Blood contains a mixture of extracellular vesicles from different cell types, primarily platelets, endothelial cells, leucocytes and erythrocytes. Erythrocytes are the most abundant cell type in blood and could, especially in certain pathologies, represent an important source of vesicles. Since erythrocytes contain the haemoglobin components iron and haem, which are potentially toxic, it is important to investigate the contribution of vesicle-associated haemoglobin to total cell-free haemoglobin levels. To our knowledge, this is the first time that cell-free plasma haemoglobin has been differentiated into vesicle-associated and molecular species. We investigated the contribution of vesicle-associated haemoglobin in residual patient material that was routinely analysed for total cell-free plasma haemoglobin. All patient samples included in the study were haemolytic with total cell-free haemoglobin concentration ranging from 80 to 2500 mg/l. In the majority of the samples, total cell-free haemoglobin concentration was between 100 and 200 mg/l. No haemoglobin could be detected in the vesicle fraction, indicating that the contribution of vesicle-associated haemoglobin to total cell free-haemoglobin levels in plasma is negligible. It is important to investigate whether erythrocyte vesicles are not formed in blood or that their production is not increased during pathologies associated with haemolysis or that the clearance rate of the vesicles surpasses the formation rate.
- differential ultracentrifugation
- extracellular vesicle
Extracellular vesicles are particles originating from cells. They have attracted tremendous scientific interest because of their (potential) role in cell–cell communication, diagnostics and drug delivery [1–3]. The term ‘extracellular vesicles’ comprises different vesicle classes, such as exosomes and microvesicles. Exosomes are considered to be formed by invagination of the endosomal membrane, leading to the formation of intracellular multivesicular bodies. When these fuse with the cellular membrane, exosomes are released. Exosomes have a size of between 30 and 100 nm. Microvesicles, on the other hand, are formed by budding off from the cell membrane and are between 100 nm and 1 μm. Some cells constitutively release extracellular vesicles, whereas others only do so when triggered with (specific) stimuli .
Vesicles consist of a phospho(lipid) bilayer surrounding an aqueous core. Within the bilayer, a variety of membrane proteins can be present, and the aqueous core can contain a diverse mix of second messengers, proteins and genetic material (DNA, mRNA and non-coding RNA, such as miRNA). Interestingly, the composition is only partly a cross-section of the vesicles’ cellular origin. Specific components are enriched and others are selectively depleted, and the enrichment/depletion is dependent on the stimulus by which they are formed [5,6]. It is thought that the specific composition, thus generated, can communicate detailed information on cell status to cells in the vicinity and at distant sites.
Taken together, extracellular vesicles encompass a wide range of submicron membrane particles, whose composition forms a ‘fingerprint’ of the route of formation, the producing cell type and the stimulus for formation.
Extracellular vesicles in blood
Blood contains a mixture of extracellular vesicles produced by various cells. The most prominent cell types from which extracellular vesicles are recovered in healthy individuals are the various blood cells and endothelium (Table 1).
Despite the differences between the vesicles of different cellular origin, there are certain similarities. Most prominent is the presence of negatively charged phospholipids on many (if not all) vesicle surfaces. These lipids are involved in coagulation and inflammatory reactions, putting vesicles at the crossroads of many disorders. Also, a consensus appears to have been reached on the presence of certain markers, such as CD63 on exosomes.
Platelets were the first cells shown to shed vesicles. As early as 1967, Wolf  detected extracellular vesicles from platelets, and demonstrated that these had the same effect as whole activated platelets on blood coagulation. Platelet vesicles are known for their pro-coagulant activity, which is caused by the presence of anionic phospholipids, particularly phosphatidylserine and the glycoprotein tissue factor. Phosphatidylserine is a phospholipid which normally preferentially resides at the inner layer of the cell membrane. It is capable of forming an electrostatic interaction between γ-carboxyglutamic acid residues that have bound calcium. These modified amino acids are present in clotting proteins such as Factor VIIa . Tissue factor, in turn, catalyses (in complex with Factor VIIa) the conversion of Factor X. Phosphatidylserine-positive vesicles can create a large surface area to collect these proteins in close vicinity and facilitate their interaction. These and other effects of platelet vesicles are described in a recent review by Aatonen et al. .
Endothelial-cell-derived vesicles have received much attention in relation to angiogenesis. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, plays an important role in cancer and other diseases, and involves endothelial cell survival, proliferation, migration and differentiation. Endothelial vesicles have been shown to impair endothelial function in vitro, diminishing acetylcholine-induced vasorelaxation and nitric oxide production . In addition, it affects vascular network formation of human umbilical vein endothelial cells in culture.
A recent study by Hergenreider et al.  showed that extracellular vesicles, secreted by Krüppel-like factor-2 (a transcription factor regulating endothelial gene expression) expressing endothelial cells in culture are enriched in miR-143/miR-145 and these vesicles can control target gene expression in co-cultured smooth muscle cells. Extracellular vesicles derived from these endothelial cells also reduced atherosclerotic lesion formation in the aorta of apolipoprotein E-knockout mice .
A study by de Jong et al.  showed that the protein and RNA profile in endothelial vesicles is dependent on the stimulus, with TNFα (tumour necrosis factor α) and hypoxia having a strong influence on vesicle composition, whereas hyperglycaemia has only limited effects . The picture that emerges of these and other studies is that endothelial cells respond readily to activation with secretion of extracellular vesicles with diverse effects on other endothelial cells and cells in close vicinity, altogether generating a complex picture. These effects and their significance in the many diseases in which the endothelium is affected have been reviewed in [18–20].
