Platelet-derived Microparticles Research Articles

Ex Vivo Evaluation of Red Blood Cell Adhesion and Whole Blood Thrombosis in Pyruvate Kinase Deficiency

IntroductionPyruvate Kinase Deficiency (PKD) is an inherited glycolytic enzymopathy that is characterized by a life-long chronic hemolytic anemia with severe comorbidities. Hypercoagulability due to increased platelet activity caused by nitric oxide sequestration by cell free hemoglobin has been well-described not just in PKD, but in other hemolytic anemias as well, such as e.g., sickle cell disease (SCD). Hypercoagulability is often accompanied by a cascade of pathophysiological events leading to cell oxidative damage, endothelial activation, and changes in both cell stability and adhesive properties. Increased red blood cell (RBC) adhesion and hypercoagulability may impair microvascular blood flow. Despite these well-recognized rheological changes that are similar to those that occur in other hemolytic anemias, the relationship between baseline erythrocyte adhesion and thrombosis potential have not been well-studied in PKD.Methods10 PKD subjects and 5 healthy controls were recruited under the IRB-approved protocol from Wayne State University. Flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM) was performed by flowing whole blood (1:1 dilution) through a microfluidic channel for 3 minutes (1 dyne/cm 2 shear stress, 1.67Hz pulse frequency). Flow adhesion avidity of the whole blood sample to VCAM-1 (FAAv-WB-VCAM), representing the strength of the RBC-VCAM-1 adhesive interactions, was assessed by quantifying adhesion following sequential increase in shear (5, 10, 20 dyne/cm 2). Thrombin generation assay was conducted using platelet poor plasma with and without thrombomodulin and microparticles (MP) as previously published [1]. Clotting time - reported as lag time (LT), time to peak (ttPeak) and peak height (velocity and amount of net thrombin production), and endogenous thrombin potential (ETP), representing number of substrates potentially convertible by thrombin, were measured. Significance was at p < 0.05.ResultsFA-WB-VCAM at baseline sample hematocrit was significantly elevated (Figure 1) in PKD subjects (808±377 cells/mm², n=10) compared to healthy controls (6±4 cells/mm², n=4) and even to our previously reported steady state levels in sickle cell samples (290±50 cells/mm² [2]. Thrombin generation profiles were similar between PKD subjects and healthy controls with the exception of the thrombin generation index (PPP+TP/PPP)*100ETP that was significantly (p<<0.01) elevated in citrated plasma of PKD subjects (92.9±6.8) as compared to healthy controls (68.6±11.9). For PKD subjects, FA-WB-VCAM correlated significantly with platelet counts (R²=0.81, p<0.05), and FAAv-WB-VCAM was negatively correlated with platelet (P=0.03, R 2=0.5), but not with erythrocyte-derived microparticles (MP). Platelet-derived MP strongly correlated with thrombin generation (ETP, p<0.01, R 2=0.76) but not with LT or ttPeak of thrombin generation. Red blood cell MP were significantly (p=0.02) decreased in splenectomized patients (200±170, n=7) vs. non-splenectomized subjects (2090±1860, n=3). LT and ttPeak were significantly longer in PKD subjects with thrombosis history than without.ConclusionsPKD subjects in this study had elevated RBC adhesive properties similar to that observed in SCD, confirming that pathologic RBC membrane damage resulting in increased adhesion is a common feature of hemolytic anemias. The hemoglobin level of 7.8±1.1 g/dL (mean±SD) for PKD patients was within 6 to 11 g/dl range of hemoglobin levels typical for SCD. There was no significant difference in any other measured parameters (thrombin generation, adhesion avidity, microparticles data). Thrombin generation in PKD subjects was not consistent with hypercoagulability. Based on these observations, pathologic RBC adhesion may be both a novel a mechanism driving hypercoagulability in individuals with PKD. Further studies to determine whether RBC-modifying therapies may decrease thrombosis risk in PKD are warranted.1. Zia A, Callaghan MU, Callaghan JH, et al. Hypercoagulability in adolescent girls on oral contraceptives - global coagulation profile and estrogen receptor polymorphisms. Am J Hematol, 2015;90:725-312. Pittman DD, Hines PC, Beidler D, et al. Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers. Blood. 2021;137(15):2010-20 [Display omitted] DisclosuresHines: Functional Fluidics: Current holder of stock options in a privately-held company. Gao: Functional Fluidics: Current Employment. Herppich: Functional Fluidics: Ended employment in the past 24 months. Kwiatkowski: Imara: Consultancy, Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Sangamo: Research Funding; Bioverativ: Research Funding; Vertex: Research Funding; Silence Therapeutics: Consultancy; bluebird bio: Consultancy, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Chiesi: Research Funding; CRISPR: Research Funding. Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company.

