Augueste Sturk

Classification, Functions, and Clinical Relevance of Extracellular Vesicles

Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discussed.

Elevated Numbers of Tissue-Factor Exposing Microparticles Correlate With Components of the Metabolic Syndrome in Uncomplicated Type 2 Diabetes Mellitus

Background—Type 2 diabetes is associated with accelerated atherosclerosis. Because cell-derived microparticles support coagulation and inflammation, they may be involved in atherogenesis. We characterized circulating microparticles both in patients with uncomplicated, well-regulated type 2 diabetes and in healthy subjects, as well as their relationship with coagulation and metabolic control. Methods and Results—Microparticles were isolated from plasma, stained with annexin V, cell-specific monoclonal antibodies (MoAbs) and a MoAb directed against tissue factor (TF), and analyzed by flow cytometry. Microparticle numbers and origin were comparable in the two groups, but the median number of TF-positive microparticles was twice as high in patients than in controls (P =0.018). Patients had higher percentages of TF-positive microparticles from T-helper cells (P =0.045), granulocytes (P =0.004), and platelets (P =0.002). Subpopulations of TF-positive microparticles from platelets and T-helper cells exposed granulocytic markers. Correlations were found between the numbers of various TF-positive microparticle subpopulations and body mass index, fasting plasma glucose and insulin, or tumor necrosis factor-&agr; and serum HDL cholesterol. Microparticles from patients generated less thrombin in vitro (P =0.007). Microparticle numbers did not correlate with in vivo coagulation markers prothrombin fragment F1+2 and thrombin-antithrombin complexes. Conclusions—TF, possibly of granulocytic origin, is exposed on microparticle subpopulations in asymptomatic patients with well-regulated type 2 diabetes. TF-positive microparticles are associated with components of the metabolic syndrome but not with coagulation. Thus, TF on microparticles may be involved in processes other than coagulation, including transcellular signaling or angiogenesis.

Cell-Derived Microparticles Generated in Patients During Cardiopulmonary Bypass Are Highly Procoagulant

BACKGROUND Microparticles from platelets and other cells have been extensively studied and characterized in vitro. Although the level of platelet-derived microparticles is elevated in a variety of diseases, including cardiac surgery, virtually nothing is known about their functions in vivo. The aim of the present study was to investigate the procoagulant properties of microparticles generated in vivo. METHODS AND RESULTS In 6 patients at the end of cardiopulmonary bypass, 14.8 x 10(9)/L (median; range, 9.7 to 27.4 x 10(9)/L) platelet-derived microparticles were present in pericardial blood, whereas blood obtained from the systemic circulation contained 1.6 x 10(9)/L (median; range, 0.4 to 8.9 x 10(9)/L) of such microparticles, as determined by flow cytometry. Microparticles stained positively for phosphatidylserine as determined with labeled annexin V. In contrast to systemic blood, pericardial blood contained not only microparticles of platelet origin but also microparticles that originated from erythrocytes, monocytes, or granulocytes, and other hitherto unknown cellular sources. Plasma prepared from pericardial blood and to a lesser extent plasma from systemic blood obtained at the same time, stimulated formation of thrombin in vitro. This activity of pericardial plasma was lost after removal of its microparticles by high-speed centrifugation, whereas the corresponding microparticle pellet was strongly procoagulant. The generation of thrombin in vitro involved a tissue factor/factor VII-dependent and factor XII-independent pathway. CONCLUSIONS This study is the first to demonstrate that microparticles generated in vivo can stimulate coagulation.

Value of Myoglobin, Troponin T, and CK-MBmass in Ruling Out an Acute Myocardial Infarction in the Emergency Room

BACKGROUND Ruling out acute myocardial infarction (AMI) on the basis of rapid assays for cardiac markers will allow early triage of patients and cost-effective use of available coronary care facilities. METHODS AND RESULTS We studied the value of myoglobin, creatine kinase (CK)-MBmass, and troponin T in ruling out an AMI in the emergency room in 309 consecutive patients presenting with chest pain. The gold standard for AMI was the combination of history, ECG, and a typical curve of the CK-MB activity (CK-MBact). Myoglobin was the earliest marker, and its negative predictive value (NPV) was significantly higher than for CK-MBmass and troponin T from 3 to 6 hours after the onset of symptoms (myoglobin versus CK-MBmass, P < .03; myoglobin versus troponin T, P < .01). The NPV of myoglobin reached 89% 4 hours after the onset of symptoms. The NPV of CK-MBmass reached 95% 7 hours after the onset of symptoms. Troponin T was not an early marker for ruling out AMI, and NPV changed over time, together with CK-MBact. The early NPV was higher in a subgroup of patients with a low probability of the presence of AMI for the three markers. Cardiac markers rise earlier in patients with large infarcts than in patients with small infarcts as indicated by the cumulative proportion of the marker above the upper reference limit at each time point (myoglobin, P = .04; CK-MBmass, P = .013; troponin T, P = .016). CONCLUSIONS For ruling out AMI in the emergency room, myoglobin is a better marker than CK-MBmass or troponin T from 3 until 6 hours after the onset of symptoms, but the maximal NPV reaches only 89%. At 7 hours, the NPV of CK-MBmass is 95%. The test characteristics are influenced by the probability of the presence of AMI in the patients studied and by the size of their AMI. Infarct size of AMI patients should be reported in studies evaluating cardiac markers.

