Leukocyte Extracellular Vesicles Predict Progression of Systolic Dysfunction in Heart Failure with Mildly Reduced Ejection Fraction (LYCHEE) - A Prospective, Multicentre Cohort Study.

Risk stratification in heart failure with mildly-reduced ejection fraction (HFmrEF) remains challenging. We evaluated the predictive value of advanced glycation end products (AGEs) and plasma concentrations of extracellular vesicles (EVs) for the systolic and diastolic dysfunction progression in HFmrEF patients. Skin AGE accumulation was measured using AGE Reader. Plasma EV concentrations were measured using flow cytometry. Among 74 patients enrolled, 13 (18%) had systolic dysfunction progression and 5 (7%) had diastolic dysfunction progression during 6.5 months follow-up. Leukocyte EVs concentrations were higher in patients with systolic dysfunction progression (p = 0.002) and predicted the progression with 75.0% sensitivity and 58.3% specificity, independent of other clinical variables (OR 4.72, 95% CI 0.99-22.31). Skin AGE levels and concentrations of other EV subtypes were not associated with systolic or diastolic dysfunction progression. Increased leukocyte EVs concentrations are associated with 4.7-fold higher odds of systolic dysfunction progression in HFmrEF patients.

MIBLood-EV: An Online Reporting Tool to Facilitate the Standardized Reporting of Preanalytical Variables and Quality Control of Plasma and Serum to Enhance Rigor and Reproducibility in Liquid Biopsy Research.

Pre-analytical variability significantly impacts the reproducibility of liquid biopsy research, which is critical for precision medicine and biomedical research. This report highlights the challenges and variability in the pre-analytical processes of liquid biopsies, especially regarding extracellular vesicles (EVs), which are crucial for diagnostics in oncology. The MIBlood-EV initiative aims to standardize the reporting of pre-analytical variables and the quality control of plasma and serum samples to enhance reproducibility in EV research. By providing a comprehensive and flexible reporting framework, MIBlood-EV seeks to improve the reliability of EV studies and facilitate the development of evidence-based protocols, ultimately advancing the field of liquid biopsy research.

Proteome encoded determinants of protein sorting into extracellular vesicles.

Extracellular vesicles (EVs) are membranous structures released by cells into the extracellular space and are thought to be involved in cell-to-cell communication. While EVs and their cargo are promising biomarker candidates, sorting mechanisms of proteins to EVs remain unclear. In this study, we ask if it is possible to determine EV association based on the protein sequence. Additionally, we ask what the most important determinants are for EV association. We answer these questions with explainable AI models, using human proteome data from EV databases to train and validate the model. It is essential to correct the datasets for contaminants introduced by coarse EV isolation workflows and for experimental bias caused by mass spectrometry. In this study, we show that it is indeed possible to predict EV association from the protein sequence: a simple sequence-based model for predicting EV proteins achieved an area under the curve of 0.77 ± 0.01, which increased further to 0.84 ± 0.00 when incorporating curated post-translational modification (PTM) annotations. Feature analysis shows that EV-associated proteins are stable, polar, and structured with low isoelectric point compared to non-EV proteins. PTM annotations emerged as the most important features for correct classification; specifically, palmitoylation is one of the most prevalent EV sorting mechanisms for unique proteins. Palmitoylation and nitrosylation sites are especially prevalent in EV proteins that are determined by very strict isolation protocols, indicating they could potentially serve as quality control criteria for future studies. This computational study offers an effective sequence-based predictor of EV associated proteins with extensive characterisation of the human EV proteome that can explain for individual proteins which factors contribute to their EV association.

Poloxamer-188 as a wetting agent for microfluidic resistive pulse sensing measurements of extracellular vesicles.

INTRODUCTION: Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20. METHODS: An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined. RESULTS: Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 µs, which is lower than 56 µs for BSA and 16 µs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 µs, which confirms the reliability of MRPS measurements. CONCLUSION: The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.

Extracellular Vesicles to Predict Outcomes After Transcatheter Aortic Valve Implantation - a Prospective, Multicenter Cohort Study.

INTRODUCTION: Transcatheter aortic valve implantation (TAVI) is an established treatment for aortic stenosis (AS) in patients at intermediate and high surgical risk. Circulating extracellular vesicles (EVs) are nanoparticles involved in cardiovascular diseases. We aimed to (i) determine the effect of TAVI on plasma concentrations of five EV subtypes and (ii) evaluate the predictive value of EVs for post-TAVI outcomes. METHODS: Blood samples were collected 1 day before TAVI and at hospital discharge. Concentrations of EVs were evaluated using flow cytometry. RESULTS: Concentration of leukocytes EVs decreased after TAVI, compared to the measurement before (p = 0.008). Among 123 patients discharged from the hospital, 19.5% experienced MACCE during the median of 10.3 months. Increased pre-TAVI concentration of phosphatidylserine-exposing EVs was an independent predictor of MACCE in multivariable analysis (OR 5.313, 95% CI 1.164-24.258, p = 0.031). CONCLUSIONS: Patients with increased pre-TAVI concentration of procoagulant, PS-exposing EVs have over fivefold higher odds of adverse outcomes.

