Platelet-derived Microparticles Research Articles

Megakaryocyte- and Platelet-Derived Microparticles as Novel Diagnostic and Prognostic Biomarkers for Immune Thrombocytopenia.

Altered cell-derived microparticles (MPs) have been reported in multiple autoimmune diseases. However, the roles of megakaryocyte- and platelet-derived MPs (MKMPs and PMPs) in immune thrombocytopenia (ITP) have not been investigated. In this study, we examined plasma MKMP and PMP levels in patients with ITP and evaluated their potential diagnostic values. Plasma MKMP and PMP levels were analyzed by flow cytometry in a discovery set of ITP patients (n = 78), non-immune thrombocytopenia (TP) patients (n = 69), and age- and gender-matched healthy controls (n = 88). Samples from a therapy set of ITP patients (n = 21) were used to assess the response to thrombopoietin receptor agonist (TPO-RA) treatment. Spearman correlation analysis was performed between MP levels and disease parameters. Receiver operator characteristic (ROC) curves were generated to evaluate the diagnostic values of the MPs. We found that plasma MKMP and PMP levels were significantly lower in ITP patients than those in healthy controls (p values < 0.0001) but higher than in those in TP patients (p < 0.002 and p < 0.0002, respectively). After normalization to platelet counts, PMP/Platelet ratios in ITP patients were higher than those in TP patients and healthy controls (p values < 0.001). PMP/Platelet ratios had a diagnostic value for ITP (area under the curve = 0.808, p < 0.0001) with 73.1% sensitivity and 77.3% specificity. MKMP levels can be used to discriminate ITP from TP with a cut-off value of 112.5 MPs/μL and a sensitivity of 74.4%. Moreover, both MKMP and PMP levels were elevated in ITP patients who responded to TPO-RA treatment. Plasma PMP levels positively correlated with platelet counts in the responders (r = 0.558, p < 0.01). Our results indicate that plasma MKMP and PMP levels are decreased in ITP patients and that plasma MKMP and PMP levels may serve as biomarkers for ITP diagnosis and prediction of TPO-RA treatment response.

Shear Stress Promotes Platelet Surface Desialylation, Platelet Count Drop, and Microvesiculation: Do Platelets Need to be Sugar-Coated?

Introduction. Mechanical circulatory support (MCS) devices restore hemodynamics and are lifesaving for patients with advanced heart failure and, recently, for patients with severe COVID-19. Long-term MCS is associated with bleeding complications, often requiring multiple transfusions of blood products or reoperations. To date, due to undefined etiology, device-related bleeding lacks efficient therapeutic management. We have shown that hypershear stress, existing within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, downregulation of key adhesion receptors, impaired platelet aggregation, and microvesiculation - all contributors to bleeding. Recent studies have also shown that glycosylation of platelet surface receptors, i.e. the platelet "sugar coat," plays a major role in the regulation of platelet function and lifespan. Here, we tested the hypothesis that hypershear stress promotes platelet surface desialylation, thus facilitating platelet count drop and increased microvesiculation. Methods. Human platelets were obtained from ACD-anticoagulated blood of healthy volunteers via gel filtration on Sepharose 2B. Gel-filtered platelets were exposed to neuraminidase and continuous shear stress (30 or 70 dyne/cm2,10 min) in a hemodynamic shearing device. Optionally, platelets were pretreated with oseltamivir, a common neuraminidase inhibitor. Platelets were co-stained with fluorophore-conjugated SNA lectin, binding terminal 2,6-linked sialic acid moieties, and anti-CD41 antibody. Multi-colored flow cytometry was performed to quantify platelet surface sialylation, platelets, and microparticles; fluorescent nanobeads SPHEROTM were used as a size standard. The surface density of platelet sialylation was calculated as median fluorescence intensity normalized to forward scatter indicating platelet size. The platelet neuraminidase activity was quantified using fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (MUNANA) and was presented as µM of substrate per min. Results. Platelet exposure to both hypershear stress and neuraminidase-1 induced platelet surface desialylation as indicated by the significant decrease of SNA lectins' binding density on platelets (Fig.1A). Platelet desialylation induced by shear stress and neuraminidase was associated with a notable decrease in platelet count and an increase in platelet-derived microparticles (Fig.1B). Interestingly, neuraminidase reinforced shear-mediated desialylation, platelet count drop, and microvesiculation. Oseltamivir slightly inhibited shear-mediated desialylation, preserved platelet count (30% increase vs. shear), and decreased microparticle generation (29% decrease vs. shear). Exposure to 70 dyne/cm2 shear stress resulted in a 9.5-fold increase in platelet neuraminidase activity as compared to basal level: 0.85 ± 0.61 µM/min vs 0.05 ± 0.03 µM/min, respectively (Mean ± SD, p < 0.01, one-way ANOVA). Conclusions. Shear stress promotes platelet surface desialylation with the associated reduction in platelet count and increased microvesiculation. Neuraminidase inhibition restores platelet count and decreases microparticle generation caused by hypershear. Shear-mediated increase of platelet neuraminidase activity is considered a plausible mechanism for shear-mediated platelet deglycosylation. Developing therapeutic strategies for preservation of platelet sialylation offers significant translational potential for pharmacologic management of MCS-related platelet dysfunction and bleeding coagulopathy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Circulating microparticles are associated with plaque burden and cause eNOS uncoupling in patients with carotid atherosclerosis.

