The small vesicles liberated by cells into the extracellular milieu are collectively known as extracellular vesicles. Extracellular vesicles stored in multivesicular bodies (or α-granules in platelets) and released by exocytosis have dimensions ranging between 50 and 150 nm and are called exosomes. Extracellular vesicles produced by plasma membrane budding and shedding appear larger (100–1000 nm) and are called microvesicles. Apoptotic cells also release extracellular vesicles, which are heterogeneous in size (100 nm to 5 μm) and are called apoptotic bodies or apoptotic vesicles [1▪,2].
The release of extracellular vesicles is a process conserved through evolution, supporting their essential physiological role. Hence, prokaryotic, plant, and eukaryotic cells all produce extracellular vesicles. Extracellular vesicles participate in coagulation, affect vascular function, can play roles in cellular proliferation and differentiation, are involved in inflammation and mediate cell–cell communication [1▪]. Microvesicles originating from platelets, erythrocytes, endothelial cells, and leukocytes are present in the blood circulation, with those from platelets being the most abundant followed by extracellular vesicles derived from erythrocytes [3▪▪]. Upon activation, platelets are extremely potent at producing microvesicles, which are historically known as microparticles. For this reason, platelet-derived microvesicles are commonly referred to as ‘microparticles,’ and this terminology will be used throughout this review.
Herein, the recent advances in the study of microparticles will be discussed, with emphasis on the diversity of platelet-derived microparticles in health and disease.
ACTIVATED PLATELETS AS A SOURCE OF MICROPARTICLES
The activation of platelets by physiological agonists such as collagen, thrombin, the complement membrane attack complex C5b-9, lipopolysaccharide, immune complexes and viruses triggers the release of microparticles [4–6]. Platelet storage, cryopreservation, shear stress, and apoptosis were also demonstrated to induce the generation of microparticles [7,8,9▪▪,10]. Using scanning electron microscopy to directly assess platelet activation, Yano et al. observed that microparticle formation principally occurred in the vicinity of the pseudopod terminal of adherent platelets. Loss of cytoskeleton–plasma membrane adhesion and profound cytoskeleton rearrangements are implicated in this process, as the inhibition of actin polymerization impairs formation of pseudopods and the production of microparticles [9▪▪,11]. Recent work also evidenced the contribution of the proteasome in the shedding of platelet microparticles [12▪].
Loss of membrane phospholipid asymmetry coincides with microparticle formation . In unactivated platelets, phosphatidylserine is absent from the outer leaflet and is almost uniquely comprised in the inner leaflet of the plasma membrane. Upon activation, a rise in cytosolic calcium via nonselective ion channels and the mitochondrial permeability transition pore, and enzymes such as floppases and scramblase, are involved in the promotion of phosphatidylserine exposure [14–16]. Patients with Scott syndrome are characterized by bleeding disorders because of reduced floppase activity. In these patients, cells show defects in phosphatidylserine exposure and a consistent decrease in microparticle release [16,17]. Although these observations suggest that phosphatidylserine exposure might be necessary for the liberation of microparticles, it does not necessarily preclude the release of phosphatidylserine-negative microparticles. In fact, approximately half of the circulating microparticles do not harbor surface phosphatidylserine [3▪▪].
Recent study of platelets under physiologic flow revealed that activated platelets could form very long membrane strands (up to 250 μm in length) emerging downstream of the site of platelet adherence [18▪▪]. Mitochondrial calcium efflux, the protease calpain, and disassembly of F-actin and microtubule cytoskeletal proteins are necessary for the formation of these impressive structures built from membranes of the open canalicular system. As these strands harbor surface phosphatidylserine and disintegrate into microparticles, the formation of platelet strands, called flow-induced protrusion, is suggested to participate in the generation of phosphatidylserine-positive microparticles under physiologic flow [18▪▪].
Platelet activation is a prerequisite in the aforementioned mechanisms leading to microparticle production. Hence, higher concentrations of platelet microparticles have been documented in diseases in which platelets play a role, such as heparin-induced thrombocytopenia, thrombosis, idiopathic thrombocytopenic purpura, sickle cell disease, uremia, cancer, multiple sclerosis, rheumatoid arthritis, antiphospholipid syndrome, and systemic lupus erythematosus [19–31]. Nonetheless, approximately 104 platelet microparticles/μl, identified by the presence of CD41 molecules, circulate throughout the blood of healthy individuals [3▪▪]. As CD41+ microparticles are the most abundant in the blood of healthy individuals, this points to constitutive generation of platelet microparticles in vivo, potentially contributing to the maintenance of hemostasis.