M1-derived extracellular vesicles polarize recipient macrophages into M2-like macrophages and alter skeletal muscle homeostasis in a hyper-glucose environment.

BACKGROUND: Macrophages release not only cytokines but also extracellular vesicles (EVs). which are small membrane-derived nanovesicles with virus-like properties transferring cellular material between cells. Until now, the consequences of macrophage plasticity on the release and the composition of EVs have been poorly explored. In this study, we determined the impact of high-glucose (HG) concentrations on macrophage metabolism, and characterized their derived-EV subpopulations. Finally, we determined whether HG-treated macrophage-derived EVs participate in immune responses and in metabolic alterations of skeletal muscle cells. METHODS: THP1-macrophages were treated with 15mM (MG15) or 30mM (MG30) glucose. Then, M1/M2 canonical markers, pro- and anti-inflammatory cytokines, activities of proteins involved in glycolysis or oxidative phosphorylation were evaluated. Macrophage-derived EVs were characterized by TEM, NTA, MRSP, and 1H-Nuclear magnetic resonance spectroscopy for lipid composition. Macrophages or C2C12 muscle cells were used as recipients of MG15 and MG30-derived EVs. The lipid profiles of recipient cells were determined, as well as proteins and mRNA levels of relevant genes for macrophage polarization or muscle metabolism. RESULTS: Untreated macrophages released small and large EVs (sEVs, lEVs) with different lipid distributions. Proportionally to the glucose concentration, glycolysis was induced in macrophages, associated to mitochondrial dysfunction, triacylglycerol and cholesterol accumulation. In addition, MG15 and MG30 macrophages had increased level of CD86 and increase release of pro-inflammatory cytokines. HG also affected macrophage sphingolipid and phospholipid compositions. The differences in the lipid profiles between sEVs and lEVs were abolished and reflected the lipid alterations in MG15 and MG30 macrophages. Interestingly, MG15 and MG30 macrophages EVs induced the expression of CD163, Il-10 and increased the contents of triacylglycerol and cholesterol in recipient macrophages. MG15 lEVs and sEVs induced insulin-induced AKT hyper-phosphorylation and accumulation of triacylglycerol in myotubes, a state observed in pre-diabetes. Conversely, MG30 lEVs and sEVs induced insulin-resistance in myotubes. CONCLUSIONS: As inflammation involves first M1 macrophages, then the activation of M2 macrophages to resolve inflammation, this study demonstrates that the dialog between macrophages through the EV route is an intrinsic part of the inflammatory response. In a hyperglycemic context, EV macrophages could participate in the development of muscle insulin-resistance and chronic inflammation.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Lipoprotein apheresis affects the concentration of extracellular vesicles in patients with elevated lipoprotein (a).

Lipoprotein apheresis (LA) is a therapeutic option for hyperlipoproteinemia(a) (hyper-Lp(a)) and atherosclerotic cardiovascular disease (ASCVD). LA improves blood rheology, reduces oxidative stress parameters and improves endothelial function. The underlying molecular mechanisms of LA beneficial effects are unknown, but it has been suggested that LA exhibits multiple activities beyond simply removing lipoproteins. We hypothesized that LA removes not only lipoproteins, but also extracellular vesicles (EVs). To test this hypothesis, we performed a prospective study in 22 patients undergoing LA for hyper-Lp(a) and ASCVD. Different EVs subtypes were measured before and directly after LA, and after 7 days. We used calibrated flow cytometry to detect total particle concentration (diameter > ~ 100 nm), total lipoproteins concentration (diameter > 200 nm, RI > 1.51), total EV concentration (diameter > 200 nm, RI < 1.41), concentrations of EVs derived from erythrocytes (CD235a+; diameter > 200 nm, RI < 1.41), leukocytes (CD45+; diameter > 200 nm, RI < 1.41) and platelets (CD61+, PEVs; diameter > 200 nm, RI < 1.41). LA reduced the concentrations of all investigated EVs subtypes and lipoproteins. Lp(a) concentration was lowered by 64.5% [(58% - 71%); p < 0.001]. Plasma concentrations of EVs > 200 nm in diameter derived from platelets (CD61 +), leukocytes (CD45+) and erythrocytes (CD235a+) decreased after single LA procedure by 42.7% [(12.8-54.7); p = 0.005], 42.6% [(29.7-54.1); p = 0.030] and 26.7% [(1.0-62.7); p = 0.018], respectively, compared to baseline. All EV subtypes returned to the baseline concentrations in blood plasma after 7 days. To conclude, LA removes not only Lp(a), but also cell-derived EVs, which may contribute to LA beneficial effects.