Aims: The study aimed to evaluate the correlation of different microparticle (MP) phenotypes with plaque burden and their diagnostic value and preliminarily explore the role of MPs in atherosclerosis (AS). Methods: Carotid intima-media thickness (CIMT) and maximal plaque area in 23 patients with carotid atherosclerosis (CAS) and 22 healthy subjects were measured by ultrasound. Transmission electron microscopy, nanoparticle tracking analysis and western blot were used to identify MPs. Flow cytometry assay measured absolute number of MPs, and receiver operating characteristic (ROC) analysis was used to assess the relationship between plaque burden and MPs. To study the preliminary mechanism of MPs in AS, MPs were administered to 32 male Kunming mice, which were randomly divided into control, CAS, healthy, and tetrahydrobiopterin (BH4) groups. Hematoxylin-eosin staining, immunohistochemistry staining, and Western blot were adopted to detect relevant indexes 24h after the injection. Results: The plasma levels of CD45+ leukocyte-derived microparticle (LMP), CD11a+ LMP, CD11a+/CD45+ LMP, and CD31+/CD42b+ platelet-derived microparticle (PMP) in CAS patients were significantly higher than those in healthy subjects, and were positively correlated with the maximal plaque area. Moreover, the levels of CD11a+ LMP, CD11a+/CD45+ LMP were also positively correlated with CIMT. The area under the ROC curve of the four MPs was 0.689, 0.747, 0.741, and 0.701, respectively. Compared with healthy subjects, MPs from CAS patients resulted in a significantly lower expression of endothelial nitric oxide synthase (eNOS) dimer/monomer, and BH4 could improve eNOS uncoupling. Moreover, the level of VCAM-1 in intima in the CAS group was significantly higher than in the other three groups. Conclusion: CD11a+ LMP and CD11a+/CD45+ LMP might be potential biomarkers for CAS prediction. BH4-related eNOS uncoupling occurs in CAS patients, and circulating MPs from them lead to endothelial dysfunction through eNOS uncoupling.

Platelet-Derived Exosomes in Atherosclerosis.