MEGAKARYOCYTES AS A SOURCE OF MICROPARTICLES
The mechanisms underlying platelet formation by megakaryocytes have been studied in detail . During platelet formation, cytoplasmic extensions, called proplatelet processes, spread from the megakaryocyte and protrude into the interior of vascular sinusoids. Proplatelets are subsequently released into the blood as either globular preplatelets or as barbell-shaped proplatelets. Close examination of mouse megakaryocytes further revealed that, concomitant with the production of proplatelets, microparticles were generated from micropodia and the cytoplasmic membrane of megakaryocytes . Shear could enhance the production of megakaryocyte-derived microparticles, which were shown to promote hematopoietic stem cell differentiation into megakaryocytes [34▪]. Organelles were excluded from these megakaryocyte-derived microparticles, and studies confirmed that they fulfilled the definition of microparticles as their size was limited to less than 1 μm (0.2–0.5 μm in diameter), and they expressed canonical ‘platelet’ surface markers CD41, CD42b, glycoprotein VI (GPVI), and phosphatidylserine . These observations suggested that circulating microparticles, originally thought to originate from platelets, might in fact emanate from megakaryocytes.
PLATELET-DERIVED VERSUS MEGAKARYOCYTE-DERIVED MICROPARTICLES
The demonstration that megakaryocytes could produce copious levels of microparticles sharing important features with those from activated platelets prompted the quest for distinguishing markers. Although microparticles released from activated mouse platelets (using collagen and thrombin as stimuli) maintained CD62P and lysosomal-associated membrane protein-1 (LAMP-1) expression, these two markers were absent in megakaryocyte-derived microparticles. Of note was that the majority of the CD41+ microparticles circulating in the blood of healthy mice also lacked expression of CD62P and LAMP-1, suggesting that most circulating microparticles actually originate from megakaryocytes . However, it is currently unknown whether unactivated platelets constitutively shed microparticles into the blood circulation, and if so, whether these microparticles share similarities with microparticles from resting megakaryocytes or with those from activated platelets.
Platelet microparticle levels in blood are increased in diseases such as rheumatoid arthritis [28–30]. Thus, a recent study determined whether this increase was because of enhanced production of microparticles from platelets or from megakaryocytes. Platelets express three receptors that signal through immunoreceptor tyrosine-based activation motifs (ITAM) : Fc receptor γIIA (a low affinity receptor for immunoglobulin G immune complexes); Fc receptor γ-chain, which is noncovalently associated with GPVI and is necessary for GPVI function; and the C-type lectin 2 (CLEC-2), a receptor for podoplanin. Activation of ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain of Fc receptor γIIA, whereas CLEC-2 remains on the cell surface [36▪▪]. Consequently, microparticles induced by platelet activation express CLEC-2 as the unique ITAM receptor. Conversely, megakaryocyte-derived microparticles express both GPVI and CLEC-2. It was found that in rheumatoid arthritis, circulating CD41+ microparticles express CLEC-2, but not GPVI, suggesting that these microparticles were indeed generated from activated platelets during disease [36▪▪]. As platelet microparticles in rheumatoid arthritis were found to express the inflammatory cytokine interleukin-1β , this suggests that circulating platelet microparticles could amplify inflammation systemically.
PHOSPHATIDYLSERINE-EXPRESSING AND PHOSPHATIDYLSERINE-NONEXPRESSING MICROPARTICLES
Platelet microparticles were originally identified with functional assays, as they could support the coagulation cascade even in the absence of platelets . This procoagulant function of platelets is mediated through the exposure of anionic phosphatidylserine . It has long been assumed that all platelet-derived microparticles expressed surface phosphatidylserine. Although earlier flow cytofluorometric analyses suggested that only a fraction of microparticles expressed phosphatidylserine , the recent examination of platelet microparticles present in the plasma of healthy volunteers and in the synovial fluid of rheumatoid arthritis patients convincingly clarified that both microparticle subpopulations exist [3▪▪,40]. Using electron microscopy and annexin-V conjugated gold nanobeads (a probe used to recognize phosphatidylserine-expressing microparticles) to directly visualize microparticles, it was found that approximately 50% of the microparticles do not harbor phosphatidylserine [3▪▪]. As surface phosphatidylserine-positive microparticles participate in the coagulation process, this suggests that the remaining subpopulation, negative for phosphatidylserine, might play distinct roles, other than the maintenance of primary hemostasis. Furthermore, phosphatidylserine-positive microparticles are rapidly eliminated from the circulation, mainly through binding to the developmental endothelial locus-1 expressed by endothelial cells . Thus, a subset of microparticles may transmit their contents (e.g., microRNA)  to endothelial cells through phosphatidylserine recognition, whereas the phosphatidylserine-negative microparticles may circulate in stealth for a longer period of time after their release.
