Blood Microvesicles: from Proteomics to Physiology

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

Available online at www.sciencedirect.com

jou rnal homepage: http://www.elsevier.com/locate/trprot

Review

Blood microvesicles: From proteomics to physiology

a,∗ a a b

Jean-Daniel Tissot , Giorgia Canellini , Olivier Rubin , Anne Angelillo-Scherrer ,

a a a

Julien Delobel , Michel Prudent , Niels Lion

Service Régional Vaudois de Transfusion Sanguine, Switzerland

Service and Central Laboratory of Hematology, Lausanne University Hospital, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Phospholipid vesicles of less than 1 ␮m are present in blood in physiological state and their

Received 22 March 2013 concentration may vary under pathological conditions. Various names such as exosomes

Received in revised form (EXS) and microparticles (MPS) have been used to designate these extracellular vesicles

17 April 2013 (EVS). Although EXs and MPS possibly arise from separate mechanisms, they share numer-

Accepted 30 April 2013 ous similarities representing a challenge for their puriﬁcation and characterization. These

vesicles generally originate from various types of cells such as red blood cells, platelets,

Keywords: leukocytes or endothelial cells but also from tumor cells. They participate in numerous

Aging biological processes including hemostasis. It is therefore of major scientiﬁc interest to char-

Blood cells acterize the protein content of these different types of EVS and that of their membranes in

Exosomes order to elucidate the essential functions of these dynamic vesicular compartments. Pro-

Microparticles teomics has been shown to be a particularly adequate tool in this study ﬁeld. This review

Microvesicles attempts to link proteomic data with physiological roles and functions of blood EVS.

Contents

1. Introduction ...... 39

2. Quantiﬁcation and isolation of blood vesicles ...... 39

3. Proteomics of blood vesicles ...... 41

3.1. Proteomics of red blood cell-derived vesicles ...... 41

3.2. Proteomics of platelet-derived vesicles ...... 43

3.3. Proteomics of leukocyte-derived vesicles ...... 43

3.4. Proteomics of endothelial cell-derived vesicles...... 44

Abbreviations: EEVS, endothelial-derived microvesicles; ELISA, enzyme linked immune-sorbent assays; EVS, extracellular vesicles; EXS,

exosomes; DSL, dynamic light scattering; LEVS, leukocyte-derived microvesicles; MPS, microparticles; NTA, nanoparticle tracking analysis;

PEVS, platelet-derived microvesicles; RBC, red blood cell; REVS, red blood cell-derived microvesicles; ROS, reactive oxygen species.

∗

Corresponding author at: Route de la Corniche 2, CH-1066 Epalinges, Switzerland. Tel.: +41 21 314 65 89; fax: +41 21 314 65 78.

E-mail address: [email protected] (J.-D. Tissot).

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 39

4. Biology of blood vesicles ...... 44

4.1. Red blood cell-derived vesicles ...... 44

4.2. Platelet-derived vesicles ...... 46

4.3. Leukocyte-derived vesicles ...... 46

4.4. Endothelial cell-derived vesicles ...... 47

5. Conclusions and perspectives ...... 47

Conﬂict of interest ...... 47

References ...... 47

A model of MPS formation including translocases, lipid rafts,

1. Introduction

various protein modiﬁcations and irreversible membrane rear-

rangements has been proposed (Fig. 2) [24,25]. MPS are not

Proteomics is of major interest for the study of blood and blood

cell fragments or “dust” without any biological function [26].

diseases [1–11]. Plasma proteins and their modiﬁcation in var-

They play a role in various broad biological functions such as

ious conditions have been extensively evaluated over the last

thrombosis and hemostasis [20,27,28], inﬂammation [27,29] or

decades in search of speciﬁc biomarkers of human diseases

immunosuppression [30,31]. However, numerous similarities

[12], particularly in cancer patients [13,14]. Proteomics rep-

exist between EXS and MPS with respect to their physical char-

resents nowadays the technique of choice – if not the gold

acteristics and compositions. These similarities frequently

standard – to characterize amyloidosis in tissue and plasma

hampered the separation and puriﬁcation of these EVS in body

samples obtained from patients with protein deposition syn-

ﬂuids and brought confusion in the scientiﬁc literature. In this

dromes [15,16].

review, we will mainly focus on blood EVS, with a particular

The proteome of many blood cells has been well charac-

emphasis on platelet and RBC EVS, as well as on MPS released

terized, especially that of red blood cells (RBCs) and platelets.

during storage of blood units. For clarity purposes, the term

The interest of applying proteomic technology to such cells is

EVS will be used in the following sections, grouping both MPS

mainly related to the fact that they share a limited capacity to

and EXS.

synthesize new proteins. In this context, there is a rising value

Quantiﬁcation, proteomic analysis as well as the biol-

of proteomics compared to genomics and it is not surprising

ogy of RBC-derived EVS (REVS), platelet-derived EVS (PEVS),

that it has also proved effective in determining the protein

leukocyte-derived EVS (LEVS), and of endothelial cell-derived-

content of extracellular vesicles (EVS). Release of membrane

EVS (EEVS) are different, even if they share many common

vesicles, a process conserved in both prokaryotes and eukary-

determinants. This review will present proteomic data that are

otes, represents an evolutionary link, and suggests essential

“speciﬁc” for each type of EVS and then, will give insights onto

functions of this dynamic vesicular compartment [17]. Recent

the physiology of the various forms of EVS that are normally

studies provided support for the concept of EVS as vectors for

present in the blood or in blood products.

the intercellular exchange of biological signals and informa-

tion [18]. EVS are able to transfer part of their components and

content to selected target cells, allowing cell activation, phe-

notypic modiﬁcation, and reprogramming of cell function [19]. 2. Quantiﬁcation and isolation of blood

Because EVS circulate in the blood ﬂow, they serve as shut- vesicles

tle modules and signaling transducers not only in their local

environment but also at distance from their site of origin. A variety of techniques is commonly used to study EVS (Fig. 3).

Classiﬁcation of membrane vesicles, protocols of their iso- Traditionally, a limited set of technologies has been used

lation and detection, molecular details of vesicular release, to characterize micro- and nano-vesicles with more tech-

clearance and biological functions are still under intense niques being available to the micro sized particles [32]. These

investigation. EVS have been identiﬁed in the blood circula- include: ﬂow cytometry, dynamic light scattering (DLS), elec-

tion for a long time, and have been ﬁrst considered as cell tron microscopy [33,34] or enzyme linked immune-sorbent

fragments. In fact, EVS are quite heterogeneous and at least assays (ELISA) [35–37]. Most widespread is ﬂow cytometry;

two main distinct types have been identiﬁed: exosomes (EXS) commercial ﬂow cytometry typically has a lower practi-

and microparticles (MPS). Both EXS and MPS are detected in cal size limit (for polystyrene beads) of around 300 nm at

blood ﬂow, and arose out of cells such as platelets, leukocytes which point the signal is indistinguishable from the base-

and endothelial cells [20]. EXS are small (40–100 nm in diam- line noise level. Whilst this detection limit can be extended

eter), spherical vesicles of endocytic origin that are secreted with the use of ﬂuorescent labels, at lower sizes the ability

upon fusion of the limiting membrane of multivesicular bod- to accurately size such particles is quite limited. Dynamic

ies with the plasma membrane. Red blood cell (RBC)-derived light scattering has also been used in this application,

vesicles (REVS) have been also described in blood samples but being an ensemble measurement, the results comprise

obtained from patients with many different diseases as well as either a simple z-average (intensity weighted) particle size

a storage lesion from red blood cell preparations dedicated for and polydispersity, or a very limited-resolution particle size

transfusion [21,22]. EXS contain subproteome cytosolic pro- distribution proﬁle [38,39]. Electron microscopy is a useful

teins, mRNAs and miRNAs, and are involved in intercellular research tool for studying micro- and nanovesicles but at the

signaling. In contrast, MPS bud directly from the plasma mem- expense of capital running costs, extensive sample prepara-

␮ brane and their size ranges from 100 nm to 1 m (Fig. 1) [23]. tion [40].