Atherosclerosis (AS), the main cause of many cardiovascular diseases (CVDs), is a progressive inflammatory disease characterized by the accumulation of lipids, fibrous elements, and calcification in the innermost layers of arteries. The result is the thickening and clogging of these vessel walls. Several cell types are directly involved in the pathological progression of AS. Among them, platelets represent the link between AS, inflammation, and thrombosis. Indeed, besides their pivotal role in hemostasis and thrombosis, platelets are key mediators of inflammation at injury sites, where they act by regulating the function of other blood and vascular cell types, including endothelial cells (ECs), leukocytes, and vascular smooth muscle cells (VSMCs). In recent years, increasing evidence has pointed to a central role of platelet-derived extracellular vesicles (P-EVs) in the modulation of AS pathogenesis. However, while the role of platelet-derived microparticles (P-MPs) has been significantly investigated in recent years, the same cannot be said for platelet-derived exosomes (P-EXOs). For this reason, this reviews aims at summarizing the isolation methods and biological characteristics of P-EXOs, and at discussing their involvement in intercellular communication in the pathogenesis of AS. Evidence showing how P-EXOs and their cargo can be used as biomarkers for AS is also presented in this review.

Correlations between the Circulating Level of Cell-Derived Microparticles and Surgical Variables in Heart Valve Surgery with Cardiopulmonary Bypass.

Cell-derived microparticles (MPs) as membrane vesicles are procoagulant. They play a role in surgical hemostasis. In this study, the correlations between the circulating level of cell-derived MPs and surgical variables in heart valve surgery were investigated. The present prospective case-series study was conducted in Rajaie Cardiovascular Medical and Research Center from January through March 2021. Forty patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled. Before the induction of anesthesia and 30 minutes after the administration of protamine sulfate, venous blood samples were collected. After MP isolation, the concentration of MPs was determined via the Bradford method. Flow cytometry analysis was performed to determine the MP count and phenotype. Intraoperative variables and postoperative routine coagulation tests were defined as surgical variables. Postoperative coagulopathy was defined as an activated partial thromboplastin time (aPTT) ≥48 seconds or an international normalized ratio (INR) >1.5. The total concentration of MPs and the MP count increased significantly after surgery compared with before surgery. The postoperative concentration of MPs was positively correlated with the CPB time (P=0.030, ρ=0.40). The preoperative concentration of MPs was significantly lower in patients with higher postoperative aPTT and INR (P=0.003, P= -0.50 and P=0.020, P= -0.40, respectively). In multivariate logistic regression analysis, the preoperative MP concentration (OR, 1.00; 95% CI, 1.00 to 1.01; P=0.017) was considered a risk factor for postoperative coagulopathy. The levels of MPs, especially platelet-derived MPs, rose after surgery, in correlation with the CPB time. Given the role of MPs in the induction of coagulation and inflammation, they can be considered therapeutic goals for preventing postoperative complications. In addition, the preoperative levels of MPs are a risk factor for predicting the occurrence of postoperative coagulopathy in heart valve surgery.

Annexin V− and tissue factor+ microparticles as biomarkers for predicting deep vein thrombosis in patients after joint arthroplasty

ObjectiveVenous thromboembolism (VTE) is a common and severe complication of joint arthroplasty. Microparticles (MPs) containing phosphatidylserine (PS) and tissue factor (TF) can trigger coagulation in VTE. This study aims to measure and compare MP levels in joint arthroplasty patients with and without VTE. MethodsThis prospective cohort study enrolled 181 patients who underwent joint arthroplasty. Ultrasound examination was used to diagnose VTE on preoperative day 0 and postoperative day 6. MPs were analysed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The levels of platelet-derived microparticles (PMPs), endothelial cell-derived microparticles (EMPs), granulocyte-derived microparticles (GMPs), red cell-derived microparticles (RMPs), monocyte-derived microparticles (MMPs), Annexin V+ MPs (AV+ MPs), and tissue factor+ MPs (TF+ MPs) derived from five kinds of MPs were measured on day 0 (before surgery), 1, 2, 3, 4, 5, and 6 after surgery. ResultsThe levels of AV-TF+ EMPs and AV-TF+ MMPs were significantly increased in patients with VTE on postoperative day 5 compared to those without VTE (P = 0.031 and P = 0.031, respectively). ConclusionAV-TF+ MPs may indicate the development of VTE and serve as predictive markers in joint arthroplasty patients.