Traceable characterization of hollow organosilica beads as potential reference materials for extracellular vesicle measurements with optical techniques.

The concentration of cell-type specific extracellular vesicles (EVs) is a promising biomarker for various diseases. However, concentrations of EVs measured by optical techniques such as flow cytometry (FCM) or particle tracking analysis (PTA)  in clinical practice are incomparable. To allow reliable and comparable concentration measurements suitable reference materials (RMs) and SI-traceable (SI-International system of units) methods are required. Hollow organosilica beads (HOBs) are promising RM candidates for concentration measurements of EVs based on light scattering, as the shape, low refractive index, and number concentration of HOBs are comparable to EVs of the respective size range that can be detected with current optical instrumentation. Here, we present traceable methods for measuring the particle size distribution of four HOB types in the size range between 200 and 500 nm by small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), as well as the number concentration by single-particle inductively coupled plasma mass spectrometry (spICP-MS). Based on the size and shape results, traceable reference values were obtained to additionally determine the refractive index of the shell of the HOB samples by FCM. Furthermore, the estimated refractive indexes of the HOBs plausibly agree with the refractive indexes of EVs of corresponding size. Due to their narrow size distribution and their similar shape, and low refractive index, all HOB samples studied are suitable RM candidates for calibration of the measured sample volume by optical methods within the photon wavelength range used, and thus for calibration of number concentration measurements of EVs in the size range indicated. This was confirmed as the number concentration values obtained by PTA and two independent flow cytometric measurements agreed with the concentration reference values obtained by two independent spICP-MS measurements within the calculated uncertainty limits.

A beginner's guide to study extracellular vesicles in human blood plasma and serum.

Blood is the most commonly used body fluid for obtaining and studying extracellular vesicles (EVs). While blood is a standard choice for clinical analysis, using blood as a source of EVs introduces multiple layers of complexity. At the Blood Extracellular Vesicle Workshop organized by the International Society for Extracellular Vesicles in Helsinki (2022), it became evident that beginner researchers lack trustworthy information on how to initiate their research and avoid common pitfalls. This educational guide explains the composition and frequently used terminology of blood, provides guidelines for blood collection, and the preparation of plasma and serum. It also introduces the basic principles of isolating and detecting blood EVs while considering blood-related factors. The goal of this guide is to assist beginners by offering a concise and evidence-based introduction to the current knowledge and available resources to study blood EVs.

Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

MIBlood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor-dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre-analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood-EV), a tool to record and report information about pre-analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre-analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence-based optimization of pre-analytical protocols through meta-analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter-laboratory comparative EV studies, and (iv) improve the peer review process.

Plasma extracellular vesicle test sample to standardize flow cytometry measurements.

Background: Extracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. Objectives: To improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry-compatible, and iv) is stable. Methods: The PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at -80 °C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). Results: We demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at -80 °C for 12 months when stored in trehalose. Conclusion: The PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry-compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures.

Extracellular Vesicles in Coronary Artery Disease.

Coronary artery disease (CAD) is the leading cause of death and disability worldwide. Despite recent progress in the diagnosis and treatment of CAD, evidence gaps remain, including pathogenesis, the most efficient diagnostic strategy, prognosis of individual patients, monitoring of therapy, and novel therapeutic strategies. These gaps could all be filled by developing novel, minimally invasive, blood-based biomarkers. Potentially, extracellular vesicles (EVs) could fill such gaps. EVs are lipid membrane particles released from cells into blood and other body fluids. Because the concentration, composition, and functions of EVs change during disease, and because all cell types involved in the development and progression of CAD release EVs, currently available guidelines potentially enable reliable and reproducible measurements of EVs in clinical trials, offering a wide range of opportunities. In this chapter, we provide an overview of the associations reported between EVs and CAD, including (1) the role of EVs in CAD pathogenesis, (2) EVs as biomarkers to diagnose CAD, predict prognosis, and monitor therapy in individual patients, and (3) EVs as new therapeutic targets and/or drug delivery vehicles. In addition, we summarize the challenges encountered in EV isolation and detection, and the lack of standardization, which has hampered real clinical applications of EVs. Since most conclusions are based on animal models and single-center studies, the knowledge and insights into the roles and opportunities of EVs as biomarkers in CAD are still changing, and therefore, the content of this chapter should be seen as a snapshot in time rather than a final and complete compendium of knowledge on EVs in CAD.