ENDOTOXAEMIA: AN EARLY PREDICTOR OF SEPTICAEMIA IN FEBRILE PATIENTS

In 473 consecutive febrile patients a sensitive and rapid chromogenic limulus assay was used to assess the value of endotoxaemia versus bacteraemia for predicting development of the syndrome of septicaemia. In each patient three blood specimens for culture and endotoxin testing were obtained at the onset of fever. Blood pressure, urinary output, and the occurrence of thrombocytopenia and metabolic acidosis were recorded prospectively during three days of follow-up. Septicaemia developed in 19 patients (4%). The sensitivity, specificity, and likelihood ratio for a positive result with the endotoxin assay were 79%, 96%, and 20, respectively. The corresponding indices for bacteraemia were 89%, 78%, and only 4. The results suggest that endotoxaemia is a clinically valid indicator for impending gram-negative septicaemia (positive predictive value 48%) and that the absence of endotoxaemia virtually rules out the risk that septicaemia will ensue (negative predictive value 99%).

Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner

Summary.  Background: Circulating microparticles of various cell types are present in healthy individuals and, in varying numbers and antigenic composition, in various disease states. To what extent these microparticles contribute to coagulation in vivo is unknown. Objectives: To examine the in vivo thrombogenicity of human microparticles. Methods: Microparticles were isolated from pericardial blood of cardiac surgery patients and venous blood of healthy individuals. Their numbers, cellular source, and tissue factor (TF) exposure were determined using flow cytometry. Their in vitro procoagulant properties were studied in a fibrin generation test, and their in vivo thrombogenicity in a rat model. Results: The total number of microparticles did not differ between pericardial samples and samples from healthy individuals (P = 0.786). In both groups, microparticles from platelets, erythrocytes, and granulocytes exposed TF. Microparticle‐exposed TF antigen levels were higher in pericardial compared with healthy individual samples (P = 0.036). Pericardial microparticles were strongly procoagulant in vitro and highly thrombogenic in a venous stasis thrombosis model in rats, whereas microparticles from healthy individuals were not [thrombus weights 24.8 (12.2–41.3) mg vs. 0 (0–24.3) mg median and range; P < 0.001]. Preincubation of pericardial microparticles with an inhibitory antibody against human TF abolished their thrombogenicity [0 (0–4.4) mg; P < 0.01], while a control antibody had no effect [19.6 (12.6–53.7) mg; P > 0.05]. The thrombogenicity of the microparticles correlated strongly with their TF exposure (r = 0.9524, P = 0.001). Conclusions: Human cell‐derived microparticles promote thrombus formation in vivo in a TF‐dependent manner. They might be the direct cause of an increased thromboembolic tendency in various patient groups.

Enhanced thrombin generation in normal and hypertensive pregnancy.

We investigated the plasma levels of thrombin-antithrombin III complexes in women with uncomplicated pregnancy, patients with preeclampsia, gestational hypertension, and nonpregnant control subjects. In addition, we measured the coagulation inhibitors antithrombin III, protein C, and protein S. In normal pregnancy we observed a progressive increase in plasma thrombin-antithrombin III levels, and a decrease in protein S levels. In preeclampsia we observed increased thrombin-antithrombin III levels, reduced antithrombin III and protein C levels, and no further reduction of protein S compared with normal pregnancy. These new methods provide solid evidence for a prethrombotic state in normal pregnancy, especially in preeclampsia.

Genetic influence on cytokine production in meningococcal disease.

1912 Vol 349 • June 28, 1997 production with low interleukin-10 production. The figure shows that in healthy volunteers the maximum production of interleukin-10 is independent of the production of TNFwhen determined in a whole blood stimulation assay in vitro. TNFis a double-edged sword, it is a necessary mediator in the host response to infection but high circulating concentrations can be harmful. We propose a complete switch in the understanding of proinflammatory and anti-inflammatory cytokines that contribute to sepsis syndrome. Our data suggest that patients with low production of TNFand high production of interleukin10 early in fulminant meningococcal disease have an insufficient host response to infection and end up with a large bacterial burden, high circulating concentrations of cytokines, and a poor outcome.

Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease

It has long been known that patients with sickle cell disease have ongoing activation of their coagulation system, which is exacerbated during painful occlusive crises. In this paper, the authors explore the role of the increased numbers of erythrocyte derived microparticles in this phenomenon and suggest that a surprisingly large proportion of this is dependent on Factor XI. See related perspective article on page 1481. Background Sickle cell disease is characterized by a hypercoagulable state as a result of multiple factors, including chronic hemolysis and circulating cell-derived microparticles. There is still no consensus on the cellular origin of such microparticles and the exact mechanism by which they may enhance coagulation activation in sickle cell disease. Design and Methods In the present study, we analyzed the origin of circulating microparticles and their procoagulant phenotype during painful crises and steady state in 25 consecutive patients with sickle cell disease. Results The majority of microparticles originated from platelets (GPIIIa,CD61) and erythrocytes (glycophorin A,CD235), and their numbers did not differ significantly between crisis and steady state. Erythrocyte-derived microparticles strongly correlated with plasma levels of markers of hemolysis, i.e. hemoglobin (r=−0.58, p<0.001) and lactate dehydrogenase (r=0.59, p<0.001), von Willebrand factor as a marker of platelet/endothelial activation (r=0.44, p<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52, p<0.001 and r=0.59, p<0.001, respectively) as markers of fibrinolysis and coagulation activation. Thrombin generation depended on the total number of microparticles (r=0.63, p<0.001). Anti-human factor XI inhibited thrombin generation by about 50% (p<0.001), whereas anti-human factor VII was ineffective (p>0.05). The extent of factor XI inhibition was associated with erythrocyte-derived microparticles (r=0.50, p=0.023). Conclusions We conclude that the procoagulant state in sickle cell disease is partially explained by the factor XI-dependent procoagulant properties of circulating erythrocyte-derived microparticles.

Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes.

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.