Platelet detection as a new liquid biopsy tool for human cancers.

Cancer is still a leading cause of death worldwide and liquid biopsy is a powerful tool that can be applied to different stages of cancer screening and treatment. However, as the second most abundant cell type in the bloodstream, platelets are isolated through well-established and fast methods in clinic but their value as a BioSource of cancer biomarkers is relatively recent. Many studies demonstrated the bidirectional interaction between cancer cells and platelets. Platelets transfer various proteins (e.g., growth factors, cytokine, chemokines) and RNAs (e.g., mRNA, lncRNA, miRNA, circRNA) into the tumor cells and microenvironment, leading the stimulation of tumor growth and metastasis. In turn, the platelet clinical characteristics (e.g., count and volume) and contents (e.g., RNA and protein) are altered by the interactions with cancer cells and this enables the early cancer detection using these features of platelets. In addition, platelet-derived microparticles also demonstrate the prediction power of being cancer biomarkers. In this review, we focus on the clinical applications of platelet detection using the platelet count, mean platelet volume, platelet RNA and protein profiles for human cancers and discuss the gap in bringing these implementations into the clinic.

Increased Platelet-derived Microparticles Counts is Correlated with Elevated Blood LDL Cholesterol in Acute Myocardial Infarction

BACKGROUND: Platelet-derived microparticles (PDMPs) and low-density lipoprotein (LDL) cholesterol are contributing factors to acute myocardial infarction (AMI). However, the association between LDL cholesterol and PDMPs in AMI has not fully discovered. This study assessed the correlation between these two parameters in patients diagnosed with AMI.METHODS: This was an observational cross-sectional study involving 95 subjects with AMI. The blood measurement of PDMPs counts and LDL cholesterol levels were conducted concomitantly within 24 hours of admission. PDMPs count was analyzed by flow-cytometry method, meanwhile the LDL cholesterol was measured with enzymatic and colorimetric methods. For further analysis, subjects were further divided into LDL cholesterol level ≥130 mg/dL and &lt;130 mg/dL. A statistical test was conducted for a correlative and comparative analyses.RESULTS: A correlative analysis to assess the association between PDMPs counts and LDL cholesterol level depicted a low but significant positive correlation (r=0.231, p=0.024). Furthermore, mean PDMPs counts was significantly higher in subjects with LDL cholesterol level ≥130 mg/dL compared to LDL cholesterol level &lt;130 mg/dL (12,499.59 (95% CI: 8,507.44-16,491.74) counts/μL vs. 9,267.23 (95% CI: 4,445.45-14,089.01) counts/μL; p=0.039).CONCLUSION: There was a significant correlation between PDMPs counts and LDL cholesterol levels in AMI. A significantly increased PDMPs counts were found in subjects with LDL cholesterol level ≥130 mg/dL. Therefore, it is recommended to measure PDMPs in patients with high LDL cholesterol levels as both might be significant AMI biomarkers.KEYWORDS: acute myocardial infarction, LDL-cholesterol, platelet microparticles, platelet activation

Identification and ultrasensitive photoelectrochemical detection of LncNR_040117: a biomarker of recurrent miscarriage and antiphospholipid antibody syndrome in platelet-derived microparticles