40 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

Fig. 1 – (A) Formation of extracellular vesicles from spicules of echinocytes (confocal differential interference contrast

microscopy, magniﬁcation 4000×). (B) A single red blood cell derived (micro-)vesicle surrounded by nanovesicles

(transmission electron microscopy, magniﬁcation 67 000×).

Most frequently, EVS are counted in biological samples by Chandler et al. evaluated methods to number PEVS in fresh

ﬂow cytometry [21,41,42]. Several authors have pointed out and frozen aliquots of plasma as well as in fresh and frozen

that despite ﬂow cytometry is the technique of choice for aliquots of platelet-rich plasma [50]. They measured platelet

evaluation of EVS, it is limited by lack of adequate standardiza- (CD41+) and annexin V+, and were able to determine PEVS in

tion [43]. Preanalytical as well as analytical issues have been blood samples from normal individuals. Platelet-rich plasma

evaluated in detail by Yuana et al. [44]. Preanalytical parame- from healthy individuals contained 730 000/␮l total EVS based

ters were also studied in our laboratory, when measuring EVS on light-scattering measurements, and a median of 27 000/␮l

in stored blood products: in addition to the ﬂow cytometry of those EVS were of platelet origin. They also provided

parameters chosen for the numbering of EVS, we observed that emphasis on the importance of preanalytical issues show-

many preanalytical variables have to be taken into account ing that freeze–thawing has variable effects on EVS counts,

(diluents used, temperature, vortexing duration, etc.) [45,46]. depending on the sample preparation used. For instance, it

In addition, Mullier et al. recently evaluated many aspects of has been reported that the centrifugation protocols inﬂuence

the prenalytical conditions as well as pending issue that has the EV counts [45,48].

to be considered when measuring EVS in blood samples [47]. Strasser et al. reported a comparison analysis of three

Because REVS express transmembrane proteins such as different methods for the quantiﬁcation and characteriza-

band 3, glycophorins, or blood group antigens, it is quite con- tion of PEVS [51]. The authors, in their study, analyzed

venient to use speciﬁc antibodies raised against glycophorin A, PEVS from 31 healthy blood donors and compared pre-

CD47 or other blood group speciﬁc antigens for ﬂow cytometric and postdonation results of donors with data of platelet-

purposes. The expression of negatively charged phospholipids pheresis products by three different methods; PEVS counts

(surface phosphatidylserine) at the external part of the vesi- were analyzed by ﬂow cytometry using calibrated beads of

cles membrane can be explored by using annexin V as a deﬁned diameter and annexin V-ﬂuorescein isothiocyanate

ligand. However, standardization is mandatory in order to and CD41-phycoerythrin staining, whereas PEVS activity was

be able to compare data from different sets of experiments, tested by prothrombinase assay and, ﬁnally, a procoagulant

to compare normal individuals with patients and to com- phospholipid-dependent clotting time assay was used. The

pare data from different laboratories. Recently, Xiong et al. results showed a concentration of PEVS that was more than

presented a standardized approach based on quantitative threefold higher in single-platelet units compared to double-

ﬂow cytometric technique [48]. The enumeration of REVS is platelet units. The prothombinase assay and the procoagulant

made possible by using a ﬁxed number of different-sized cal- clotting assay also revealed a signiﬁcant higher PEVS activ-

ibration beads spiked into each sample. The master bead ity in single platelet units compared to double platelet units.

mixture allowed deﬁning the relative side and forward light These results are important for the transfusion medicine com-

scatter properties of the EVS events and enhanced the accu- munity; they conﬁrm that various procedures may results in

racy of the enumeration process. Such an approach certainly the production of different products.

may also prove useful to measure EVS that derives from An alternative approach for measuring EVS is nanoparti-

endothelial cells, leukocytes or platelets. Numbering of PEVS cle tracking analysis (NTA) [44,52,53]. In nanoparticle tracking

is a challenge. Validation of the usefulness of cytometry to analysis, the size is derived from the measure of Brownian

analyze PEVS was provided by Leong et al. who combined motion of EVS in a liquid suspension [44,52]. This technol-

ﬂow cytometry and atomic force microscopy [49]. In addi- ogy has been successfully applied for the analysis of EVS

tion, the authors demonstrated that atomic force microscopy derived from placenta, and allows speciﬁc EXS and EVS in

allows performing nanoscale measurements of individual the range of 50–1000 nm in liquid suspension. On the con-

PEVS events isolated by ﬂow cytometry. This method provided trary of DLS that measures all the particles at the same

the ﬁrst quantitative nanoscale images of PEVS ultrastructure. time, NTA visualizes individual particle and counts them in

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 41

real-time. NTA also provides high-resolution particle size dis-

tribution proﬁles and concentration measurements. However,

NTA is time consuming and the detection of small particles

is underestimated when larger particles are present [44]. The

technique is commercially available (NanoSight Ltd., Ames-

bury, UK; www.nanosight.com).

Various approaches have been developed for isolating

blood EVS. The particles can be immuno-adsorbed on sur-

faces using speciﬁc antibodies, using different centrifugation

approaches with or without density gradients, or as recently

reported using cell sorting [54]. In their study, Bosman et al.

isolated EVS from whole blood by differential centrifuga-

tion followed by ﬂuorescence-activated ﬂow cytometry. They

removed intact cells by low speed centrifugation from cit-

rated blood, and vesicles were isolated from the supernatant,

concentrated and washed with phosphate-buffered saline by

centrifugation. The EVS were then stained with speciﬁc anti-

bodies in order to label REVS and PEVS, respectively. Finally,

EVS were sorted on a ﬂow cytometer, and analyzed using pro-

teomic tools.

3. Proteomics of blood vesicles

Proteomics is an ideal tool to study EVS [55,56]. The number

of papers published on this topic is rapidly increasing. Pro-

teomics has been used to evaluate EVS from mesenchymal

stem cells [57], from tumor cells [58,59], in ascite of patients

presenting with colon cancer [60], from HIV-infected lympho-

cytes [61], in saliva, in urine [62,63], in amniotic ﬂuid [64]

or human cerebrospinal ﬂuid [65], just to cite the expend-

ing ﬁeld of research covered by different groups interested in

the study of EVS. The technique has been also successfully

applied to the evaluation of blood EVS [66,67] as performed

by Bastos-Amador et al. who analyzed EVS from plasma of

healthy donors and showed a remarkably high variability in

the protein content of EVS from different donors [68].

Fig. 2 – Model of formation of microparticles. The

phospholipid organization of the membrane is under the

control of three enzymes: ﬂippase, ﬂoppase and 3.1. Proteomics of red blood cell-derived vesicles

scramblase. (A) In resting cells, ﬂippase internalizes

negatively charged phospholipids and maintains the Differentiation of erythroblasts into mature RBC is a com-

asymmetry of the phospholipid bilayer. Floppase and plex mechanism, and many steps have been described. The

scramblase are inactive, and the cytoplasmic calcium ﬁnal pathway leads to the transformation of reticulocytes into

concentration is low. (B) Upon activation, intracellular circulating RBCS. Carayon et al. analyzed the composition of

calcium concentration increases, ﬂippase is inhibited while EXS released by reticulocytes during their differentiation [69].

ﬂoppase and scramblase are activated. Floppase Several mechanisms are involved in the process leading to

externalizes phosphatidyserine, a negative phospholipid, maturation of reticulocytes into mature RBCS and resulting

and scramblase translocates phospholipids non-speciﬁcally in the synthesis of large amounts of hemoglobin as well as in

through the membrane resulting in the loss of the elimination of numerous cellular components. By combin-

phospholipid asymmetry. (C) Increased intracellular ing proteomic and lipidomic approaches, the authors observed

calcium also activates proteases that cleave the alterations in the composition of the EXS retrieved over the

cytoskeleton; the membrane become less rigid and can bleb course of a 7-day in vitro differentiation protocol, and pro-

resulting in the formation and release of vesicles posed a model in which EXS are involved in speciﬁc pathways

Reprinted from [22], with permission from S. Karger. of cellular differentiation and maturation.