Subclinical markers of cardiovascular toxicity of benzene inhalation in mice

Benzene is a ubiquitous environmental pollutant. Recent population-based studies suggest that benzene exposure is associated with an increased risk for cardiovascular disease. However, it is unclear whether benzene exposure by itself is sufficient to induce cardiovascular toxicity. We examined the effects of benzene inhalation (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on the biomarkers of cardiovascular toxicity in male C57BL/6J mice. Benzene inhalation significantly increased the biomarkers of endothelial activation and injury including endothelial microparticles, activated endothelial microparticles, endothelial progenitor cell microparticles, lung endothelial microparticles, and activated lung and endothelial microparticles while having no effect on circulating levels of endothelial adhesion molecules, endothelial selectins, and biomarkers of angiogenesis. To understand how benzene may induce endothelial injury, we exposed human aortic endothelial cells to benzene metabolites. Of the metabolites tested, trans,trans-mucondialdehyde (10 μM, 18h) was the most toxic. It induced caspases-3, -7 and -9 (intrinsic pathway) activation and enhanced microparticle formation by 2.4-fold. Levels of platelet-leukocyte aggregates, platelet macroparticles, and a proportion of CD4+ and CD8+ T-cells were also significantly elevated in the blood of the benzene-exposed mice. We also found that benzene exposure increased the transcription of genes associated with endothelial cell and platelet activation in the liver; and induced inflammatory genes and suppressed cytochrome P450s in the lungs and the liver. Together, these data suggest that benzene exposure induces endothelial injury, enhances platelet activation and inflammatory processes; and circulatory levels of endothelial cell and platelet-derived microparticles and platelet-leukocyte aggregates are excellent biomarkers of cardiovascular toxicity of benzene.

Platelets Derived Microparticles in COVID-19

BACKGROUND: Evidences indicate that COVID-19 infection causes hypercoagulable state with micro and macrovascular thrombosis. Platelet-derived microparticles (PDMPs) have inflammatory and diverse coagulant roles. AIM: The aim of the study was to assess PDMPs in patients with active and convalescent post COVID-19 infection and correlate PDMPs with clinical, radiological and laboratory findings used in follow up. PATIENTS AND METHODS: The study enrolled 25 patients during active COVID-19 (Group A), of them five patients had thromboembolic events (TEE); and Group B including 32 patients during post COVID-19 stages. Clinical and radiological assessment, routine biomarkers, and detection of PDMPs levels, using enzyme-linked immunosorbent assay method, were done for all patients. RESULTS: In addition to significant differences detected regarding hemoglobin level, total leukocyte count, absolute neutrophil count, absolute lymphocyte count, C-reactive protein level, D-dimer, and serum ferritin, and high significant differences in PDMPs levels were elicited between groups A and B (mean ± standard deviation: 38.7 ± 10.6 IU/mL, and 18.9 ± 15.3 IU/mL) respectively, with discriminative level at 20.5 IU/mL. PDMPs showed nonsignificant difference between patients with and without TEE and no correlation was detected between PDMPs and clinical or radiological severity in post-COVID-19 patients. CONCLUSION: In COVID-19 infection, PDMPs are related to viral activity, and their major role is inflammatory associated.

Endothelial dysfunction and hypercoagulability in severe sickle-cell acute chest syndrome

RationaleAcute pulmonary hypertension (PH) may develop during sickle-cell acute chest syndrome (ACS), and is associated with an increased mortality. Its mechanisms remain poorly known. We questioned whether there is endothelial dysfunction and hypercoagulability in severe ACS, with and without acute PH.MethodsIn a prospective monocentre cohort follow-up study, all sickle-cell adult patients with ACS admitted to the intensive care unit underwent transthoracic echocardiography and measurement of biomarkers of coagulation, endothelial activation and platelet and erythrocyte activation. Acute PH was defined as a high echocardiographic probability of PH. The biological profiles of sickle-cell patients were analysed at the time of ACS, contrasting with the existence of acute PH, and compared with steady-state and with non-sickle-cell controls (healthy subjects and community-acquired pneumonia).ResultsMost patients (36 patients with 39 ACS episodes; 23 males; median age 27 years) had thoracic pain, dyspnoea and computed tomography scan lung consolidation. Acute PH was diagnosed in seven (19%) patients. Erythrocyte- and platelet-derived microparticles and the pro-coagulant activity of microparticles were higher in ACS patients with acute PH, compared with their counterparts. Compared with healthy controls, ACS patients had higher levels of tissue factor, fibrin monomers, D-dimer, release of pro-coagulant microparticles and erythrocyte- and platelet-derived microparticles. Compared with community-acquired pneumonia patients, ACS patients had increased levels of fibrin monomers and erythrocyte- and platelet-derived microparticles.ConclusionsSevere ACS is characterised by endothelial dysfunction and hypercoagulability, with a marked pro-coagulant profile in cases of associated PH.