The abnormal expression of long non-coding RNAs (LncRNAs) in platelet-derived microparticles (PMPs) is closely related to immune disorders and may lead to antiphospholipid antibody syndrome and recurrent miscarriage. To understand the association between the LncRNAs in PMPs and RM/APS, the differences in the expression of LncRNAs in RM/APS patients and healthy controls were analyzed. Microarray analysis and RT-qPCR detection proved that RM/APS patient exhibited high levels of LncNR_040117 expression. The lentiviral silent expression transfection of HTR-8/SVneo cells indicated that LncNR_040117 downregulation decreased the activity of HTR-8/SVneo cells and inhibited the MAPK signaling pathway, further confirming the biomarker proficiency of LncNR_040117 for RM/APS. After that, we proposed a β-In2S3@g-C3N4 nanoheterojunction-based photoelectrochemical (PEC) biosensor to achieve the ultrasensitive detection of LncNR_040117. The nanoheterojunction aids in the effective separation of photogenerated carriers and significantly improve the photocurrent response of the biosensor. The conjugation of LncNR_040117 onto the PEC biosensing platform increased the steric hindrance between electrolyte and electrode, subsequently decreasing the photocurrent signal. The PEC biosensor showed a wide detection range of 0.1–106 fM and a low limit of detection of 0.025 fM. For clinical sample testing, the results of the PEC and RT-qPCR were highly consistent. Overall, LncNR_040117 in PMPs was identified as an effective biomarker for RM/APS and could be accurately detected by the proposed PEC biosensor, which is expected to provide a reliable diagnostic platform for RM/APS.

Deposition of platelet-derived microparticles in podocytes contributes to diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the developed world. Podocyte injury is a critical cellular event involved in the progression of DN. Our previous studies demonstrated that platelet-derived microparticles (PMPs) mediated endothelial injury in diabetic rats. This study aimed to investigate whether PMPs are deposited in podocytes and to assess their potential effects on podocyte injury in DN. The deposition of PMPs in podocytes was assessed by immunofluorescent staining and electron microscopy. The changes in renal pathology and ultra-microstructure were assessed by periodic acid-Schiff staining and electron microscopy, respectively. The expression of inflammatory cytokines and extracellular matrix proteins was measured by immuno-histochemical staining and western blot. PMPs were widely deposited in podocytes of glomeruli in diabetic patients and animal models and closely associated with DN progression. Interestingly, aspirin treatment significantly inhibited the accumulation of PMPs in the glomeruli of diabetic rats, alleviated mesangial matrix expansion and fusion of foot processes, and decreased the protein expression of inflammatory cytokines and extracellular matrix secretion. An in vitro study further confirmed the deposition of PMPs in podocytes. Moreover, PMP stimulation induced the phenotypic transition of podocytes through decreased podocin protein expression and increased protein expression of α-SMA and fibronectin, which was correlated with increased production of inflammatory cytokines. Our findings demonstrated for the first time that the deposition of PMPs in podocytes contributed to the development of DN.

An Overview of the Latest Applications of Platelet-Derived Microparticles and Nanoparticles in Medical Technology 2010-2020.

Today, Platelets and platelet-derived nanoparticles and microparticles have found many applications in nanomedical technology. The results of our review study show that no article has been published in this field to review the current status of applications of these platelet derivatives so far. Therefore, in the present study, our goal is to compare the applications of platelet derivatives and review their latest status between 2010 and 2020 to present the latest findings to researchers. A very interesting point about the role of platelet derivatives is the presence of molecules on their surface, which makes them capable of hiding from the immune system, reaching different target cells, and specifically attaching to different cell types. According to the results of this study, most of their applications include drug delivery, diagnosis of various diseases, and tissue engineering. However, their application in drug delivery is limited due to heterogeneity, large size, and the possibility of interference with cellular pathways in microparticles derived from other cells. On the other hand, platelet nanoparticles are more controllable and have been widely used for drug delivery in the treatment of cancer, atherosclerosis, thrombosis, infectious diseases, repair of damaged tissue, and photothermal therapy. The results of this study show that platelet nanoparticles are more controllable than platelet microparticles and have a higher potential for use in medicine.

DNA Origami-Platelet Adducts: Nanoconstruct Binding without Platelet Activation.