Bosman et al. presented pioneering proteomic investiga-

tions of EVS isolated from RBCS [70–72], and of EVS isolated

from plasma [54]. In these series of studies, RBC membrane

and EVS membrane were compared, allowing the identiﬁca-

tion of several proteins differentially expressed between the

two types of samples. Furthermore, the authors were able

42 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

Fig. 3 – Technical approaches for microparticle investigation. Combination of these methods are most frequently used for

vesicle characterization.

to characterize the effects of cellular aging on RBCS in vivo. et al., accumulation of band 3 aggregates is observed, espe-

They compared the proteome of REVS with that of the RBCS cially at the end of the storage period 54. Moreover, they probed

membrane separated according to cell age. They observed the the level of protein oxidation (carbonylation) that was signiﬁ-

presence of band 3 and actin in the REVS but the absence of cantly higher in vesicles, compared to originated membranes,

almost all other integral membrane and cytoskeletal proteins. up to 21 days of storage. Then, the level of oxidation drasti-

They also identiﬁed speciﬁc alterations in band 3 aggregation cally decreased, which has been attributed to the depletion of

and degradation related to aging and compatible with a unique highly carbonylated proteins. They concluded on the ability of

RBC aging process, the mechanism of which being speciﬁ- RBCs to get rid of harmful materials by vesiculation.

cally band 3-centered. Finally, their results pointed out that In our laboratory, we evaluated REVS from RBC stored

the age-related recruitment of plasma proteins, proteins of in blood banking conditions [74] and analyzed their oxi-

the ubiquitin–proteasome system, and small G proteins to the dation patterns by evaluating carbonylation as a hallmark

RBC membrane supports the hypothesis that changes and/or of protein oxidative lesions [75]. In order to improve global

degradation of band 3 is involved in vesiculation [54]. RBC protein carbonylation assessment, subcellular fraction-

Under the same period, Kriebardis et al. have followed the ation has been performed, allowing to study four protein

proteome of REVS during storage of EC [73]. They found that populations that were (i) soluble hemoglobin, (ii) hemoglobin-

microparticles contained Hb and modiﬁed Hb, and mainly depleted soluble fraction, (iii) integral membrane and (iv)

proteins with MW lower than 70 kDa. REVS are depleted of cytoskeleton membrane protein fractions. In addition, car-

spectrins and cytoskelateal proteins such as proteins 4.1 and bonylation in REVS has been investigated. We observed

4.2, and contain lipid raft proteins. Because of the absence of that carbonylation in the cytoskeletal membrane fraction

protein 4.2, they suggested that the subpopulation observed increased remarkably between day 29 and day 43, and that

concerns proteins that are not band3-cytoskeletal linked (or protein carbonylation within MPS released during storage

we may also speculate that this subpopulation contains mem- showed a two-fold increase along the storage period. Taken

brane proteins originally linked to the cytoskeleton and that together, a scheme of protein oxidation has been proposed

were released after various lesions). As shown by Bosman (Fig. 4), suggesting a cumulative aspect of the storage lesion: a

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 43

cellular compartment-based evolution of global carbonylation with modiﬁed levels in these patients (increased levels of

in stored RBCS, an increasing elimination of harmful car- peroxiredoxin 6, apolipoprotein E, cyclophilin A and heat

bonylated proteins through microvesiculation and a potential shock protein 90), suggesting that the oxidative damage in

role of hemichrome-induced cytoskeleton oxidation in this RBC and platelets potentially induces production of EVS

elimination process. The oxidative pattern in REVS observed with altered proteome that may facilitate thromboembolic

in this study (level of carbonylation higher at the end of the complications.

storage) differs from the one published by Kriebardis et al.

[73]. In their study, they normalized the oxidation level to 3.2. Proteomics of platelet-derived vesicles

the corresponding RBC membranes, which may explain the

differences in case of accumulation of oxidized proteins at State of the art of platelet proteomics has been recently

the membranes. Delobel et al. also identiﬁed mechanisms reviewed [79–82]. A number of investigations focused on stud-

of oxidation of proteins and postulated that REVS may have ies using subproteomic strategies to analyze speciﬁc platelet

a role, as cargos leading to their elimination [75]. Finally, conditions (resting or activated), compartments (membrane,

the group of Bosman provided very recently interesting data granules and MPS) or fractions (phosphoproteome or glyco-

dealing with the physiology of RBC removal and alloimmu- proteome) [83–85]. More speciﬁcally, the proteome of PEVS has

nization [76]. The authors investigated if RBC storage leads to been the object of proteomic studies. Gracia et al. found that

the increased expression of non-physiological antigens, and PEVS contain membrane surface proteins such as GPIIIa, GPIIb,

for that purpose, used immunoprecipitation, testing RBCS and P-selectin, as well as other platelet proteins such as the

and REVS from blood units of increasing storage periods chemokines CXCL4 and CXCL7 [86]. In another study, Jin et al.

with patient plasma containing RBC autoantibodies. The compared the proteome of PEVS with that of plasma using

immunoprecipitate was analyzed using proteomic tools. With two-dimensional gel electrophoresis and mass spectrometry

such an approach, the authors observed that patient plasma [87]. They were able to identify 83 different proteins that were

autoantibody binding increased with RBC storage time, not reported in the plasma proteome. Dean et al. presented

which was not the case with healthy volunteer plasma, and results of proteomic studies evaluating PEVS released by acti-

showed that pathology-associated antigenicity changes with vated platelets [88]. In this study, PEVS were separated by gel

storage. They also identiﬁed several membrane proteins as ﬁltration chromatography into 4 size classes to facilitate iden-

candidate antigens (namely band 3, adducing, ankyrin, band tiﬁcation of active protein and lipid components, and proteins

4.1, band 4.2 and other protein of the band 3 complex) for were separated using two-dimensional gel electrophoresis,

immunization. The protein complexes that were precipitated liquid chromatography, and identiﬁed by tandem mass spec-

by the patient autoantibodies were different whether RBCS or trometry. The authors observed that PEVS of different sizes

vesicles formed during RBC storage were used, indicating that signiﬁcantly differ in the content of plasma membrane recep-

the storage-associated REVS have a different immunization tors and adhesion molecules, chemokines, growth factors and

potential. Soluble immune mediators including complement protease inhibitors. The thousands of platelet proteins and

factors were present in the patient plasma immunoprecipi- interactions discovered so far by these different powerful pro-

tates, but not in the controls. The fascinating observations teomic approaches represent a precious source of information

made by the group of Bosman support the theory that dis- for both basic science and clinical applications in the ﬁeld of

turbed RBC aging during storage of blood units contributes platelet biology.

to transfusion-induced alloantibody and autoantibody

formation. 3.3. Proteomics of leukocyte-derived vesicles

Using ﬂow cytometry analysis, Almizraq et al. observed a

signiﬁcant increase in the mean number of EVS per mL during The protein characterization of LEVS is still largely unex-

storage, with signiﬁcant decreases in the amount of phospho- plored. Furthermore, many preanalytical difﬁculties should be

lipids and total lipids within the RBC membrane. These results taken into account, because of the great diversity of leukocytes

clearly indicate that lipidomics of the storage lesion of RBCS in blood circulation. It is therefore mandatory to purify each

is certainly a new avenue of research [77]. different type of LEVS using speciﬁc expressed CD antigens. A

The proteome of REVS from patients with beta- ﬁrst attempt of deciphering the proteome of B-cell LEVS has

thalassemia/hemoglobin E has been explored by been published by Wubbolts et al., ten years ago [89]. In this

Chaichompoo et al. [78]. In these patients, aggregability study, highly puriﬁed human B cell-derived EVS were analyzed

and oxidative damage of RBCS, combined with platelet activa- by mass spectrometry which revealed the presence of major

tion and increased amount of REVS may provoke thrombotic histocompatibility complex class I and class II, heat shock cog-

events. The authors evaluated the number of EVS, the oxida- nate 70, heat shock protein 90, integrin alpha 4, CD45, moesin,

tive stress status, the procoagulant activity, as well as the tubulin (alpha and beta), actin, G(i)alpha(2), and a multitude

proteome of EVS obtained from patients. By ﬂow cytometric of other proteins. EVS appeared to be enriched in cholesterol,

analysis, the number of phosphatidylserine-bearing EVS was sphingomyelin, and ganglioside GM3, lipids that are typically

signiﬁcantly higher as compared to controls. The high levels concentrated in detergent-resistant membranes.

of EVS did not only correlate with the increase of procoagulant LEVS of HIV-1-infected and -uninfected lymphocytic H9

activity but also with the increase of platelet counts. These cells were evaluated by Li et al. [61]. Using the technique of

EVS corresponded to two major populations: REVS and PEVS. stable isotope labeling by amino acids in cell culture (SILAC),

Proteome analysis (two-dimensional gel electrophoresis the authors compared protein expression patterns in the EVS

followed by mass spectrometry) identiﬁed about 30 proteins compartment of HIV-1-infected and -uninfected lymphocytes.