Rivaroxaban compared with low-dose aspirin in individuals with type 2 diabetes and high cardiovascular risk: a randomised trial to assess effects on endothelial function, platelet activation and vascular biomarkers

Aims/hypothesisIndividuals with type 2 diabetes mellitus and subclinical inflammation have stimulated coagulation, activated platelets and endothelial dysfunction. Recent studies with the direct factor Xa inhibitor rivaroxaban in combination with low-dose aspirin demonstrated a significant reduction of major cardiovascular events, especially in individuals with type 2 diabetes and proven cardiovascular disease. Therefore, we asked the question of whether treatment with rivaroxaban could influence endothelial function, arterial stiffness and platelet activation.MethodsWe conducted a multi-centre, prospective, randomised, open-label trial in 179 participants with type 2 diabetes (duration 2–20 years), subclinical inflammation (high-sensitivity C-reactive protein 2–10 mg/l) and at least two traits of the metabolic syndrome to compare the effects of the direct factor Xa inhibitor rivaroxaban (5 mg twice daily) vs aspirin (100 mg every day) on endothelial function (assessed by forearm occlusion plethysmography), skin blood flow (assessed by laser-Doppler fluxmetry), arterial stiffness (assessed by pulse wave velocity) and serum biomarkers of endothelial function and inflammation. Furthermore, we investigated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in platelets, the concentration of platelet-derived microparticles (PMPs) and the effects of isolated PMPs on HUVEC proliferation in vitro.ResultsRivaroxaban treatment for 20 weeks (n = 89) resulted in a significant improvement of post-ischaemic forearm blood flow (3.6 ± 4.7 vs 1.0 ± 5.2 ml/100 ml, p = 0.004), a numerically increased skin blood flow and reduced soluble P-Selectin plasma level vs aspirin. We did not find significant differences of arterial stiffness or further biomarkers. Neither rivaroxaban nor aspirin influenced VASP phosphorylation of platelets. The number of PMPs increased significantly with both rivaroxaban (365.2 ± 372.1 vs 237.4 ± 157.1 μl−1, p = 0.005) and aspirin (266.0 ± 212.7 vs 201.7 ± 162.7 μl−1, p = 0.021). PMPs of rivaroxaban-treated participants stimulated HUVEC proliferation in vitro compared with aspirin. Rivaroxaban was associated with a higher number of bleeding events.Conclusions/interpretationOur findings indicate that the direct factor Xa inhibitor rivaroxaban improved endothelial function in participants with type 2 diabetes and subclinical inflammation but also increased the risk of bleeding.Trial registration:ClinicalTrials.gov NCT02164578.FundingThe study was supported by a research grant from Bayer Vital AG, Germany.Graphical abstract

Predicting disease progression in advanced non-small cell lung cancer with circulating neutrophil-derived and platelet-derived microparticles

BackgroundMicroparticles (MPs) are extracellular vesicles that are associated with cancer development and progression. Advanced non-small cell lung cancer (NSCLC) still shows disease progression after multiple lines of treatment. Therefore, the objective of this study was to explore the correlation between circulating MPs and disease progression in advanced NSCLC, and to find a new method for concise and rapid determination of disease progression.MethodsPatients with advanced NSCLC admitted to hospital between October 2019 and October 2020 were included and divided into objective remission (OR) and progressive disease (PD) groups. The morphology of MPs was observed using transmission electron microscopy. The circulating total MPs, neutrophil MPs (NMPs), and platelet MPs (PMPs) before and after treatment were detected by flow cytometry, and a predictive model for disease progression in advanced NSCLC was developed.ResultsEighty-six patients were included; 60 in the OR group and 26 in the PD group. There was no significant difference in total MPs, NMPs, or PMPs at baseline between the two groups. After treatment, total MPs, NMPs, and PMPs were significantly higher in the PD than those in the OR group. Multivariate regression analysis showed that post-treatment NMPs≥160 events/μL(OR,3.748;95%CI,1.147–12.253,p = 0.029), PMPs≥80 events/μL(OR,10.968;95%CI,2.973–40.462,p < 0.0001) and neutrophil/lymphocyte ratio (NLR) ≥3.3 (OR,4.929;95%CI,1.483–16.375,p = 0.009) were independently associated with progression of advanced NSCLC. Post-treatment NMPs and PMPs combined with NLR were used to build a predictive model for progression of advanced NSCLC. The area under the curve was 0.825 (95%CI,0.715–0.934, p < 0.0001), optimal cut-off value was 16, sensitivity was 80.8%, and specificity was 88.3%.ConclusionNMPs and PMPs are associated with progression of advanced NSCLC. The predictive model for progression of advanced NSCLC, established combining NMPs, PMPs, and NLR, can screen out 80.8% of patients with PD. This is helpful for real-time accurate, concise and rapid assessment of disease progression and timely adjustment of drug therapy.Trial registrationChinese Clinical Trial Registry, ChiCTR1800020223. Registered 20 December 2018, http://www.chictr.org.cn/index.aspx.

Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes.

Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

Avatrombopag, a Novel Thrombopoietin Receptor Agonist, Increases Platelet Counts without Increasing Platelet Activation in Patients with Thrombocytopenia Due to Chronic Liver Disease

Background: The thrombopoietin (TPO) receptor c-mpl is expressed on the surface of megakaryocytes and platelets. In addition to its effect on increasing megakaryocyte growth and maturation, TPO also lowers the threshold for platelet activation in vivo and in vitro . TPO receptor agonists (TPO-RA) may, perhaps to differing extents, also lead to platelet activation. Indeed, in a recent study of patients with chronic liver disease (CLD), the TPO-RA eltrombopag reduced the need for platelet transfusions, but was associated with an increased incidence of thrombosis. Therefore, the goal of this study was to evaluate the effect of avatrombopag, a novel TPO-RA, on in vivo platelet activation and in vitro platelet reactivity in response to agonists in patients with CLD.Methods: CLD patients participating in the Phase 3 E5501-G000-310 and -311 studies (Randomized, Global, Double-blind, Placebo-controlled, Parallel-group Studies to Evaluate the Efficacy and Safety of Once-daily Oral Avatrombopag for the Treatment of Adults with Thrombocytopenia Associated with Liver Disease Prior to an Elective Procedure) could participate in a substudy to evaluate the effects of avatrombopag on platelet function. IRB-approved informed consent was obtained prior to sample collection. Patients (n = 30) were randomized to treatment on Days 1 - 5 with placebo or avatrombopag: 60 mg daily in patients with platelet counts <40 x 109/L, and 40 mg daily in patients with platelet counts 40 to ≤50 x 109/L. Samples were collected at Baseline (Day 1, pre-drug) and after treatment (Day 4 and Day 10). No patient received a platelet transfusion within the 10 days prior to these blood draws.Platelet activation was evaluated by whole blood flow cytometry (which, unlike other methods, is accurate in samples with low platelet counts) with and without in vitro stimulation with adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP). Endpoints included platelet surface activated glycoprotein (GP) IIb-IIIa (as reported by the activation-dependent monoclonal antibody PAC1), platelet surface P-selectin, platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles. Citrate-anticoagulated whole blood was incubated (15 min, room temperature) with an antibody cocktail containing fluorescein isothiocyanate (FITC)-conjugated PAC1, phycoerythrin (PE)-conjugated anti-P-selectin, and PE-Cy5-conjugated anti-CD42b (GPIb) with or without ADP 0.5 µM or 20 µM or TRAP 1.5 µM or 20 µM. Samples were fixed with 1% formaldehyde, then shipped cold together with the remaining blood sample (for analysis of phosphatidylserine expression and microparticles) to the Center for Platelet Research Studies for analysis. Phosphatidylserine expression on platelets was determined by staining with FITC-conjugated annexin V. Platelet-derived microparticles were identified by light scattering properties and positive staining for CD41 and CD42b. Changes from Baseline for drug-treated (60 mg and 40 mg avatrombopag cohorts combined, n=20) vs. placebo-treated patients (n=10) were compared using a mixed effects model with p<0.05 being considered significant.Results: Avatrombopag, 60 mg and 40 mg treatment, but not placebo treatment, resulted in significant increases in platelet counts at Day 10 (Table). Circulating activated platelets were not increased in avatrombopag-treated patients compared with placebo-treated patients (Table, “no agonist” data), as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. Platelet reactivity, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin after low and high concentrations of ADP and TRAP, was also not increased in avatrombopag-treated patients compared with placebo-treated patients (Table). Similarly, there was no evidence of increased platelet activation in avatrombopag-treated patients compared with placebo-treated patients as assessed by platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles.Conclusions: In this randomized, double-blind, placebo-controlled, parallel-group study of patients with thrombocytopenia due to CLD, treatment with avatrombopag resulted in increased platelet counts, but did not increase either platelet activation in vivo or platelet reactivity in vitro . [Display omitted] DisclosuresFrelinger:Ionis: Research Funding; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; GLSynthesis: Research Funding; Baxalta: Research Funding. Michelson:Elsevier: Patents & Royalties; Eisai: Research Funding; GLSynthesis: Research Funding; Instrumentation Laboratory: Consultancy; Baxalta: Research Funding; Ionis: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding.

Levels of GP-Iib/Iiia Positive Platelet Microparticles (PMP) but Not CD31+/CD42+ PMP Predict Adverse Outcomes in Troponin-Negative Patients with Chest Pain.