Objective. Platelets are small, mechanosensitive blood cells responsible for maintaining vascular integrity and activatable on demand to limit bleeding and facilitate thrombosis. While circulating in the blood, platelets are exposed to a range of mechanical and chemical stimuli, with the platelet membrane being the primary interface and transducer of outside-in signaling. Sensing and modulating these interface signals would be useful to study mechanochemical interactions; yet, to date, no methods have been defined to attach adducts for sensor fabrication to platelets without triggering platelet activation. We hypothesized that DNA origami, and methods for its attachment, could be optimized to enable nonactivating instrumentation of the platelet membrane. Approach and Results. We designed and fabricated multivalent DNA origami nanotile constructs to investigate nanotile hybridization to membrane-embedded single-stranded DNA-tetraethylene glycol cholesteryl linkers. Two hybridization protocols were developed and validated (Methods I and II) for rendering high-density binding of DNA origami nanotiles to human platelets. Using quantitative flow cytometry, we showed that DNA origami binding efficacy was significantly improved when the number of binding overhangs was increased from two to six. However, no additional binding benefit was observed when increasing the number of nanotile overhangs further to 12. Using flow cytometry and transmission electron microscopy, we verified that hybridization with DNA origami constructs did not cause alterations in the platelet morphology, activation, aggregation, or generation of platelet-derived microparticles. Conclusions. Herein, we demonstrate that platelets can be successfully instrumented with DNA origami constructs with no or minimal effect on the platelet morphology and function. Our protocol allows for efficient high-density binding of DNA origami to platelets using low quantities of the DNA material to label a large number of platelets in a timely manner. Nonactivating platelet-nanotile adducts afford a path for advancing the development of DNA origami nanoconstructs for cell-adherent mechanosensing and therapeutic agent delivery.

P78: Detection of Platelet-Derived Microparticles via Dynamic Light Scattering

Purpose: Implantable cardiac devices, while effective therapeutically, often generate supraphysiologic shear forces which activate platelets and generate platelet-derived microparticles (PDMPs), all increasing overall thrombotic risk. While a variety of methods exist to measure platelet activation, limited methods exist for ready detection and quantitation of PDMPS. Identifying a core methodology that is useful for translation and development of a portable PDMP assay system will aid in the management of implanted patients reducing both thrombotic and bleeding risks. Here we examine laser light scattering as a method of microparticle detection. Methods: Spherical polystyrene latex microparticles (100-1000 nm in diameter) were suspended in saline and exposed to 520 nm laser light. An avalanche photodiode was used as a light/scatter detector. Signals were processed as to frequency and amplitude. Intensity signals were processed using an autocorrelation function to create calibration curves that vary with particle size. Calibration curves were used to size and quantify the microparticles within the sample, with particle size and concentration displayed on a GUI. The entire system is housed in a dark enclosure to ensure a reliable environment for optical analysis. Results: This system configuration successfully detected particle-induced light scattering. A change in light intensity over time was measured between samples of differing microparticle sizes and concentrations. An autocorrelation function is useful to create different calibration curves that are unique to size and concentration. Conclusion: Laser light scattering is effective as a method for microparticle detection. Our initial system demonstration reveals that both concentration and particle size may be measured via comparison to reference calibration curves. Further advancement and translation of this approach is targeted to yield a portable instrument for clinical utility.

Depletion of tumor associated macrophages enhances local and systemic platelet-mediated anti-PD-1 delivery for post-surgery tumor recurrence treatment

Immunosuppressive cells residing in the tumor microenvironment, especially tumor associated macrophages (TAMs), hinder the infiltration and activation of T cells, limiting the anti-cancer outcomes of immune checkpoint blockade. Here, we report a biocompatible alginate-based hydrogel loaded with Pexidartinib (PLX)-encapsulated nanoparticles that gradually release PLX at the tumor site to block colony-stimulating factor 1 receptors (CSF1R) for depleting TAMs. The controlled TAM depletion creates a favorable milieu for facilitating local and systemic delivery of anti-programmed cell death protein 1 (aPD-1) antibody-conjugated platelets to inhibit post-surgery tumor recurrence. The tumor immunosuppressive microenvironment is also reprogrammed by TAM elimination, further promoting the infiltration of T cells into tumor tissues. Moreover, the inflammatory environment after surgery could trigger the activation of platelets to facilitate the release of aPD-1 accompanied with platelet-derived microparticles binding to PD-1 receptors for re-activating T cells. All these results collectively indicate that the immunotherapeutic efficacy against tumor recurrence of both local and systemic administration of aPD-1 antibody-conjugated platelets could be strengthened by local depletion of TAMs through the hydrogel reservoir.