As microparticles encapsulate cytoplasm during vesiculation, they represent a means of cellular communication upon internalization by recipient cells. Indeed, the microparticle cargo is vast and is reported to contain cytokines, functional enzymes, messenger RNA, and noncoding RNA originating from platelets .
The organelle in charge of oxidative phosphorylation, and the source of energy for the cell, is the mitochondrion. This organelle is implicated in cell death and in the formation of the inflammasome. Platelets contain an average of four mitochondria in the cytoplasm, making them the most important mitochondrial reservoir in the bloodstream [9▪▪]. Thus, it was hypothesized that activated platelets could release their mitochondria encapsulated in microparticles. Accordingly, proteomic studies of platelet microparticles, separated on the basis of size using gel filtration chromatography, showed that the larger microparticles contained mitochondrial proteins whereas the smaller microparticles did not . It was recently confirmed that in addition to mitochondria-deficient microparticles, platelets could release mitochondria-containing microparticles. Of particular note is that extracellular mitochondria were shown to be respiratory competent, as they could mediate oxidative phosphorylation [9▪▪]. These observations suggest that upon cellular internalization, mitochondria-containing microparticles might impact the cellular bioenergetics of the recipients. Furthermore, as mitochondria are not present in microparticles shed from megakaryocytes, these findings might provide another means of discriminating between platelet-derived versus megakaryocyte-derived microparticles.
Damaged cells, activated mast cells, neutrophils, and lymphocytes were shown to release free mitochondria (i.e., not encapsulated in microparticles), possibly because of inadequate mitophagy that occurs under activation conditions and potentially in certain diseases [44–47]. This phenomenon appears conserved through cellular lineages, as activated platelets also release free mitochondria [9▪▪]. According to the theory of evolution, mitochondria are thought to be descendants of the primitive Alphaproteobacteria . Thus, the presence of extracellular mitochondria is an important source of damage-associated molecular pattern, capable of initiating potent innate immune responses. The identification of extracellular mitochondria in inflammatory synovial fluid and murine lungs in a model of transfusion-induced acute lung injury [9▪▪,49] suggested that platelets could release diverse types of microparticles (including mitochondria) under these conditions, thereby contributing to inflammation. In addition, several studies determined that extracellular mitochondria were abundant in platelet concentrates prepared for transfusion [9▪▪,50,51]. Of clinical relevance is that extracellular mitochondria were present at higher levels in platelet concentrates that had triggered adverse inflammatory reactions in human recipients [9▪▪]. This points to extracellular mitochondria as potent inflammatory mediators, and suggests that they might be used as a biomarker to assess the quality of blood transfusion products.
The peptidylarginine deiminase enzyme mediates the modification of arginine into citrulline, a process that occurs during inflammation and notably during NETosis . Citrullination is highly active in rheumatoid arthritis and generates the neo-epitopes for the autoantibodies characteristic of this autoimmune disease. It was found that the majority of the immune complexes present in the synovial fluid of rheumatoid arthritis patients comprised platelet microparticles [40,53]. Platelet microparticles were also present in the synovial fluid of psoriatic arthritis patients, a disease in which limited citrullination occurs, but were not associated with antibodies, pointing to citrullination as a process implicated in the formation of microparticle-containing immune complexes [37,40]. Hence, platelet microparticles were found to be susceptible to citrullination, and surface citrullinated fibrinogen appeared as a relevant factor contributing to the formation of microparticle and autoantibody scaffolds. Of importance was the demonstration that microparticles, once coated with autoantibodies, were highly potent at activating immune cells, possibly through Fc receptors . As NETosis and citrullination processes are highly active in the arthritic joint , these suggest that microparticles might undergo citrullination upon accumulation in the diseased tissue. How platelet microparticles egress the blood circulation is unclear, but it was proposed that this might occur through the more permeable vasculature that characterizes the arthritic joint during arthritis . The diverse subtypes of platelet microparticles that have been discussed herein are summarized in Fig. 1.