Background: Cell derived microparticles (MP) are useful biomarkers in various thrombotic and inflammatory disorders. However, MP are heterogeneous in their phenotypes, functional properties, and origin. Platelet-derived microparticles (PMP) have been shown to be elevated in acute coronary syndromes but different species of PMP have not been compared. We performed a study to compare two subspecies of PMP, those identified by anti-GP-IIb/IIIa vs. those by CD31+/CD42+, for predicting adverse outcomes in patients with chest pain of possible ischemic cause.Methods: The study population consisted of 105 patients who presented to the emergency department with chest pain, non-diagnostic electrocardiograms and negative serial cardiac troponin I levels. Blood was obtained after the third negative troponin result for determination of levels of GP-IIb/IIIa positive PMP (PMPIIb/IIIa) and CD31+/CD42+ PMP (PMP31). All treatment decisions were left at the caregiver's discretion. Telephone follow-up was done at 6 months for the development of a combined endpoint of death, myocardial infarction or need for cardiac catheterization or revascularization. The mean age of our patients was 57.5 years. The age, diabetes, hypertension, smoking and history of coronary artery disease was not significantly different between patients who reached an endpoint at 6 months versus those who did not. Overall 19% reached an endpoint at 6 months. The endpoint was largely driven by the need for cardiac catheterization due to positive stress test results.Results: On univariate analysis, patients who reached an endpoint at 6 months had higher levels of PMPIIb/IIIa than those who did not (geometric means 2059.6 vs. 924.4 counts/μL; p=0.04; medians 2157.5 vs 1764.0 counts/μL; p=0.009). When adjusted for age, diabetes, hypertension, dyslipidemia, aspirin use and current smoking by analysis of covariance, the difference persisted (adjusted geometric means: 2484.9 vs. 829.5 counts/μL; p=0.01). In contrast, PMP31 levels were not significantly different between patients who reached an endpoint at 6 months versus those who did not (5908 ±7990 vs. 3812 ±3109 counts/μL; p=0.26).Conclusions: Our findings suggest that different subspecies of PMPs may reflect different pathophysiological phenomena. PMP31 levels did not correlate with the risk of adverse outcomes in this population. In contrast, GpIIb/IIIa-positive PMP appear to be promising prognostic indicators in chest pain patients with negative troponins and nondiagnostic EKGs. Our results are consistent with prior studies showing association between PMP and thrombosis, but suggest that not all PMP are equal. Further research is needed to investigate a possible role of PMP in the pathogenesis of ACS, and as prognostic markers in current stratification strategies. Assay of different species of MP is likely to provide more discriminating diagnostic and prognostic information.

Effect of DDAVP on Platelet Activation and Platelet-Derived Microparticle Generation.

The way by which 1-deamino-8-D-arginine vasopressin (DDAVP) acts on platelets remains unclear. Data from the literature tend to show that there is no definite effect on platelet activation, but recent work has suggested that a subtype of platelets, activated by the combined action of collagen and thrombin, was triggered by DDAVP. Moreover, platelet microparticles (PMPs), which have been shown to be procoagulant, have rarely been studied in this context. The goal of this study was to analyze the effects of DDAVP on PMPs' release through platelet activation. Fifteen out of 18 consecutive patients undergoing a therapeutic test with DDAVP were included. They were suffering from factor VIII deficiency or from von Willebrand disease. The expression of P-selectin and PAC-1 binding on platelets and the numbers of circulating PMPs were evaluated ex vivo before and after DDAVP infusion. Peripheral blood was collected on CTAD to limit artifactual platelet activation. DDAVP induced a significant decrease of platelet counts and volume. Only small changes of P-selectin expression and PAC-1 binding were observed. Considering PMPs, two populations of patients could be defined, respectively, with (120%, n = 6) or without (21%, n = 7) an increase of PMPs after DDAVP. The decrease in platelet counts and volume remained significant in the group of responders. This study shows that DDAVP induces the generation/release of PMPs in some patients with factor VIII deficiency and von Willebrand disease 1 hour after DDAVP infusion.

CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling

Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. However, the expression pattern of circulating extracellular vesicle (EV)-incorporated lncRNAs in patients with coronary artery disease (CAD) is still poorly investigated. A human lncRNA array revealed that certain EV-lncRNAs are significantly dysregulated in CAD patients. Circulating small EVs (sEVs) from patients with (n = 30) or without (n = 30) CAD were used to quantify PUNISHER (also known as AGAP2-antisense RNA 1 [AS1]), GAS5, MALAT1, and H19 RNA levels. PUNISHER (p = 0.002) and GAS5 (p = 0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Fluorescent labeling and quantitative real-time PCR of sEVs demonstrated that functional PUNISHER was transported into the recipient cells. Mechanistically, the RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK), interacts with PUNISHER, regulating its loading into sEVs. Knockdown of PUNISHER abrogated the EV-mediated effects on endothelial cell (EC) migration, proliferation, tube formation, and sprouting. Angiogenesis-related gene profiling showed that the expression of vascular endothelial growth factor A (VEGFA) RNA was significantly increased in EV recipient cells. Protein stability and RNA immunoprecipitation indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Loss of PUNISHER in EVs abolished the EV-mediated promotion of VEGFA gene and protein expression. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.

Platelet Microparticles Enriched in miR-223 Reduce ICAM-1-Dependent Vascular Inflammation in Septic Conditions.