Study on the impact of the drug (doxorubicin)-loaded platelet microparticles on cancer cells; the Daudi and Vero cell lines

Platelet-derived microparticles (PMPs) may be used as drug carriers for cancer treatments. Doxorubicin with fluorescent properties as a chemotherapeutic drug is available to treat many cancers. This study aims to compare different drug loading methods into PMPs and evaluate their effects on the viability and apoptosis of cancer cells. The platelet concentrates were taken from the Iranian Blood Transfusion Organization. PMPs were isolated on day 5 of platelet storage, and doxorubicin was loaded into PMPs through incubation, cell-penetrating peptides (CPP), and sonication methods. The rate of drug loading into PMPs was measured by the flow cytometry method. Finally, the effect of the drug-loaded microparticles on Daudi and Vero cells was investigated by determining the survival percentage (MTT) and caspase-3 gene expression levels (Real-time PCR). The average fluorescence light calculated in each of the incubation, sonication, and CPP methods was determined to be 79.09% ± 11.37%, 47.48% ± 25.12%, and 56.69% ± 23.24%, respectively. After 48 and 72 h of treating the Vero cells with drug-loaded microparticles, the survival rate decreased, and the expression of the caspase-3 gene was increased. In Daudi cells, similar results were obtained after 72 h of treatments. Also, the incubation method provided a higher rate of drug loading into PMPs. Therefore, this method can be used for drug loading in PMPs. As a result, as drug carriers, PMPs can lower cell viability in cancer cells.

Upregulation of cytokine signalling in platelets increases risk of thrombophilia in severe COVID-19 patients

Abnormal coagulation dynamics, including disseminated intravascular coagulopathy, pulmonary embolism, venous thromboembolism and risk of thrombosis are often associated with the severity of COVID-19. However, very little is known about the contribution of platelets in above pathogenesis. In order to decipher the pathophysiology of thrombophilia in COVID-19, we recruited severely ill patients from ICU, based on the above symptoms and higher D-dimer levels, and compared these parameters with their asymptomatic counterparts. Elevated levels of platelet-derived microparticles and platelet-leukocyte aggregates suggested the hyperactivation of platelets in ICU patients. Strikingly, platelet transcriptome analysis showed a greater association of IL-6 and TNF signalling pathways in ICU patients along with higher plasma levels of IL-6 and TNFα. In addition, upregulation of pathways like blood coagulation and hemostasis, as well as inflammation coexisted in platelets of these patients. Further, the increment of necrotic pathway and ROS-metabolic processes in platelets was suggestive of its procoagulant phenotype in ICU patients. This study suggests that higher plasma IL-6 and TNFα may trigger platelet activation and coagulation, and in turn aggravate thrombosis and hypercoagulation in severe COVID-19 patients. Therefore, the elevated IL-6 and TNFα, may serve as potential risk factors for platelet activation and thrombophilia in these patients.

Ozonation of Whole Blood Results in an Increased Release of Microparticles from Blood Cells.

