Extracellular vesicles and coagulation in blood from healthy humans revisited.

: In 2001, we studied the presence and coagulant properties of "microparticles" in the blood of healthy humans. Since then, multiple improvements in detection, isolation and functional characterization of the now called "extracellular vesicles" (EVs) have been made, and shortcomings were identified. : To revisit the presence and function of EVs in blood from healthy humans.

Randomized controlled trial protocol to investigate the antiplatelet therapy effect on extracellular vesicles (AFFECT EV) in acute myocardial infarction.

Activated platelets contribute to thrombosis and inflammation by the release of extracellular vesicles (EVs) exposing P-selectin, phosphatidylserine (PS) and fibrinogen. P2Y12 receptor antagonists are routinely administered to inhibit platelet activation in patients after acute myocardial infarction (AMI), being a combined antithrombotic and anti-inflammatory therapy. The more potent P2Y12 antagonist ticagrelor improves cardiovascular outcome in patients after AMI compared to the less potent clopidogrel, suggesting that greater inhibition of platelet aggregation is associated with better prognosis.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

Background: Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.

Objective: This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.

Comparison of Generic Fluorescent Markers for Detection of Extracellular Vesicles by Flow Cytometry.

Background: Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry.

Absolute sizing and label-free identification of extracellular vesicles by flow cytometry.

Blood contains extracellular vesicles (EVs), which are biological nanoparticles with clinical applications. In blood plasma, EVs are outnumbered by similar-sized lipoprotein particles (LPs), leading to controversial data such as non-specific binding of antibodies to LPs. Flow cytometry is a clinically applicable technique to characterize single EVs in body fluids.

Development of Peritoneal Carcinomatosis in Epithelial Ovarian Cancer: A Review.

Epithelial ovarian cancer (EOC) metastasizes intra-abdominally with often numerous, superficial, small-sized lesions. This so-called peritoneal carcinomatosis is difficult to treat, and peritoneal recurrences are frequently observed, leading to a poor prognosis. Underlying mechanisms of interactions between EOC and peritoneal cells are incompletely understood.

Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles.

Background: Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface.

Methods: We investigated the potential of a 48-multiplex surface plasmon resonance imaging (SPRi) system to perform EV phenotyping. Antigen surface density of 11 antigens was measured on the human breast cancer cell lines HS578T, MCF7, and SKBR3 and their EVs by use of both SPRi and the widely used flow cytometry (FCM).

Handling and storage of human body fluids for analysis of extracellular vesicles.

Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze-thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated.

Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.

Background: Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research.

Clinical Significance of Tissue Factor-Exposing Microparticles in Arterial and Venous Thrombosis.

Microparticles (MP) are small extracellular vesicles (30-1,000 nm) that are released from activated cells or platelets. Exposure of negatively charged phospholipids and tissue factor (TF) renders MP procoagulant. Normal plasma levels of intravascular TF-exposing MP (TFMP) are low, but their number may rise in pathological conditions, including cancer and infectious disease.

Single-step isolation of extracellular vesicles by size-exclusion chromatography.

Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively.

Aim: To develop a single-step protocol to isolate vesicles from human body fluids.

Refractive index determination of nanoparticles in suspension using nanoparticle tracking analysis.

The refractive index (RI) dictates interaction between light and nanoparticles and therefore is important to health, environmental, and materials sciences. Using nanoparticle tracking analysis, we have determined the RI of heterogeneous particles <500 nm in suspension. We demonstrate feasibility of distinguishing silica and polystyrene beads based on their RI.

Effects of cancer on platelets.

The main function of circulating platelets is to stop bleeding upon vascular injury by the formation of a hemostatic plug. The presence of cancer results in numerical and functional abnormalities of platelets. Thrombocytosis is commonly observed in cancer patients and is associated with decreased survival.

Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.

Extracellular vesicles (EVs) facilitate intercellular communication by carrying bioactive molecules such as proteins, messenger RNA, and micro (mi)RNAs. Recently, high-density lipoproteins (HDL) isolated from human plasma were also reported to transport miRNA to other cells. HDL, when isolated from human plasma, ranges in density between 1.

Laboratory evaluation of the Coasys® Plus C coagulation analyzer.

Introduction: The Coasys® Plus C (Behnk Elektronik, distributed by Roche Diagnostics) is a coagulation analyzer for small to midsize clinical chemistry laboratories. We performed a laboratory evaluation.

Materials And Methods: After a familiarization period the dead volume, carry-over, capacity, within-assay reproducibility and imprecision were determined for the tests aPTT (STA APTT en STA Cephascreen), PT (STA Neoplastin Plus, STA Neoplastin R), INR (Hepato Quick), fibrinogen (STA Fibrinogen), antithrombin (Antithrombin III) and D-Dimer (Tina-quant D-Dimer Gen.

Extracellular vesicles in physiological and pathological conditions.

Body fluids contain surprising numbers of cell-derived vesicles which are now thought to contribute to both physiology and pathology. Tools to improve the detection of vesicles are being developed and clinical applications using vesicles for diagnosis, prognosis, and therapy are under investigation. The increased understanding why cells release vesicles, how vesicles play a role in intercellular communication, and how vesicles may concurrently contribute to cellular homeostasis and host defense, reveals a very complex and sophisticated contribution of vesicles to health and disease.

Microparticles in vascular disorders: how tissue factor-exposing vesicles contribute to pathology and physiology.

Coagulation is initiated by tissue factor (TF). Coagulant TF is constitutively expressed by extravascular cells, but there is increasing evidence that TF can also be present within the blood, in particular during pathological conditions. Such TF is exposed on circulating cell-derived vesicles, and its presence has been associated with development of disseminated intravascular coagulation and venous thrombosis.

Coagulation activation and microparticle-associated coagulant activity in cancer patients. An exploratory prospective study.

Cancer increases the risk of venous thromboembolism (VTE). Here, we investigated the contribution of microparticle (MP)-dependent procoagulant activity to the prothrombotic state in these patients. In 43 cancer patients without VTE at study entry and 22 healthy volunteers, markers of in vivo and MP-dependent coagulation were measured and patients were prospectively followed for six months for the development of VTE.

The functions of microparticles in pre-eclampsia.

Pre-eclampsia (P-EC), a heterogenic multisystem disorder characterized by hypertension and proteinuria, usually develops in the second half of pregnancy. The incidence is 2 to 5%, and P-EC is therefore a major cause of maternal and perinatal morbidity and mortality. Although the exact etiology is unknown, placental factors released into the maternal circulation lead to systemic maternal inflammation and endothelial dysfunction.

Cell-derived vesicles exposing coagulant tissue factor in saliva.

On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown.

Platelet microparticles contain active caspase 3.

During storage, platelets undergo processes resembling apoptosis, including microparticle release, aminophospholipid exposure, and procaspase 3 processing. Recently, we showed that microparticles from endothelial cells contain caspase 3, one of the executioner enzymes of apoptosis. In this study we determined whether platelet-derived microparticles (PMP) contain caspase 3 in vitro (stored platelet concentrate) and ex vivo (plasma from healthy humans).