In the process of sepsis, activated platelets shed microvesicles containing microRNAs (miRNAs), which can be internalized by distinct recipient cells in circulation, consequently eliciting a potent capability to regulate their cellular functions in different diseases. In the present study, activated human platelets transferring miR-223 into endothelial cells via platelet-derived microparticles (PMPs) was investigated in vitro during septic conditions with a proposed mechanism involving in downregulation of the enhanced expression of intercellular adhesion molecule-1 (ICAM-1). The uptake of PMPs encasing miR-223 and the adhesion of peripheral blood mononuclear cells (PBMCs) on human coronary artery endothelial cells (HCAECs) were observed by immunofluorescence microscopy upon co-culture with PMPs isolated from sepsis or control plasma. The expression of miR-223-3p and its gene target ICAM1 in HCAECs were quantified by RT-qPCR and ELISA after the cells were incubated with septic or control PMPs, whose levels were induced with thrombin-receptor activating peptide (TRAP). Leukocyte-depleted platelets (LDPs) from septic patients showed a decreased miR-223 level, while septic plasma and PMPs revealed an elevated miRNA level compared to control samples. Similarly, TRAP-activated LDPs demonstrated a reduced intracellular miR-223 expression, while increased levels in the supernatant and PMP isolates were observed vs. untreated samples. Furthermore, TNF-α alone resulted in decreased miR-223 and elevated ICAM1 levels in HCAECs, while PMPs raised the miRNA level that was associated with downregulated ICAM1 expression at both mRNA and protein levels under TNF-α treatment. Importantly, miR-223 was turned out not to be newly synthesized as shown in unchanged pre-miR-223 level, and mature miR-223 expression was also elevated in the presence of PMPs in HCAECs after transfection with Dicer1 siRNA. In addition, septic PMPs containing miR-223 decreased ICAM1 with a reduction of PBMC binding to HCAECs. In conclusion, septic platelets released PMPs carrying functional miR-223 lower ICAM1 expression in endothelial cells, which may be a protective role against excessive sepsis-induced vascular inflammation.

FC 005DEPOSITION OF PLATELET-DERIVED MICRO-PARTICLES IN PODOCYTES CONTRIBUTES TO DIABETIC NEPHROPATHY

Abstract Background and Aims Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the developed world. Podocyte injury has been shown to be a critical cellular event in the progression of DN. Our previous studies demonstrated that platelet-derived microparticles (PMPs) mediated endothelial injury in diabetic rats. Therefore, this study aimed to investigate whether the PMPs deposit in podocytes and its potential effects on podocyte injury in DN. Method Deposition of PMPs in podocytes was checked by immunofluorescent staining and electron microscopy. Renal pathological change and ultra-microstructure were respectively checked by periodic acid-schiff staining and electron microscopy. The protein expression of inflammatory cytokines and extracellular matrix was measured by immunohistochemical staining and Western blot. Results The PMPs were found to be widely deposited in podocytes of glomeruli in diabetic patients and animal models and closely associated with progression of DN. Interestingly, aspirin treatment significantly inhibited accumulation of PMPs in glomeruli of diabetic rats, accompanied with alleviated mesangial matrix expansion, fusion of foot processes, decreased protein expression of inflammatory cytokines and extracellular matrix secretion. The in vitro study further confirmed the deposition of PMPs in podocytes. Moreover, PMPs stimulation induced phenotype transition of podocytes through decreased protein expression of podocin and increased protein expression of α-SMA and fibronectin, which was correlated with increased production of inflammatory cytokines. Conclusion Our findings for the first time demonstrated that deposition of PMPs in podocytes contributed to the development of DN.

Protective Role of Platelets in Myocardial Infarction and Ischemia/Reperfusion Injury.

Thrombotic occlusion of the coronary artery is a key component in the pathogenesis of myocardial ischemia and myocardial infarction (MI). The standard therapy for ischemia is revascularization and restoration of blood flow to previously ischemic myocardium. Paradoxically, reperfusion may result in further tissue damage called ischemia/reperfusion injury (IRI). Platelets play a major role in the pathogenesis of MI and IRI, since they contribute to the thrombus and microthrombi formation, inflammation, release of immunomodulatory mediators, and vasoconstrictive molecules. Antiplatelet therapies have proven efficacy in the prevention of thrombosis and play a protective role in cardiac IRI. Beyond the deterioration effect of platelets in MI and IRI, in the 90s the first reports on a protective effect of molecules released from platelets during MI appeared. However, the role of platelets in cardioprotection is still poorly understood. This review describes the involvement of platelets in MI, IRI, and inflammation. It mainly focuses on the protective role of platelets in MI and IRI. Platelets are involved in cardioprotection based on platelet-releasing molecules and antiplatelet therapy, apart from antiaggregatory effects. Additionally, the use of platelet-derived microparticles as possible markers of MI, with and without comorbidities, and their role in cardioprotection are discussed. This review is aimed at illustrating the present knowledge on the role of platelets in MI and IRI, especially in a context of cardioprotection.