44 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

Fourteen proteins were found to be differentially expressed in (discocyte), to stomatocyte and to echinocyte (the form of RBC

the LEVS fraction of HIV-1-infected cells versus -uninfected leading to the formation of EVS) [98].

controls. Three immunomodulatory molecules were repro- Under normal conditions, REVS account for approximately

ducibly identiﬁed and included ADP-ribosyl cyclase 1 (CD38), 7.3% of EVS found in whole blood. The other populations

-lactate dehydrogenase B chain, and annexin V. This study consist of particles derived from platelets (38.5%) and EVS

revealed that LEVS released from HIV-1-infected cells are resulting from endothelial cells (43.5%) [48]. Many compre-

composed of a unique and quantitatively different protein hensive studies have been published this last decade on the

signature and harbor regulatory molecules that impact the various aspects of the biology of blood EVS [99], and their

processes of cellular apoptosis. roles in physiology as well as in physiopathology have been

In patients with B-cell malignancy, accumulation of LEVS explored in details.

have been observed and analyzed using proteomic tools by Here, a brief summary of the accumulating knowledge on

Miguet et al., in order to identify speciﬁc biomarker capa- blood EVS will be presented.

ble of diagnosing difﬁcult cases such as leukemic phase of

non-Hodgkin lymphoma [90,91]. These studies allowed the 4.1. Red blood cell-derived vesicles

identiﬁcation CD148, a membrane receptor with phosphatase

activity, as a discriminating biomarker candidate. For conﬁr- REVS formation has been described as part of RBC senes-

mation purposes, ﬂow cytometry analyses were performed on cence [71] and also proposed as a part of an apoptosis-like

158 patients and 30 controls revealing that CD148 was overex- form in these cells [100]. This “ageing” process of RBC was

pressed in mantle cell lymphoma as compared to other B-cell observed during storage in blood bank condition [22,74,101].

neoplasms. During their 120 days of lifespan, RBCS lose approximately

20% of their volume through vesicles emission whereas their

hemoglobin concentration increases by 14% [102]. Vesicula-

3.4. Proteomics of endothelial cell-derived vesicles

tion would be a mean for RBCS to get rid of speciﬁc harmful

agents such as denatured hemoglobin, C5b-9 complement

Until now, a few proteomic studies have been published

attack complex, band 3 neoantigen and IgG that tend to accu-

on the proteome of EEVS. Nevertheless, EEVS have been

mulate in RBCS or on their membrane during their lifespan

characterized at the proteome level by Banﬁ et al. [92].

[71,101,103]. The release of REVS plays a protective role that

Mass spectrometry analyses revealed the presence of newly

allows RBCS to clear away dangerous molecules, such as oxi-

described proteins such as metabolic enzymes, proteins

dized proteins [75], and thus, preventing their early removal

involved in adhesion and fusion processes, members of

from blood ﬂow. In the other hand, REVS could promote

protein folding event, cytoskeleton associated proteins and

removal of RBCS by accumulating CD47 which is an inte-

nucleosome. In an interesting study, Liu et al. provided infor-

gral membrane protein present on RBC’s surface, acting as

mation not only on proteomics of EEVS but also on the changes

a marker of self. Thanks to CD47, normal RBCS are recog-

of the protein content in the endothelial cells after stimulation

nized as self by macrophages (through their signal regulatory

and EEVS release [93]. A direct correlation between the pro-

protein ␣) and phagocytosis is inhibited. Senescent or dam-

teins that form EEVS and tumor necrosis factor-␣-activated

aged RBCS whose CD47 expression is reduced by shedding

endothelial cells was observed.

of REVS enriched in CD47 would no longer be recognized as

self and thus be eliminated by macrophages [104–106]. Still in

the context of RBC aging process, two main models result-

4. Biology of blood vesicles ing in microvesiculation have been proposed, the eryptosis

model and the band 3 clustering. The term “eryptosis” has

The biology of REVS, PEVS, LEVS, and of EEVS is depend- been introduced a few years ago by Lang’s group [100]. It

ent of the cells from which they originate. Nevertheless, describes mechanism similar to apoptosis of nucleated cells

many physiological properties, such as their role in ﬁbrino- in response to various stresses but applied to RBCS. Inﬂux of

lysis, may be shared by EVS deriving from different cellular ionic calcium through nonspeciﬁc cation channels leads to

origin, as demonstrated for LEVS and EEVS. However, when activation of several enzymes such as calpain or scramblase.

generation of plasmin is analyzed in more details, it was Reasons triggering this calcium intake are largely unknown

possible to demonstrate that LEVS expressed urokinase-type but alteration of nonspeciﬁc cation channels is often men-

plasminogen activator activity whereas EEVS are presenting tioned as a possible cause [107,108]. The band 3 clustering

tissue plasminogen activator and tissue plasminogen activa- model is characterized by protein oxidation. The oxidation of

tor/inhibitor complexes [94]. hemoglobin contributes to hemichrome formation, which is

The forces that maintain cellular and adhesive forces of constituted of Hb derived products (likely met-hemoglobin)

the cellular membrane has been studied in details, both the- linked to the inner leaﬂet, followed by the clustering and

oretically [95] and in physiological condition [96]. Thus, the aggregation of band 3 multimers in the membrane [109]. Band

remodeling of the RBC membrane that maintains its bicon- 3 clustering forms or uncovers senescent neoantigens, prob-

cave shape has been deciphered [97]. Finally, the physical ably because of relatively small structural modiﬁcations that

forces involved in the membrane structure has been stud- are recognized by naturally occurring autologous IgG with sub-

ied, and a model resulting from different dynamic forces has sequent complement activation [110]. Both models share the

been evaluated allowing to better understand the ﬂuctuations same ﬁnal outcome, which is phosphatidylserine external-

of the membrane leading to the formation of a normal RBC ization on RBCS membrane and degradation of cytoskeleton

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 45

Fig. 4 – Hypothesis of oxidation-driven vesiculation in stored RBCS. (1) RBC antioxidant defenses prevent protein

carbonylation by reducing reactive oxygen species (ROS) into less reactive intermediates. After a certain storage period,

these defenses are exceeded by the oxidative stress and protein carbonylation occurs on both soluble and membrane

proteins. (2) Carbonylated proteins are recognized and degraded by the 20S proteasome. (3) Over-oxidized proteins crosslink

and cannot be degraded by proteasome anymore, inhibiting degradation of fairly oxidized proteins as well as other

damaged proteins due to steric obstruction. (4) Oxidized hemoglobin aggregates as hemichromes and binds to and alters

band 3 conformation. (5) Hemichromes heme autoxidation produces ROS which induces cytoskeleton and membrane

protein oxidation. (6) Membrane vesiculation allows elimination of altered band 3 and oxidized membrane proteins.

