreased mitochondrial dehydrogenase activities and altered cell cycle progression without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the distribution in the different phases of the cell cycle. In all the analyzed breast cancer cells, PEVs triggered a sustained increase of intracellular Ca2+, but only in MDA-MB-231 cells, this was associated to the stimulation of selected signaling proteins implicated in migration, including p38MAPK and myosin light chain. Importantly, inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results demonstrate that PEVs are versatile regulators of cancer cell behavior and elicit a variety of different responses depending on the specific breast cancer cell subtype.

The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.

Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells.

AimsPlatelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs).MethodsEVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca2+) content.ResultsOsteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response.ConclusionPL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations.Cite this article: Bone Joint Res 2020;9(10):667–674.

Platelet-derived extracellular vesicles infiltrate and modify the bone marrow during inflammation

AbstractDuring inflammation, steady-state hematopoiesis switches to emergency hematopoiesis to repopulate myeloid cells, with a bias toward the megakaryocytic lineage. Soluble inflammatory cues are thought to be largely responsible for these alterations. However, how these plasma factors rapidly alter the bone marrow (BM) is not understood. Inflammation also drives platelet activation, causing the release of platelet-derived extracellular vesicles (PEVs), which package diverse cargo and reprogram target cells. We hypothesized that PEVs infiltrate the BM, providing a direct mode of communication between the plasma and BM environments. We transfused fluorescent, wild-type (MPL+) platelets into recipient cMpl−/−mice before triggering systemic inflammation. Twenty hours postinfusion, we observed significant infiltration of donor platelet-derived particles in the BM, which we tracked immunophenotypically (MPL+ immunohistochemistry staining) and quantified by flow cytometry. To determine if this phenomenon relates to humans, we extensively characterized both megakaryocyte-derived and PEVs generated in vitro and in vivo, and found enrichment of extracellular vesicles in bone marrow compared with autologous peripheral blood. Last, BM from cMpl−/− mice was cultured in the presence or absence of wild-type (MPL+) PEVs. After 72 hours, flow cytometry revealed increased megakaryocytes only in cultures with added PEVs. The majority of CD41+ cells were bound to PEVs, suggesting a PEV-mediated rescue of megakaryopoiesis. In conclusion, we report for the first time that plasma-residing PEVs infiltrate the BM. Further, PEVs interact with BM cells in vivo and in vitro, causing functional reprogramming that may represent a novel model of inflammation-induced hematopoiesis.

Calming the Cytokine Storm in Pneumonia by Biomimetic Nanoparticles

Recently, the Wang group at Soochow University and the Gu group at the University of California, Los Angeles demonstrated the targeting ability of platelet-derived extracellular vesicles to deliver anti-inflammatory drug [5-(p-fluorophenyl)-2-ureido] thiophene-3-carboxamide (TPCA-1) to pneumonia for calming the local cytokine storm in acute lung injury.

Normothermic Ex Vivo Liver Perfusion Prevents Intrahepatic Platelet Sequestration After Liver Transplantation

The detrimental role of platelets in sinusoidal endothelial cell (SEC) injury during liver transplantation (LT) has been previously addressed after static cold storage (SCS), however, it is currently unknown after normothermic ex vivo liver perfusion (NEVLP). Pig LT was performed with livers from heart-beating donors or donation after circulatory death (DCD) donors subjected to SCS or NEVLP (n = 5/group). All pigs except for 1 (DCD-SCS-group) survived 4 days. The heart-beating donor- and DCD-NEVLP-groups showed significantly lower aspartate transaminase-levels compared with the SCS-groups 3 hours post-LT (P = 0.006), on postoperative day (POD) 2 (P = 0.005), POD3 (P = 0.007), and on POD4 (P = 0.012). Post-LT total platelet count recovered faster in the NEVLP than in the SCS-groups at 12 hours (P = 0.023) and 24 hours (P = 0.0038). Intrahepatic sequestration of platelets was significantly higher in the SCS-groups 3 hours postreperfusion and correlated with severity of SEC injury. In both SCS-groups, levels of tumor growth factor-β were higher 3 hours post-LT, on POD1 and on POD3. Moreover, platelet factor 4 levels and platelet-derived extracellular vesicles were increased in the SCS-groups. Hyaluronic acid levels were significantly higher in the SCS-groups, indicating a higher grade of endothelial cell dysfunction. Platelet inhibition achieved by pretreatment with clopidogrel (n = 3) partly reversed the detrimental effects on SEC injury and therefore provided further evidence of the important role of platelets in ischemia/reperfusion injury and SEC injury. Normothermic perfusion of liver grafts before transplantation effectively reduced platelet aggregation and SEC injury, which translated into an improved posttransplant organ function.