Disrupting tumour vasculature and recruitment of aPDL1-loaded platelets control tumour metastasis

Although therapies of cancer are advancing, it remains challenging for therapeutics to reach the sites of metastasis, which accounts for majority of cancer associated death. In this study, we have developed a strategy that guides an anti-programmed cell death-ligand 1 (aPDL1) antibody to accumulate in metastatic lesions to promote anti-tumour immune responses. Briefly, we have developed a combination in which Vadimezan disrupts tumour blood vessels of tumour metastases and facilitates the recruitment and activation of adoptively transferred aPDL1-conjugated platelets. In situ activated platelets generate aPDL1-decorated platelet-derived microparticles (PMP) that diffuse within the tumour and elicit immune responses. The proposed combination increases 10-fold aPDL1 antibody accumulation in lung metastases as compared to the intravenous administration of the antibody and enhances the magnitude of immune responses leading to improved antitumour effects.

Platelet-Derived Procoagulant Microparticles as Blood-based Biomarker of Breast Cancer.

Objective:Breast cancer is the main cause of cancer death in women worldwide. Elevated plasma levels of circulating cell-derived microparticles (MPs) have been reported in various types of cancer, including breast cancer, with the ability to mediate inflammation and thrombosis. Microparticles are bioactive agents, and it has been suggested that MPs can be used as a diagnostic, prognostic, or therapeutic biomarker in various diseases. The aim of this study was to investigate the levels of platelet-derived MPs (PMPs) in breast cancer patients. Materials and Methods:In this case-control study, 30 patients with breast cancer and 20 normal subjects were sampled after obtaining written consent. MPs were isolated from blood samples by centrifugation technique. CD42b and annexin V markers were used respectively for counting PMPs and procoagulant MPs with flow cytometry. Results:Flow cytometry results showed that the number of PMPs and procoagulant annexin V positive MPs was significantly higher in the breast cancer patients than normal subjects (p<0.001). The number of the annexin V MPs differed significantly in patients with high tumor size (T2) compared to the patients with low tumor size (T1) and controls (p<0.001). Significant and positive correlations were found between PMP levels and tissue-based biomarkers, tumor grading, and distant metastasis (P<0.05). Tumor histological type did not correlate with the numbers of PMPs (p=0.065). Conclusion:Increased levels of PMPs and activity in terms of hemostasis and having a positive and significant relationship with tumor grading and metastasis may indicate the effective role of PMPs in the pathogenesis and prognosis of breast cancer.

Breast Cancer Processes Are Modulated by Platelet‐Derived Microparticles

Cancer is one of the leading causes of morbidity and mortality among women worldwide. Although mortality rates have been declining for years, metastasis accounts for the majority of deaths associated with the disease. An increasing number of studies show that platelets as well as platelet-derived microparticles (PMPs) play a significant role in cancer malignancy and disease progression. PMPs are small vesicles released into the circulatory system and the extracellular environment during platelet activation. PMP generation involves a process where bioactive material is transferred from platelets to the PMPs. Interestingly, PMPs have the capacity to interact with target recipient cells and transfer their bioactive cargo into these cells upon internalization. Accordingly, studies have shown that PMPs allow intercellular exchange and trafficking of bioactive material to modulate signaling and activation processes of recipient cells. We believe that PMPs represent an important source of breast cancer modulators leading to malignant features and disease progression. The objective of this study was to evaluate the impact of PMPs on breast cancer processes. More specifically, we investigated the modulating effects of PMPs on breast cancer metabolism and phenotypic processes involved in breast cancer metastasis. First, we characterized and validated the capacity of breast cancer models to internalize PMPs and their cargo using confocal microscopy and flow cytometry. Interestingly, we observed that the levels of PMPs internalization vary and are dependent of the type of breast cancer recipient cells. Using a series of biochemical and cell-based assays, we also demonstrated that the cargo of PMPs is biologically active which results in the modulation of breast cancer cell metabolism, viability and migration properties of recipient cells. Overall, we demonstrate that PMPs modulate cancer cell processes reminiscent of disease malignancy. These findings provide a better understanding of the role of PMPs and their influence as cancer disease modulators. The knowledge gained from these studies will thus foster the development of potentially new strategic interventions to help mitigate the morbidity and mortality associated with cancer disease.

Circulating endothelial microparticles: a promising biomarker of acute kidney injury after cardiac surgery with cardiopulmonary bypass