Reprinted from [75], with permission from Elsevier.

proteins followed by modiﬁcation in the phosphorylation sta- and in the subsequent loss of modulation of calcium homeo-

tus of band 3 (Fig. 4) [75]. Based on recent data, a clearer stasis [119]. Increased intracellular levels of calcium appeared

picture of the molecular mechanisms underlying this pro- to promote vesiculation, even if the eryptosis-like phenomena

cess is emerging: oxidative damage induces the binding of is also induced by a calcium-independent fashion by starva-

hemoglobin to band 3, activation of calcium-dependent per- tion [120].

meable channels, phosphorylation of key player proteins and EVS have been observed in blood storage as well as in differ-

aggregation of band 3 leading to vesiculation. This process ent hematological diseases that have been recently reviewed

gives sufficient membrane flexibility resulting in REVS for- [21,22]. Briefly, REVS are involved in clinical situations char-

mation and release. The hypothesis of an oxidative stress acterized by hemolysis or endothelial activation. In sickle

being involved in RBCS storage lesions has been reinforced cell disease, the abnormal hemoglobin S adds to the mem-

by the studies of Stowell et al. [111]. By adding ascorbic acid brane instability and favors the development of EVS [121]. In

solution to RBCs during storage, the authors observed a ben- this disease, EVS number correlates to the rate of intravas-

eﬁcial effect on recovery and immunogenicity of RBCs after cular hemolysis and the degree of coagulation activation

transfusion, but not cytokine induction. They also demon- [122,123] and deﬁnes subclinical phenotypes with enhanced

strated a signiﬁcant decrease in EVS formation. Thus, the complications [124]. Phosphatidylserine-positive REVS poten-

addition of ascorbic acid (or other antioxidants) to human tiate thrombin generation [122,123,125] through factor XI

RBCs may have beneﬁcial effects. Almizraq et al., by assessing activation [28,122,126]. However, other investigators identiﬁed

RBCs throughout storage also observed signiﬁcant increases activated factor XII as being the key player in the coagula-

in percent hemolysis, while signiﬁcant decreases in ATP con- tion cascade [127]. In paroxysmal nocturnal hemoglobinuria,

centrations as well as the mean corpuscular hemoglobin complement activation may be involved in EVS generation

concentration [77,112]. The metabolic deregulation has been which contributes to the thrombotic proﬁle of these patients

identiﬁed during RBC aging in vivo [113–116], and in vitro [21,128–130]. In stem cell transplants, REVS may be useful in

[117,118], resulting in the impairment of the potassium pumps distinguishing acute graft-versus-host disease from infection

46 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

or sepsis [131]. However, other types of EVS are elevated in 4.3. Leukocyte-derived vesicles

such patients [132,133]. In malaria, rates of REVS are also cor-

related with the degree of parasitemia and the severity of the LEVS represent only a small proportion of blood EVS, but

infection [133,134]. bear important physiological properties. As mentioned, LEVS

During RBC storage, many biochemical changes happen, express markers from their parental cells (neutrophils, mono-

referred as “storage lesions” including RBCS membrane mod- cytes/macrophages, and lymphocytes) and are therefore quite

iﬁcations (it becomes more rigid), lipid rafts rearrangement, heterogeneous. They harbor membrane and cytoplasmic pro-

disruption of phospholipids asymmetry and EVS release teins as well as bioactive lipids notably related to coagulation

[4,71,135]. The accumulation of EVS during blood storage is and inﬂammation. They may carry tissue factor or coagulation

well described [74]. Recently, we identiﬁed that these EVS have inhibitors and, as a result, may participate in hemosta-

factor XI dependent procoagulant properties and that they are sis and pathological thrombosis [148]. LEVS also have both

able to initiate and propagate thrombin generation [28]. EVS pro-inﬂammatory and anti-inﬂammatory properties and are

from stored RBCS also carry blood group antigens that may clearly involved in a number of biological processes. EVS

play a role in alloimmunization [41]. derived are released from polymorphonuclear neutrophils EVS

upon activation. These EVS interfere with the maturation of

monocyte-derived dendritic cells [149] and down-modulate

the inﬂammatory response of human macrophages and den-

4.2. Platelet-derived vesicles dritic cells exposed to TLR-2 and -4 ligands [150]. This

down-modulation appeared to be mediated via the engage-

Platelet production is a complex process [136], beginning ment and activation of the Mer receptor tyrosine kinase

within the bone marrow. Different molecular mechanisms are (MerTK), as well as by an immediate Ca ﬂux and a rapid

involved in the formation of platelets from megakaryocytes release of TGF-beta1.

[137], leading to the splitting of large platelets which can LEVS show a complex relation with endothelial cells, at

also be considered as megaparticles. Platelets in circulation the same time improving the endothelial function or on the

release both large vesicles that are plasma membrane-derived contrary inducing an endothelial dysfunction. Consequently

microparticles and small ones that represent multivesicular LEVS are largely implicated in all stages of atherosclerosis

body-derived EXS [138]. The distinction between these two and circulate at a high level in the bloodstream of patients

populations of PEVS is difﬁcult because of an overlap in their with high atherothrombotic risk. LEVS modify the endothelial

molecular properties and in their sizes. Furthermore, as PEVS function and promote the recruitment of inﬂammatory cells

are released through several induction pathways, different in the vascular wall both representing necessary processes for

types of PEVS may be produced, each one bearing its mech- the progression of the atherosclerotic lesion. In addition, LEVS

anism of action. In a paper dealing with the biology of PEVS, favor the neovascularization within the vulnerable plaque

Varon et al. reviewed the extra-hemostatic effects of PEVS and, when the plaque is ruptured, take part in coagulation

and presented the possible roles of PEVS other than participa- and platelet activation. LEVS also participate in angiogene-

tion in blood coagulation [139]. In summary, they showed that sis [65]. LEVS, as well as other types of EVS – notably those

PEVS express and transfer functional receptors from platelet deriving from tumor cells – bind plasminogen and vectorize

membranes, increase expression of adhesion molecules on plasminogen activators, leading to an efﬁcient plasmin gen-

cells, stimulate the release of cytokines, activate intracellular eration and matrix metalloproteinases activation [151]. Thus,

signaling pathways, alter vascular reactivity, induce angio- LEVS behave as an efﬁcient catalytic surface involved in vascu-

genesis, and are involved in cancer metastasis. They also lar and matrix proteolysis-related biological processes, acting

mentioned that a high PEVS level is highly correlated with in many biological processes such as ﬁbrinolysis, cell survival,

aggressive tumors and a poor clinical outcome. PEVS also matrix remodeling, angiogenesis, and tumor metastasis.

have important physiopathological role in patients presenting Circulating levels of LEVS appear to be increased in adults

with type-II heparin induced thrombocytopenia [140]. PEVS with chronic B-cell lymphoproliferations (chronic lymphocytic

participate in several homeostatic multi-cellular processes leukemia, small cell lymphoma and mantle cell lymphoma)

[141], such as hemostasis, maintenance of vascular integrity, [91,152] and in children with B-cell neoplasm [153]. Finally,

and immunity, but they also have tasks in thrombotic [142], LEVS derived from leukemic cells probably play a pathological

inﬂammatory diseases [143] and cancer progression [144]. As role by participating to the coagulopathy that is some-

perfectly coined by Mause in an invited editorial, PEVS have times observed in patients with acute myeloblastic leukemia

a hegemonic role in atherogenesis [145]. PEVS are also gen- [154,155]. In a study evaluating patients with acute promyelo-

erated when platelets are prepared for transfusion [51], and cytic leukemia (a situation characterized by serious bleeding

investigators recently demonstrated that these PEVS are not and thrombotic complications), Ma et al. analyzed LEVS

removed by the various ﬁlters that are used for leukoreduction from 30 patients and healthy controls [156]. The morphol-