Calming Cytokine Storm in Pneumonia by Targeted Delivery of TPCA-1 Using Platelet-Derived Extracellular Vesicles

SummaryPneumonia can cause high morbidity and mortality because of uncontrolled inflammation in the lung tissue. Calming the cytokine storm may be one key to saving the life of patients with severe pneumonia. Here, inspired by the intrinsic affinity of platelets to the site of inflammation, we have engineered platelet-derived extracellular vesicles (PEVs) for pneumonia-targeted drug delivery. It is demonstrated that PEVs that are easily generated from the activated platelets can selectively target pneumonia in the mouse model with acute lung injury (ALI). By loading with [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1), which can inhibit the production of inflammatory factors, the PEVs significantly improve therapeutic benefits by inhibiting the infiltration of pulmonary inflammatory cells and calming local cytokine storm compared with the free drug-treated group. Furthermore, we find that PEVs could serve as a broad platform that can selectively target various inflammatory sites, including chronic atherosclerotic plaque, rheumatoid arthritis, and wounds associated with skin.

Mediating effects of platelet-derived extracellular vesicles on PM2.5-induced vascular endothelial injury.

At present, PM2.5 exposure has been considered as a major risk factor for cardiovascular disease. Most studies have focused on the toxic mechanism of PM2.5 in direct contact with cells or biomolecules, only few studies have reported the toxic mechanism of PM2.5 mediated by intercellular communication. Extracellular vesicles are the main carriers of intercellular communication and signal transduction in vivo, and play a vital role in the occurrence and development of cardiovascular disease. Therefore, the present research aimed to determine whether platelets-derived extracellular vesicles (P-EVs) secreted from PM2.5-exposed platelets are transferred into the human umbilical vein endothelial cells (HUVECs) and mediated the PM2.5-induced vascular endothelial injury by affecting normal cellular function. The result showed that P-EVs secreted from PM2.5-exposed platelets significantly reduced the proliferation promoting effect of normal P-EVs on vascular endothelium by decreasing the effective factors promoting vascular endothelial growth. Meanwhile, the levels of intercellular adhesion molecules, proinflammatory factors (ICAM-1, IL-6, and TNF-α) and the ROS level of HUVECs were markedly elevated. In addition, the apoptotic rate was increased via up-regulating the protein level of cytochrome-C(Cyt C), Bax, cleaved caspase-3 and down-regulating Bcl-2 in HUVECs, indicating that mitochondrial apoptotic pathway was activated by P-EVs secreted from PM2.5-exposed platelets. Further, the expression level of P-EVs targeted miRNAs in HUVECs was altered, indicating that miRNAs released from P-EVs were transferred to HUVECs and regulated the cellular function, while PM2.5 could inhibit this regulatory effect. In summary, these results demonstrate that the P-EVs secreted from PM2.5-exposed platelets can enter the HUVECs, which mediate the PM2.5-induced vascular endothelial injury. These findings provide a new perspective and theoretical basis for further exploring the mechanism of cardiovascular damage caused by PM2.5 exposure.

Biological insight into the extracellular vesicles in women with and without gestational diabetes.

Gestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy, with increasing prevalence worldwide and still unclear pathogenic mechanisms. Extracellular vesicles (EVs) are emerging as potential biomarkers of disease-specific pathways in metabolic disorders, but their potential role in GDM is not fully understood. Therefore, the main aim of this study was to evaluate the link between EVs and hyperglycaemia during pregnancy. We assessed 50 GDM women and 50 controls at the third trimester of pregnancy in whom we collected demographic characteristics and clinical and anthropometric parameters. In addition, the circulating total EVs (tEVs) and their subpopulations were assessed using flow cytometry. The levels of tEVs and EVs subtypes, expressed as median and interquartile range, were not significantly different between two groups; however, adipocyte-derived EVs (aEVs) concentration, expressed as percentage, was higher in controls than in GDM women (p = 0.045). In addition, a significant correlation was observed between aEVs (%) and third trimester total cholesterol (p = 0.022) within the GDM group. Furthermore, a significant correlation between endothelial-derived EVs (eEVs) and platelet-derived EVs (pEVs) within both groups was found, as well as a significant relation between aEVs and pEVs. These data, although preliminary, represent the starting point for further studies to determine the role of circulating EVs in GDM.

Extracellular Vesicles as Mediators of Cellular Crosstalk Between Immune System and Kidney Graft

Extracellular vesicles (EVs) are known immune-modulators exerting a critical role in kidney transplantation (KT). EV bioactive cargo includes graft antigens, costimulatory/inhibitory molecules, cytokines, growth factors, and functional microRNAs (miRNAs) that may modulate expression of recipient cell genes. As paracrine factors, neutrophil- and macrophage-derived EVs exert immunosuppressive and immune-stimulating effects on dendritic cells, respectively. Dendritic cell-derived EVs mediate alloantigen spreading and modulate antigen presentation to T lymphocytes. At systemic level, EVs exert pleiotropic effects on complement and coagulation. Depending on their biogenesis, they can amplify complement activation or shed complement inhibitors and prevent cell lysis. Likewise, endothelial- and platelet-derived EVs can exert procoagulant/prothrombotic effects and also promote endothelial survival and angiogenesis after ischemic injury. Kidney endothelial- and tubular-derived EVs play a key role in ischemia–reperfusion injury (IRI) and during the healing process; additionally, they can trigger rejection by inducing both alloimmune and autoimmune responses. Endothelial EVs have procoagulant/pro-inflammatory effects and can release sequestered self-antigens, generating a tissue-specific autoimmunity. Renal tubule-derived EVs shuttle pro-fibrotic mediators (TGF-β and miR-21) to interstitial fibroblasts and modulate neutrophil and T-lymphocyte influx. These processes can lead to peritubular capillary rarefaction and interstitial fibrosis–tubular atrophy. Different EVs, including those from mesenchymal stromal cells (MSCs), have been employed as a therapeutic tool in experimental models of rejection and IRI. These particles protect tubular and endothelial cells (by inhibition of apoptosis and inflammation–fibrogenesis or by inducing autophagy) and stimulate tissue regeneration (by triggering angiogenesis, cell proliferation, and migration). Finally, urinary and serum EVs represent potential biomarkers for delayed graft function (DGF) and acute rejection. In conclusion, EVs sustain an intricate crosstalk between graft tissue and innate/adaptive immune systems. EVs play a major role in allorecognition, IRI, autoimmunity, and alloimmunity and are promising as biomarkers and therapeutic tools in KT.

Platelet-derived- Extracellular Vesicles Promote Hemostasis and Prevent the Development of Hemorrhagic Shock