BackgroundCurrent diagnostic strategies for acute kidney injury (AKI) after cardiac surgery with cardiopulmonary bypass (CPB) are nonspecific and limited. Previously, we demonstrated that circulating microparticles (MPs) in patients with valve heart disease (VHD) and congenital heart diseases (CHD) induce endothelial dysfunction and neutrophil chemotaxis, which may result in kidney injury. We also found that circulating MPs increase after cardiac surgery with CPB and are related to cardiac function. However, the relationship between circulating MPs and AKI after CPB is unknown.MethodsEighty-five patients undergoing cardiac surgery with CPB were enrolled. Patients were divided into AKI and non-AKI groups based on the serum creatinine levels at 12 h and 3 d post-CPB. Circulating MPs were isolated from plasma, and their levels including its subtypes were detected by flow cytometer. Independent risk factors for the CPB-associated AKI (CPB-AKI) were determined by multivariate logistic regression analysis. Receiver operating characteristic (ROC) analysis was used to measure the prognostic potential of CPB-AKI.ResultsThe morbidity of AKI at 12 h and 3 d after cardiac surgery with CPB was 40% and 31.76%, respectively. The concentrations of total MPs and platelet-derived MPs (PMP) remained unchanged at 12 h and then increased at 3 d post-CPB, while that of endothelial-derived MPs (EMP) increased at both time points. In patients with AKI, PMP and EMP were elevated compared with the patients without AKI. However, no significant change was detected on monocyte-derived MPs (MMP) at 12 h and 3 d post-CPB. The logistic regression analysis showed that EMP was the independent risk factor for AKI both at 12 h and 3 d post-CPB. The area under ROC for the concentrations of EMP at 12 h and 3 d post-CPB was 0.86 and 0.91, with the specificity up to 0.88 and 0.91, respectively.ConclusionsCirculating EMP may serve as a potential biomarker of AKI after cardiac surgery with CPB.

Impact of anemia on platelet production and function in acute coronary syndrome: results of long-term single-center registry

Abstract Funding Acknowledgements Type of funding sources: None. Anemia is the most common clinical condition known to increase both ischemic and hemorrhagic risks in patients with acute coronary syndrome (ACS). Despite it"s high frequency there are certain gaps in evidence on how to choose appropriate antiplatelet regimen in anemia, for anemia was among exclusion criteria in almost all randomized clinical trials on efficacy and safety of antiplatelet agents. Purpose of the study to evaluate prevalence of anemia and it"s impact on platelet production and function in patients admitted to ICU with ACS. Methods. To determine the prevalence and characteristics of anemia we analyzed data from the local single-center long-term registry of coronary heart disease (n total= 4776, n ACS = 765). To evaluate platelet production and function we used data obtained from those patients with ACS in whom platelet function, immature platelet fraction (IPF), thrombopoietin (TPO), stromal cell-derived factor 1 (SDF1), TPO receptors (MPL) and platelet derived microparticles (PDMP) were measured serially at admission, on the second and on the 7th day since ACS manifestation (n = 321). Results. Anemia on admission was verified in 12.5% of ACS, and there were no significant differences in antiplatelet therapy at first medical contact and on admission. Anemia in patients with ACS was accompanied with decreased platelet count, therefore TPO level at admission was higher: 336.23 (141.35; 429.85) ng/mL vs 266.04 (199.16; 341.20) pg/mL, p = 0.050. PDMP level at admission was minimal in anemic patients: 6.75 (5.31; 7.81) vs 26.45 (16.05; 39.35) ng/mL (p = 0.027). There were no significant differences in TPO levels measured on the 2nd and the 7th days, as well as SDF and MPL. IPF was also comparable in anemic patients and those with normal Hb levels. Despite similar platelet function on admission in patients with anemia there was significant increase in platelet ADP-induced aggregation and secretion with maximal level at the 7th day since ACS manifestation: 2 (1; 4) vs 0 (0; 1) Ohm, p = 0.02. Anemia in ACS was characterized with unfavorable myocardial stress and injury biomarkers dynamics: BNP at the 7th day – 1155 (867; 2251) vs 73.4 (40.3; 148.5) pg/mL, and with increase in risk of early adverse (ischemic, but not hemorrhagic) events OR 8.6 CI 1.1; 19.4, p = 0.038. Conclusion. Anemia is a common comorbidity in acute coronary syndrome increasing risk of early complications. Anemia on admission in ACS is characterized by increase of platelet aggregation and secretion despite antiplatelet therapy, and this activation does not result from possible associated changes in platelet production and turnover. Received results underline the risk of early discontinuation of antiplatelet therapy in ACS with anemia, and possible benefits of platelet function monitoring in these patients.

Inhibition of post-surgery tumour recurrence via a hydrogel releasing CAR-T cells and anti-PDL1-conjugated platelets.

The immunosuppressive microenvironment of solid tumours reduces the antitumour activity of chimeric antigen receptor T cells (CAR-T cells). Here, we show that the release-through the implantation of a hyaluronic acid hydrogel-of CAR-T cells targeting the human chondroitin sulfate proteoglycan 4, polymer nanoparticles encapsulating the cytokine interleukin-15 and platelets conjugated with the checkpoint inhibitor programmed death-ligand 1 into the tumour cavity of mice with a resected subcutaneous melanoma tumour inhibits the local recurrence of the tumour as well as the growth of distant tumours, through the abscopal effect. The hydrogel, which functions as a reservoir, facilitates the enhanced distribution of the CAR-T cells within the surgical bed, and the inflammatory microenvironment triggers platelet activation and the subsequent release of platelet-derived microparticles. The post-surgery local delivery of combination immunotherapy through a biocompatible hydrogel reservoir could represent a translational route for preventing the recurrence of cancers with resectable tumours.