[146]. A stimulating new hypothesis is potential role of PEVS as ogy of the LEVS was examined by using transmission electron

a mediator of neurogenesis. Speciﬁcally, factors from platelets microscopy and laser scanning confocal microscopy. LEVS

and their PEVS may promote neo-neurogenesis by stimulat- were quantiﬁed and analyzed for their thrombin-generating

ing endogenous neural stem cells proliferation, migration and potential. Counts of LEVS in patients with acute promyelocitic

differentiation, and by stimulating niche angiogenesis and the leukemia were elevated and were typically from promyelo-

release of neurogenic signals from endothelial cells and astro- cytic cells (CD33+, tissue factor+). The CD33+ LEVS levels

cytes [147]. correlated with patient leukocyte counts and coagulation

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 47

activation (evaluated by measuring d-dimer). Moreover, LEVS launched (Journal of Extracellular Vesicles; eISSN 2001-3078),

from patients decreased the coagulation times and induced which will be the ofﬁcial journal of the Society. The ﬁrst issue

thrombin generation; interestingly, LEVS-associated thrombin is out of press. Proteomics, as highlighted in the last part of

generation was reduced by adding an anti-human tissue factor this review, is certainly a tool of major importance to char-

antibody, but neither with anti-factor XI nor anti-tissue factor acterize the proteins that are present in EVS. Proteomics has

pathway inhibitor. shown its power in a lot of topics and applications, and EVS

is and will be one of them. However, making a list of pro-

4.4. Endothelial cell-derived vesicles teins is insufﬁcient to understand the multiple functions and

roles of EVS, and proteomics is not a unique solution in ﬁne.

Vascular homeostasis is the reﬂection of quiescent, but The challenges remain the EVS isolation to obtain homoge-

competent endothelium. EEVS are released by endothelium nous subpopulations, the fractionation for accurate proteomic

[157,158]. They are now recognized as key players in a mul- analyses and the coupling to a functional approach, includ-

titude of biological functions necessary for the maintenance ing complementary data. Deﬁnitively EVS are not the rubbish

of endothelial integrity and vascular biology. EEVS have been of the cell, and should be integrated in the cellular biology.

demonstrated to act as primary and secondary messengers The future of biomarker discovery related to speciﬁc disease

of vascular inﬂammation, thrombosis, vasomotor response, will focus on EVS release in body ﬂuids from various cells. A

angiogenesis, and endothelial survival. EEVS also induce cell fascinating ﬁeld of research is open and largely dedicated to

cycle arrest through redox-sensitive processes in endothe- specialists in proteomic sciences.

lial cells, thus having implications in vascular senescence

[159]. These often-neglected EEVS are emerging as poten-

tially useful indicators of dysfunctioning endothelium. They Conﬂict of interest

have been implicated in many different diseases such as pre-

None.

eclampsia in pregnancy [160], pulmonary hypertension [161],

chronic graft versus host disease [162], antiphospholipid syn-

drome [163], or vasculitis such as in Kawasaki’s syndrome

r e f e r e n c e s

[164]. They also have been detected in cancer patients in

whose circulating levels of MPS correlate with prognosis, and

could be used as prognostic markers for example in advanced

[1] Thadikkaran L, Siegenthaler MA, Crettaz D, Queloz PA,

non-small cell lung cancer [165]. Very recently, EEVS have

Schneider P, Tissot JD. Recent advances in blood-related

been implicated as player in mitral valve disease. In a study proteomics. Proteomics 2005;5:3019–34.

of patients with mitral valve disease, Ci et al. observed an [2] Lion N, Tissot JD. Application of proteomics to hematology:

increased level of circulating EEVS from blood samples when the revolution is starting. Expert Review of Proteomics

2008;5:375–9.

compared to controls [166]. In addition, they also analyzed

[3] Tissot JD. Blood proteomics. Journal of Proteomics

the effects of EEVS on cells derived from human mitral valve

2010;73:466–7.

endothelial cells. The authors observed modiﬁcations of the

[4] Lion N, Crettaz D, Rubin O, Tissot JD. Stored red blood cells:

phosphorylation of Akt, of endothelial nitric oxide synthase,

a changing universe waiting for its map(s). Journal of

of the association of endothelial nitric oxide synthase and Proteomics 2010;73:374–85.

heat shock protein 90, the generation of nitric oxide as well [5] Prudent M, Crettaz D, Delobel J, Tissot JD, Lion N. Proteomic

analysis of intercept-treated platelets. Journal of

as of the generation of superoxide anion. EEVS were signif-

Proteomics 2012;76:316–28.

icantly increased in patients with mitral valve disease. The

[6] Liumbruno G, D’Alessandro A, Grazzini G, Zolla L. How has

increase of EEVS also impaired the function of cells derived

proteomics informed transfusion biology so far? Critical

from human mitral valve endothelial cells by inhibiting the

Reviews in Oncology/Hematology 2010;76:153–72.

Akt/endothelial nitric oxide synthase – heat shock protein 90 [7] Liumbruno G, D’Alessandro A, Grazzini G, Zolla L.

signaling pathway. CD36+ EVS have been observed as being Blood-related proteomics. Journal of Proteomics

increased in the blood of obese patients, with or without type 2010;73:483–507.

[8] Cluitmans JC, Hardeman MR, Dinkla S, Brock R, Bosman GJ.

2 diabetes mellitus. Interestingly enough, CD36+ EVS origi-

Red blood cell deformability during storage: towards

nating from erythrocytes were identiﬁed as being increased

functional proteomics and metabolomics in the Blood

in obese type 2 diabetic patients, contrasting with the main

Bank. Blood Transfusion 2012;10(Suppl. 2):s12–8.

source of CD36+ EVS that was of endothelial origin in obese

[9] Hess JR. Red cell storage. Journal of Proteomics

non-diabetic control patients [167]. 2010;73:368–73.

[10] Hess JR, Grazzini G. Blood proteomics and transfusion

safety. Journal of Proteomics 2010;73:365–7.

5. Conclusions and perspectives [11] Lion N, Prudent M, Crettaz D, Tissot JD. Proteomics and

transfusion medicine. Transfusion Clinique et Biologique

2011;18:79–96.

Nowadays, the study of the biology of EVS, EXS, MPS and other

[12] Ray S, Reddy PJ, Jain R, Gollapalli K, Moiyadi A, Srivastava S.

extracellular vesicles is a fascinating ﬁeld of research. This

Proteomic technologies for the identiﬁcation of disease

domain is rapidly growing and the medical applications of

biomarkers in serum: advances and challenges ahead.

such studies are at our doorstep. An International Society for Proteomics 2011;11:2139–61.

Extracellular vesicles has been created in 2012, and the annual [13] Hanash S, Taguchi A. Application of proteomics to cancer

early detection. Cancer Journal 2011;17:423–8.

congress was in Boston, April 2013. A new journal has been

48 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

[14] Taguchi A, Hanash SM. Unleashing the power of [34] Aras O, Shet A, Bach RR, Hysjulien JL, Slungaard A, Hebbel

proteomics to develop blood-based cancer markers. RP, et al. Induction of microparticle- and cell-associated

Clinical Chemistry 2013;59:119–26. intravascular tissue factor in human endotoxemia. Blood

[15] Brambilla F, Lavatelli F, Di Silvestre D, Valentini V, Rossi R, 2004;103:4545–53.

Palladini G, et al. Reliable typing of systemic amyloidoses [35] Mallat Z, Hugel B, Ohan J, Leseche G, Freyssinet JM, Tedgui

through proteomic analysis of subcutaneous adipose A. Shed membrane microparticles with procoagulant

tissue. Blood 2012;119:1844–7. potential in human atherosclerotic plaques: a role for

[16] Brambilla F, Lavatelli F, Merlini G, Mauri P. Clinical apoptosis in plaque thrombogenicity. Circulation

proteomics for diagnosis and typing of systemic 1999;99:348–53.

amyloidoses. Proteomics, Clinical Applications [36] Aupeix K, Hugel B, Martin T, Bischoff P, Lill H, Pasquali JL,

2013;7:136–43. et al. The signiﬁcance of shed membrane particles during

[17] Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, Aradi B, et al. programmed cell death in vitro, and in vivo, in HIV-1

Membrane vesicles, current state-of-the-art: emerging role infection. Journal of Clinical Investigation 1997;99:1546–54.

of extracellular vesicles. Cellular and Molecular Life [37] Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG,

Sciences 2011;68:2667–88. Freyssinet JM, et al. Elevated levels of shed membrane

[18] Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular microparticles with procoagulant potential in the

organelles important in intercellular communication. peripheral circulating blood of patients with acute

Journal of Proteomics 2010;73:1907–20. coronary syndromes. Circulation 2000;101:841–3.