Polymorphonuclear neutrophilic granulocytes are the most abundant leucocyte in the circulation. They release phosphatidylserine-positive vesicles that carry L-selectin and can activate the classical pathway of complement and fix C4 and C3 fragments . When stimulated, for example with the chemotactic peptide fMLP (N-formylmethionyl-leucyl-phenylalanine) or complement factor C5a, neutrophil vesicles were formed that bind to endothelial cells and monocytes and activated them. In addition, these vesicles carry antimicrobial activity such as myeloperoxidase and proteinase 3, and several proteinases (such as matrix metalloproteinase 9 and elastase) that may assist in tissue remodelling. Apart from pro-inflammatory activity, microvesicles can convey anti-inflammatory signals to macrophages , and can inhibit the interaction between neutrophils and endothelium.
A similar dual role has been described for monocyte-derived vesicles in coagulation. Since monocyte-derived vesicles have been shown to carry tissue factor, this seems to support their pro-coagulant activity. At the same time, they contain tissue factor pathway inhibitor, which has the opposite activity. In addition, because of the presence of endothelial cell protein C receptor, activation of anticoagulant protein C occurs, inactivating activated Factors V and VIII.
Apart from their role in innate immunity and coagulation, leucocyte vesicles are also implicated in adaptive immunity. Extracellular vesicles from immature dendritic cells appear to have potent immunosuppressive properties . Vesicles from mature dendritic cells, on the other hand, have been proposed as immunostimulating vaccines . After capturing antigens, dendritic cells incorporate MHC–antigenic peptide complexes in extracellular vesicles, which may travel to stimulate immature dendritic cells, which in turn acquire the ability to stimulate CD4+ and CD8+ T-cells .
Erythrocytes are probably the only cell type that do not form exosomes. However, ironically, exosomes were first identified on the erythrocyte precursor, the reticulocyte. Reticulocytes do secrete exosomes, as exosomes are part of the remodelling process during erythrocyte maturation . However, in mature erythrocytes, only microvesicles are formed. Microvesicles shed from erythrocytes have pro-coagulant activity because of the presence of phosphatidylserine. This shedding of phosphatidylserine-positive vesicles is a continuous process and is postulated to ensure erythrocyte longevity. By removal of phosphatidylserine, macrophage recognition and clearance is delayed . Apart from phosphatidylserine, erythrocyte vesicles contain large amounts of iron and haem. These compounds need to be well processed as they may generate reactive oxygen species or intoxicate cells.
Cell-free plasma haemoglobin: partly in the form of vesicular haemoglobin?
Because of the oxidative and toxic properties of the iron-containing haem in haemoglobin, cell-free haemoglobin (when liberated by haemolysis) is immediately complexed by haptoglobin. This protein directs the clearance of plasma haemoglobin towards CD163-positive phagocytes. Haptoglobin, however, is rapidly saturated and depleted. Free haem in the circulation can also be bound to the plasma glycoprotein haemopexin . This complex is taken up by a low-density-lipoprotein-related receptor, CD91 . When the capacity of haemopexin to bind haem is saturated, excess haem may bind to albumin to form methaemalbumin . Plasma (or free) haemoglobin is used as a diagnostic marker for (the degree of) intravascular haemolysis in patients. In addition, increased concentrations of cell-free haemoglobin lead to the consumption of nitric oxide and other clinical consequences . We hypothesized that part of the cell-free plasma haemoglobin that is measured is present in the form of vesicle-associated haemoglobin. This could have important implications because the cellular distribution profile of vesicle-associated haemoglobin is likely to be different from that of free haemoglobin. We have tested this hypothesis on residual material from routinely performed plasma haemoglobin measurements.
Residual lithium heparin plasma samples stored at −80°C were thawed, and vesicles were isolated by differential centrifugation: 2000 g for 30 min at 4°C, 12000 g for 45 min at 4°C and 120000 g for 1 h at 4°C. The pellet obtained was resuspended in PBS and centrifuged at 120000 g for 1 h at 4°C. The pellet was reconstituted in a final volume of 250 μl of PBS.
Haemoglobin was quantified using a spectrophotometric method described by Kahn et al.  with minor modifications. In short, plasma samples were diluted to an appropriate concentration, and 200 μl of these plasma samples or pellet samples was transferred to a 96-well plate. Absorbance was measured at 562, 578 and 598 nm at room temperature.
Samples included in the study were haemolytic (haemoglobin >80 mg/l). In the majority of samples, plasma haemoglobin was between 100 and 200 mg/l (Figure 1). Interestingly, we did not detect haemoglobin in any of the pellets of these samples. Since the limit of detection of this analytical procedure is ~1 mg/l, it is clear that the vast majority of haemoglobin is not vesicle-associated and the free haemoglobin assay is not affected by vesicle-associated haemoglobin.
To exclude the possibility that the freeze–thaw procedure we used leads to destruction of vesicles, we subjected artificially generated haemoglobin-containing erythrocyte vesicles to the same freeze–thaw procedures as the plasma samples of the routine free haemoglobin assay. The recovery of vesicle-associated haemoglobin after freezing was >70%, excluding vesicle loss through freeze–thawing as a cause for the absence of vesicle-associated haemoglobin.
The work of R.M.S. on vesicles is supported by European Research Council Starting Researcher's Grant on Microvesicle-inspired drug delivery systems [grant number 260627].
Microvesiculation and Disease: A Biochemical Society Focused Meeting held at London Metropolitan University, London, U.K., 13–14 September 2012. Organized and Edited by Annie Bligh, Una Fairbrother, Sheelagh Heugh and Jameel Inal (London Metropolitan University, U.K.).
- © The Authors Journal compilation © 2013 Biochemical Society