Preventing swarm detection in extracellular vesicle flow cytometry: a clinically applicable procedure.

Background: Flow cytometry is commonly used to detect cell-derived extracellular vesicles in body fluids such as blood plasma. However, continuous and simultaneous illumination of multiple particles at or below the detection limit may result in the detection of a single event. This phenomenon is called swarm detection and leads to incorrect particle concentration measurements. To prevent swarm detection, sample dilution is recommended. Since the concentration of particles differs between plasma samples, finding the optimal sample dilution requires dilution series of all samples, which is unfeasible in clinical routine. Objectives: Here we developed a practical procedure to find the optimal sample dilution of plasma for extracellular vesicle flow cytometry measurements in clinical research studies. Methods: Dilution series of 5 plasma samples were measured with flow cytometry (Apogee A60-Micro), triggered on side scatter. The total particle concentration between these plasma samples ranged from 2.5 × 109 to 2.1 × 1011 mL-1. Results: Swarm detection was absent in plasma samples when diluted ≥1.1 × 103-fold or at particle count rates <3.0 × 103 events·s-1. Application of either one of these criteria, however, resulted in insignificant particle counts in most samples. The best approach to prevent swarm detection while maintaining significant particle counts was by combining minimal dilution with maximum count rate. Conclusion: To prevent swarm detection in a series of clinical samples, the measurement count rate of a single diluted plasma sample can be used to determine the optimal dilution factor. For our samples, flow cytometer, and settings, the optimal dilution factor is ≥1.1 × 102-fold, while the count rate is <1.1 × 104 events·s-1.

Standardization of extracellular vesicle concentration measurements by flow cytometry: the past, present, and future.

Concentrations of extracellular vesicles (EVs) in body fluids are being explored as disease biomarkers. Most laboratories use flow cytometry to characterize single EVs at high throughput. A flow cytometer (FCM) detects light scattering and fluorescence intensities of EVs. However, detection of EVs by flow cytometry is complicated for 2 reasons. First, EVs are small and have weak light scattering and fluorescence signals compared to cells and are, therefore, hard to detect. Second, FCMs differ in sensitivity and provide data in arbitrary units, which complicates data interpretation. Due to the mentioned challenges, the measured concentration of EVs by flow cytometry is cumbersome to compare between FCMs and institutes. To improve comparability, standardization and development of traceable reference materials to calibrate all aspects of an FCM are needed, as are interlaboratory comparison studies. Within this article, we will provide an overview of the standardization of EV concentration measurements, including the current effort to introduce robust calibration of FCMs, thereby enabling comparable concentration measurements of EVs, which in turn can be used to establish clinically relevant reference ranges of EV concentrations in blood plasma and other body fluids.

Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics.

The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; six preservation and five non-preservation) and three blood processing intervals (BPI; 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies.

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Anti-high-mobility group box-1 treatment strategies improve trauma-induced coagulopathy in a mouse model of trauma and shock.

BACKGROUND: Trauma-induced coagulopathy is associated with platelet dysfunction and contributes to early mortality after traumatic injury. Plasma concentrations of the damage molecule high-mobility group box-1 (HMGB-1) increase after trauma, which may contribute to platelet dysfunction. We hypothesised that inhibition of HMGB-1 with a monoclonal antibody (mAb) or with recombinant thrombomodulin (rTM) improves trauma-induced coagulopathy in a murine model of trauma and shock. METHODS: Male 129S2/SvPasOrlRJ mice were anaesthetised, mechanically ventilated, and randomised into five groups: (i) ventilation control (VENT), (ii) trauma/shock (TS), (iii) TS+anti-HMGB-1 mAb (TS+AB), (iv) TS+rTM (TS+TM), and (v) TS+anti-HMGB-1 mAb+rTM (TS+COMBI). Primary outcome was rotational thromboelastometry EXTEM. Secondary outcomes included tail bleeding time, platelet count, plasma HMGB-1 concentration, and platelet activation. RESULTS: Trauma and shock resulted in a hypocoagulable thromboelastometry profile, increased plasma HMGB-1, and increased platelet activation markers. TS+AB was associated with improved clot firmness after 5 min compared with TS (34 [33-37] vs 32 [29-34] mm; P=0.043). TS+COMBI was associated with decreased clot formation time (98 [92-125] vs 122 [111-148] s; P=0.018) and increased alpha angle (77 [72-78] vs 69 [64-71] degrees; P=0.003) compared with TS. TS+COMBI also reduced tail bleeding time compared with TS (P=0.007). The TS+TM and TS+COMBI groups had higher platelet counts compared with TS (P=0.044 and P=0.041, respectively). CONCLUSIONS: Inhibition of HMGB-1 early after trauma in a mouse model improves clot formation and strength, preserves platelet count, and decreases bleeding time.