Autohemotherapy with ozonated blood is used in the treatment of a broad spectrum of clinical disorders. Ozone demonstrates strong oxidizing properties and causes damage to cell membranes. The impact of whole-blood ozonation on the release of microparticles from blood and endothelial cells and the concentration of selected markers in the hemostatic system (APTT, PT, D-dimer, fibrinogen) were investigated. Venous blood, obtained from 19 healthy men, was split into four equal parts and treated with air, 15 µg/mL ozone, or 30 µg/mL ozone, or left untreated. The number and types of microparticles released were determined using flow cytometry on the basis of surface antigen expression: erythrocyte-derived microparticles (CD235+), platelet-derived microparticles (CD42+), leukocyte-derived microparticles (CD45+), and endothelial-derived microparticles (CD144+). The study is the first to demonstrate that ozone induces a statistically significant increase in the number of microparticles derived from blood and endothelial cells. Although statistically significant, the changes in some coagulation factors were somewhat mild and did not exceed normal values.

Plasma microparticles of intubated COVID-19 patients cause endothelial cell death, neutrophil adhesion and netosis, in a phosphatidylserine-dependent manner.

COVID-19 has compelled scientists to better describe its pathophysiology to find new therapeutic approaches. While risk factors, such as older age, obesity, and diabetes mellitus, suggest a central role of endothelial cells (ECs), autopsies have revealed clots in the pulmonary microvasculature that are rich in neutrophils and DNA traps produced by these cells, called neutrophil extracellular traps (NETs.) Submicron extracellular vesicles, called microparticles (MPs), are described in several diseases as being involved in pro-inflammatory pathways. Therefore, in this study, we analyzed three patient groups: one for which intubation was not necessary, an intubated group, and one group after extubation. In the most severe group, the intubated group, platelet-derived MPs and endothelial cell (EC)-derived MPs exhibited increased concentration and size, when compared to uninfected controls. MPs of intubated COVID-19 patients triggered EC death and overexpression of two adhesion molecules: P-selectin and vascular cell adhesion molecule-1 (VCAM-1). Strikingly, neutrophil adhesion and NET production were increased following incubation with these ECs. Importantly, we also found that preincubation of these COVID-19MPs with the phosphatidylserine capping endogenous protein, annexin A5, abolished cytotoxicity, P-selectin and VCAM-1 induction, all like increases in neutrophil adhesion and NET release. Taken together, our results reveal that MPs play a key role in COVID-19 pathophysiology and point to a potential therapeutic: annexin A5.

MicroRNA-363-3p/sphingosine-1-phosphate receptor 1 axis inhibits sepsis-induced acute lung injury via the inactivation of nuclear factor kappa-B ligand signaling.

Infection-associated inflammation and coagulation are critical pathologies in sepsis-induced acute lung injury (ALI). This study aimed to investigate the effects of microRNA-363-3p (miR-363-3p) on sepsis-induced ALI and explore the underlying mechanisms. A cecal ligation and puncture-induced septic mouse model was established. The results of this study suggested that miR-363-3p was highly expressed in lung tissues of septic mice. Knockdown of miR-363-3p attenuated sepsis-induced histopathological damage, the inflammation response and oxidative stress in lung tissues. Furthermore, knockdown of miR-363-3p reduced the formation of platelet-derived microparticles and thrombin generation in blood samples of septic mice. Downregulation of miR-363-3p suppressed sphingosine-1-phosphate receptor 1 (S1PR1) expression in lung tissues and subsequently inactivated the nuclear factor kappa-B ligand (NF-κB) signaling. A luciferase reporter assay confirmed that miR-363-3p directly targeted the 3’-untranslated region of the mouse S1pr1 mRNA. Collectively, our study suggests that inactivation of NF-κB signaling is involved in the miR-363-3p/S1PR1 axis-mediated protective effect on septic ALI.

Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients.

Infection with SARS‐CoV‐2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID‐19 patients. In addition, immune dysfunction occurs in patients characterised by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets and platelet‐derived microparticles (PMP), during the clinical course of COVID‐19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID‐19 diagnosis. Elevated frequencies of PS+PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition and coagulation, currently used for COVID‐19 clinical scoring. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death‐ligand 1 (PD‐L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes towards lymphocytes and cause immune dysfunction in COVID‐19.