[19] Mause SF, Weber C, Microparticles:. protagonists of a novel [38] Maurer-Spurej E, Labrie A, Pittendreigh C, Chipperﬁeld K,

communication network for intercellular information Smith C, Heddle N, et al. Platelet quality measured with

exchange. Circulation Research 2010;107:1047–57. dynamic light scattering correlates with transfusion

[20] Diamant M, Tushuizen ME, Sturk A, Nieuwland R. Cellular outcome in hematologic malignancies. Transfusion

microparticles: new players in the ﬁeld of vascular disease? 2009;49:2276–84.

European Journal of Clinical Investigation 2004;34: [39] Xu Y, Nakane N, Maurer-Spurej E. Novel test for

392–401. microparticles in platelet-rich plasma and platelet

[21] Tissot JD, Rubin O, Canellini G. Analysis and clinical concentrates using dynamic light scattering. Transfusion

relevance of microparticles from red blood cells. Current 2011;51:363–70.

Opinion in Hematology 2010;17:571–7. [40] Mrvar-Brecko A, Sustar V, Jansa V, Stukelj R, Jansa R,

[22] Rubin O, Canellini G, Delobel J, Lion N, Tissot JD. Red blood Mujagic E, et al. Isolated microvesicles from peripheral

cell micropartilces: clinical relevance. Transfusion blood and body ﬂuids as observed by scanning electron

Medicine and Hemotherapy 2012;39:342–7. microscope. Blood Cells, Molecules, and Diseases

[23] Simak J, Gelderman MP. Cell membrane microparticles in 2010;44:307–12.

blood and blood products: potentially pathogenic agents [41] Canellini G, Rubin O, Delobel J, Crettaz D, Lion N, Tissot JD.

and diagnostic markers. Transfusion Medicine Reviews Red blood cell microparticles and blood group antigens: an

2006;20:1–26. analysis by ﬂow cytometry. Blood Transfusion

[24] Morel O, Toti F, Hugel B, Bakouboula B, Camoin-Jau L, 2012;10(Suppl 2):s39–45.

Dignat-George F, et al. Procoagulant microparticles: [42] Gyorgy B, Szabo TG, Turiak L, Wright M, Herczeg P, Ledeczi

disrupting the vascular homeostasis equation? Z, et al. Improved ﬂow cytometric assessment reveals

Arteriosclerosis, Thrombosis, and Vascular Biology distinct microvesicle (cell-derived microparticle) signatures

2006;26:2594–604. in joint diseases. PLoS ONE 2012;7:e49726.

[25] Hugel B, Martinez MC, Kunzelmann C, Freyssinet JM. [43] Montoro-Garcia S, Shantsila E, Orenes-Pinero E, Lozano ML,

Membrane microparticles: two sides of the coin. Physiology Lip GY. An innovative ﬂow cytometric approach for

2005;20:22–7. small-size platelet microparticles: inﬂuence of calcium.

[26] Wolf P. The nature and signiﬁcance of platelet products in Thrombosis and Haemostasis 2012;108:373–83.

human plasma. British Journal of Haematology [44] Yuana Y, Bertina RM, Osanto S. Pre-analytical and

1967;13:269–88. analytical issues in the analysis of blood microparticles.

[27] Greenwalt TJ. The how and why of exocytic vesicles. Thrombosis and Haemostasis 2011;105:396–408.

Transfusion 2006;46:143–52. [45] Rubin O, Crettaz D, Tissot JD, Lion N. Pre-analytical and

[28] Rubin O, Delobel J, Prudent M, Lion N, Kohl K, Tucker EI, methodological challenges in red blood cell microparticle

et al. Red blood cell-derived microparticles isolated from proteomics. Talanta 2010;82:1–8.

blood units initiate and propagate thrombin generation. [46] Delobel J, Rubin O, Prudent M, Crettaz D, Tissot JD, Lion N.

Transfusion 2013 [in press]. Biomarker analysis of stored blood products: emphasis on

[29] Schifferli JA. Microvesicles are messengers. Seminars in pre-analytical issues. International Journal of Molecular

Immunopathology 2011;33:393–4. Sciences 2010;11:4601–17.

[30] Sadallah S, Eken C, Schifferli JA. Ectosomes as [47] Mullier F, Bailly N, Chatelain C, Chatelain B, Dogne JM.

immunomodulators. Seminars in Immunopathology Pre-analytical issues in the measurement of circulating

2011;33:487–95. microparticles: current recommendations and pending

[31] Sadallah S, Eken C, Schifferli JA. Ectosomes as modulators questions. Journal of Thrombosis and Haemostasis

of inﬂammation and immunity. Clinical and Experimental 2013;11:693–6.

Immunology 2011;163:26–32. [48] Xiong Z, Oriss TB, Cavaretta JP, Rosengart MR, Lee JS. Red

[32] Shet AS. Characterizing blood microparticles: technical cell microparticle enumeration: validation of a ﬂow

aspects and challenges. Journal of Vascular Health and Risk cytometric approach. Vox Sanguinis 2012;103:42–8.

Management 2008;4:769–74. [49] Leong HS, Podor TJ, Manocha B, Lewis JD. Validation of ﬂow

[33] Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. cytometric detection of platelet microparticles and

Activated platelets release two types of membrane liposomes by atomic force microscopy. Journal of

vesicles: microvesicles by surface shedding and exosomes Thrombosis and Haemostasis 2011;9:2466–76.

derived from exocytosis of multivesicular bodies and [50] Chandler WL. Microparticle counts in platelet-rich and

alpha-granules. Blood 1999;94:3791–9. platelet-free plasma, effect of centrifugation and

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 49

sample-processing protocols. Blood Coagulation and [69] Carayon K, Chaoui K, Ronzier E, Lazar I, Bertrand-Michel J,

Fibrinolysis 2013;24:125–32. Roques V, et al. Proteolipidic composition of exosomes

[51] Strasser EF, Happ S, Weiss DR, Pfeiffer A, Zimmermann R, changes during reticulocyte maturation. Journal of

Eckstein R. Microparticle detection in platelet products by Biological Chemistry 2011;286:34426–39.

three different methods. Transfusion 2013;53:156–66. [70] Bosman GJ, Willekens FL, Werre JM. Erythrocyte aging: a

[52] Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, Ferguson more than superﬁcial resemblance to apoptosis? Cellular

DJ, Hole P, et al. Sizing and phenotyping of cellular vesicles Physiology and Biochemistry 2005;16:1–8.

using Nanoparticle Tracking Analysis. Nanomedicine [71] Bosman GJ, Lasonder E, Luten M,

2011;7:780–8. Roerdinkholder-Stoelwinder B, Novotny VM, Bos H, et al.

[53] Redman CW, Tannetta DS, Dragovic RA, Gardiner C, The proteome of red cell membranes and vesicles during

Southcombe JH, Collett GP, et al. Review: does size matter? storage in blood bank conditions. Transfusion

Placental debris and the pathophysiology of pre-eclampsia. 2008;48:827–35.

Placenta 2012;33(Suppl.):S48–54. [72] Bosman GJ, Lasonder E, Groenen-Dopp YA, Willekens FL,

[54] Bosman GJ, Lasonder E, Groenen-Dopp YA, Willekens FL, Werre JM, Novotny VM. Comparative proteomics of

Werre JM. The proteome of erythrocyte-derived erythrocyte aging in vivo and in vitro. Journal of Proteomics

microparticles from plasma: new clues for erythrocyte 2010;73:396–402.

aging and vesiculation. Journal of Proteomics 2012;76 [Spec [73] Kriebardis AG, Antonelou MH, Stamoulis KE,

No.: 203-10]. Economou-Petersen E, Margaritis LH, Papassideri IS.