Every year more than 500,000 deaths are attributed to trauma worldwide and severe hemorrhage is present in most of them. Transfused platelets have been shown to improve survival in trauma patients, although its mechanism is only partially known. Platelet derived-extracellular vesicles (PEVs) are small vesicles released from platelets upon activation and/or mechanical stimulation and many of the benefits attributed to platelets could be mediated through PEVs. Based on the available literature, we hypothesized that transfusion of human PEVs would promote hemostasis, reduce blood loss and attenuate the progression to hemorrhagic shock following severe trauma. In this study, platelet units from four different donors were centrifuged to separate platelets and PEVs. The pellets were washed to obtain plasma-free platelets to use in the rodent model. The supernatant was subjected to tangential flow filtration for isolation and purification of PEVs. PEVs were assessed by total count and particle size distribution by Nanoparticle Tracking Analysis (NTA) and characterized for cells of origin and expression of EV specific-surface and cytosolic markers by flow cytometry. The coagulation profile from PEVs was assessed by calibrated automated thrombography (CAT) and thromboelastography (TEG). A rat model of uncontrolled hemorrhage was used to compare the therapeutic effects of 8.7 × 108 fresh platelets (FPLT group, n = 8), 7.8 × 109 PEVs (PEV group, n = 8) or Vehicle (Control, n = 16) following severe trauma. The obtained pool of PEVs from 4 donors had a mean size of 101 ± 47 nm and expressed the platelet-specific surface marker CD41 and the EV specific markers CD9, CD61, CD63, CD81 and HSP90. All PEV isolates demonstrated a dose-dependent increase in the rate and amount of thrombin generated and overall clot strength. In vivo experiments demonstrated a 24% reduction in abdominal blood loss following liver trauma in the PEVs group when compared with the control group (9.9 ± 0.4 vs. 7.5 ± 0.5 mL, p < 0.001>). The PEV group also exhibited improved outcomes in blood pressure, lactate level, base excess and plasma protein concentration compared to the Control group. Fresh platelets failed to improve these endpoints when compared to Controls. Altogether, these results indicate that human PEVs provide pro-hemostatic support following uncontrolled bleeding. As an additional therapeutic effect, PEVs improve the outcome following severe trauma by maintaining hemodynamic stability and attenuating the development of ischemia, base deficit, and cardiovascular shock.

Platelet-Derived Extracellular Vesicles as Target of Antiplatelet Agents. What Is the Evidence?

Platelet-derived large extracellular vesicles (often referred to as microparticles in the field of cardiovascular disease) have been identified as effector in the atherothrombotic process, therefore representing a target of pharmacological intervention of potential interest. Despite that, limited evidence is so far available concerning the effects of antiplatelet agents on the release of platelet-derived extracellular vesicles. In the present narrative review, the mechanisms leading to vesiculation in platelets and the pathophysiological processes implicated will be discussed. This will be followed by a summary of the present evidence concerning the effects of antiplatelet agents under experimental conditions and in clinical settings.

Fast isolation of highly specific population of platelet-derived extracellular vesicles from blood plasma by affinity monolithic column, immobilized with anti-human CD61 antibody

A new, fast and selective immunoaffinity chromatographic method including a methacrylate-based convective interaction media (CIM®) disk monolithic column, immobilized with anti-human CD61 antibody, was developed for the isolation of CD61-containing platelet-derived extracellular vesicles (EVs) from plasma. The isolated EVs were detected and size characterized by asymmetrical flow field-flow fractionation (AsFlFFF) with multi-angle light-scattering (MALS) and dynamic light-scattering (DLS) detection, and further confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The mean size of platelet-derived EV isolates from the anti-CD61 CIM® disk monolithic column were 174 nm (SD 60 nm) based on the NTA results. These results indicated a successful isolation of platelet-derived EVs, which was confirmed by Western blotting the isolates against the EV-specific markers CD9 and TSG101 together with transmission electron microscopy. Additional elucidation of MALS and DLS data provided detailed information of the size distribution of the isolated fractions, confirming the successful isolation of also small platelet-derived EVs ranging from 30 to 130 nm based on the hydrodynamic radii. The isolation procedure took only 19 min and the time can be even further decreased by increasing the flow rate. The same immunoaffinity chromatographic procedure, following AsFlFFF allowed also the isolation and characterization of platelet-derived EVs from plasma in under 60 min. Since it is possible to regenerate the anti-CD61 disk for multiple uses, the methodology developed in this study provides a viable substitution and addition to the conventional EV isolation procedures.

Appropriation of GPIbα from platelet-derived extracellular vesicles supports monocyte recruitment in systemic inflammation.

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-β1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo. Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-β1-stimulated cremaster muscle, while in the ApoE−/− model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.