ELECTRON MICROSCOPY TO STUDY THE DIVERSITY OF MICROPARTICLES
The various roles attributed to platelet-derived microparticles (and microparticles from other sources) account for the efforts devoted to the development of efficient and reliable methods allowing their detection, phenotyping, and quantification. The characterization of microparticles is notoriously difficult because of their small size, heterogeneity in size, cell origin, and composition. Microparticles are commonly described as vesicular objects limited by a lipid bilayer ranging in diameter from 50 nm to 1 μm. However, the structural description of microparticles has recently been revisited by cryo-electron microscopy, an approach that best preserves the native structure of complex objects. Arraud et al.[3▪▪] determined the size distribution of microparticles in plasma, confirming that spherical microparticles ranging in diameter from 50 to 500 nm comprised the major population, and that plasma also contains tubular microparticles and membrane fragments of several micrometers in size. As mentioned above, it was this strategy that demonstrated microparticle heterogeneity with respect to phosphatidylserine exposure; approximately, half of the microparticles were labeled with annexin-V gold nanobeads (Fig. 2). Furthermore, microparticles were shown to present important morphological changes in response to standard isolation and storage procedures, such as microparticle ultracentrifugation and freezing at −80°C . Cryo-electron microscopy provides a unique means of revealing the existence and diversity of individual objects in native biological fluids at nanometer resolution, such as micrometer-sized immune complexes associated with microparticles in synovial fluid in arthritis .
FLOW CYTOMETRY TO ASSESS THE DIVERSITY OF MICROPARTICLES
For more than two decades, flow cytometry has been the primary method used for microparticle characterization. However, it has long been recognized that a lack of standardized methods hampered the comparison of results between studies. Recent important progress has been made in the case of blood-derived microparticles with the identification of the major causes of variability during preanalytical procedures , and the development of standardized protocols for microparticle quantification . It is also clear that polystyrene beads were inappropriate size calibration standards for microparticles, predominantly because of differences in refractive index between polystyrene and membrane vesicles [57,58]. This raised the question of the minimal microparticle size that can be detected by conventional flow cytometry based on light scattering. Although most current generation flow cytometers can detect 200-nm polystyrene beads, conclusions from independent studies indicated that the size detection limit for microparticles was approximately 500 nm [57,59]. Consequently, as most microparticles are smaller than 400 nm [3▪▪], only a minority are detected by conventional flow cytometers. The development of custom-based flow cytometers with improved performances [59,60,61▪] is expected to enable the detection of smaller microparticles.
Recent studies presented an alternative approach of flow cytometry, in which microparticles are detected on the basis of a fluorescence signal instead of a light scattering signal [37,40,59,60,62▪,63]. The detection of microparticles by fluorescence triggering was introduced almost 30 years ago , although it is unclear why this approach fell into disuse. Microparticles equivalent to 100-nm polymer beads were detected by combining this approach with an original labeling strategy in which a lipophilic fluorophore is incorporated into microparticle membranes . In another study, Arraud et al. quantified the predominant microparticle populations present in plasma, namely those of erythrocyte or platelet origin and microparticles containing phosphatidylserine and showed that approximately 50 times more microparticles are detected by fluorescence-triggering as compared to conventional flow cytometry [62▪].
Several studies discussed the possible contribution of coincidence phenomena to microparticle detection [58,60]. Coincidence occurs when several particles that are individually undetectable are simultaneously present in the detection volume, producing false positive results. Coincidence phenomena may occur for microparticle concentrations of the order of 1010 /ml. The advantage of imaging flow cytometry, a novel technology combining flow cytometry with imaging, was recently demonstrated [64,65]. Using this approach, co-migrating entities that would be misinterpreted as double-positive events by flow cytometry can be efficiently discerned. Rousseau et al. demonstrated the possible influence of phospholipid-hydrolyzing enzymes, particularly secreted phospholipase A2 found in plasma, in the quantification of microparticles. With regard to other plasma components, for example protein aggregates , these enzymes can significantly interfere in the accurate evaluation of microparticles. In summary, significant improvements to the methodologies permitting the detection and characterization of microparticles have been achieved during recent years. When combined with novel technologies like nanoparticle tracking analyses or resistive pulse sensing aimed at investigating smaller microparticles such as exosomes, we anticipate an exponential improvement in the definition of microparticle structure and functions in the near future.
An understanding of microparticle genesis will permit the elucidation of the conditions underlying their generation. Furthermore, an appreciation of microparticle diversity through detection refinement can shed light on the specific roles played by each subtype in health and disease. Thus, platelet microparticle subtypes are useful diagnostic biomarkers, and with the confirmation of their actual contribution to disease development, they appear to be promising therapeutic targets.
Financial support and sponsorship
E.B. is recipient of an award from the Canadian Institutes of Health Research. A.-C.D. is recipient of a fellowship from the Canadian Arthritis Network.
Conflicts of interest
There are no conflicts of interest.
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