[55] Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: RBC-derived vesicles during storage: ultrastructure, protein

proteomic insights and diagnostic potential. Expert Review composition, oxidation, and signaling components.

of Proteomics 2009;6:267–83. Transfusion 2008;48:1943–53.

[56] Choi DS, Kim DK, Kim YK, Gho YS. Proteomics, [74] Rubin O, Crettaz D, Canellini G, Tissot JD, Lion N.

transcriptomics and lipidomics of exosomes and Microparticles in stored red blood cells: an approach using

ectosomes. Proteomics 2013 [in press]. ﬂow cytometry and proteomic tools. Vox Sanguinis

[57] Kim HS, Choi DY, Yun SJ, Choi SM, Kang JW, Jung JW, et al. 2008;95:288–97.

Proteomic analysis of microvesicles derived from human [75] Delobel J, Prudent M, Rubin O, Crettaz D, Tissot JD, Lion N.

mesenchymal stem cells. Journal of Proteome Research Subcellular fractionation of stored red blood cells reveals a

2012;11:839–49. compartment-based protein carbonylation evolution.

[58] Epple LM, Grifﬁths SG, Dechkovskaia AM, Dusto NL, White Journal of Proteomics 2012;76 [Spec No.: 181-93].

J, Ouellette RJ, et al. Medulloblastoma exosome proteomics [76] Dinkla S, Novotny VM, Joosten I, Bosman GJ.

yield functional roles for extracellular vesicles. PLoS ONE Storage-induced changes in erythrocyte membrane

2012;7:e42064. proteins promote recognition by autoantibodies. PLoS ONE

[59] Xiao D, Ohlendorf J, Chen Y, Taylor DD, Rai SN, Waigel S, 2012;7:e42250.

et al. Identifying mRNA, microRNA and protein proﬁles of [77] Almizraq R, Tchir JD, Holovati JL, Acker JP. Storage of red

melanoma exosomes. PLoS ONE 2012;7:e46874. blood cells affects membrane composition,

[60] Choi DS, Park JO, Jang SC, Yoon YJ, Jung JW, Choi DY, et al. microvesiculation, and in vitro quality. Transfusion 2013 [in

Proteomic analysis of microvesicles derived from human press].

colorectal cancer ascites. Proteomics 2011;11:2745–51. [78] Chaichompoo P, Kumya P, Khowawisetsut L, Chiangjong W,

[61] Li M, Aliotta JM, Asara JM, Tucker L, Quesenberry P, Lally M, Chaiyarit S, Pongsakul N, et al. Characterizations and

et al. Quantitative proteomic analysis of exosomes from proteome analysis of platelet-free plasma-derived

HIV-1-infected lymphocytic cells. Proteomics microparticles in beta-thalassemia/hemoglobin E patients.

2012;12:2203–11. Journal of Proteomics 2012;76 [Spec No.: 239-50].

[62] Dear JW, Street JM, Bailey MA. Urinary exosomes: a [79] Di Michele M, Van Geet C, Freson K. Recent advances in

reservoir for biomarker discovery and potential mediators platelet proteomics. Expert Review of Proteomics

of intra-renal signaling. Proteomics 2013 [in press]. 2012;9:451–66.

[63] Santucci L, Candiano G, Bruschi M, Bodria M, Murtas C, [80] Zufferey A, Fontana P, Reny JL, Nolli S, Sanchez JC. Platelet

Petretto A, et al. Urinary proteome in a snapshot: normal proteomics. Mass Spectrometry Reviews 2012;31:331–51.

urine and glomerulonephritis. Journal of Nephrology 2013, [81] Senis Y, Garcia A. Platelet proteomics: state of the art and

http://dx.doi.org/10.5301/jn.5000233 [in press]. future perspective. Methods in Molecular Biology

[64] Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P. Body 2012;788:367–99.

ﬂuid derived exosomes as a novel template for clinical [82] Senzel L, Gnatenko DV, Bahou WF. The platelet proteome.

diagnostics. Journal of Translational Medicine 2011;9:86. Current Opinion in Hematology 2009;16:329–33.

[65] Street JM, Barran PE, Mackay CL, Weidt S, Balmforth C, [83] Greening DW, Glenister KM, Sparrow RL, Simpson RJ.

Walsh TS, et al. Identiﬁcation and proteomic proﬁling of Enrichment of human platelet membranes for proteomic

exosomes in human cerebrospinal ﬂuid. Journal of analysis. Methods in Molecular Biology 2009;528:245–58.

Translational Medicine 2012;10:5. [84] Premsler T, Lewandrowski U, Sickmann A, Zahedi RP.

[66] Little KM, Smalley DM, Harthun NL, Ley K. The plasma Phosphoproteome analysis of the platelet plasma

microparticle proteome. Seminars in Thrombosis and membrane. Methods in Molecular Biology 2011;728:279–90.

Hemostasis 2010;36:845–56. [85] Wright B, Stanley RG, Kaiser WJ, Gibbins JM. The

[67] Ostergaard O, Nielsen CT, Iversen LV, Jacobsen S, Tanassi JT, integration of proteomics and systems approaches to map

Heegaard NH. Quantitative proteome proﬁling of normal regulatory mechanisms underpinning platelet function.

human circulating microparticles. Journal of Proteome Proteomics Clinical Applications 2013;7:144–54.

Research 2012;11:2154–63. [86] Garcia BA, Smalley DM, Cho H, Shabanowitz J, Ley K, Hunt

[68] Bastos-Amador P, Royo F, Gonzalez E, Conde-Vancells J, DF. The platelet microparticle proteome. Journal of

Palomo-Diez L, Borras FE, et al. Proteomic analysis of Proteome Research 2005;4:1516–21.

microvesicles from plasma of healthy donors reveals high [87] Jin M, Drwal G, Bourgeois T, Saltz J, Wu HM. Distinct

individual variability. Journal of Proteomics proteome features of plasma microparticles. Proteomics 2012;75:3574–84. 2005;5:1940–52.

50 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

[88] Dean WL, Lee MJ, Cummins TD, Schultz DJ, Powell DW. phosphatidylserine with ﬂow cytometry. Transfusion

Proteomic and functional characterisation of platelet 2005;45:1496–503.

microparticle size classes. Thrombosis and Haemostasis [105] Oldenborg PA. Role of CD47 and signaling protein alpha

␣

2009;102:711–8. (SIRP ) in regulating the clearence of viable or aged blood

[89] Wubbolts R, Leckie RS, Veenhuizen PT, Schwarzmann G, cells. Transfusion Medicine and Hemotherapy

Mobius W, Hoernschemeyer J, et al. Proteomic and 2012;39:315–20.

biochemical analyses of human B cell-derived exosomes. [106] Burger P, de Korte D, van den Berg TK, van Bruggen R. CD47

Potential implications for their function and multivesicular in erythrocyte ageing and clearence – the Dutch point of

body formation. Journal of Biological Chemistry view. Transfusion Medicine and Hemotherapy

2003;278:10963–72. 2012;39:348–52.

[90] Miguet L, Pacaud K, Felden C, Hugel B, Martinez MC, [107] Low PS, Willardson BM, Mohandas N, Rossi M, Shohet S.

Freyssinet JM, et al. Proteomic analysis of malignant Contribution of the band 3-ankyrin interaction to

lymphocyte membrane microparticles using double erythrocyte membrane mechanical stability. Blood

ionization coverage optimization. Proteomics 1991;77:1581–6.

2005;6:153–71. [108] Lang F, Gulbins E, Lerche H, Huber SM, Kempe DS, Foller M.

[91] Miguet L, Bechade G, Fornecker L, Zink E, Felden C, Gervais Eryptosis, a window to systemic disease. Cellular

C, et al. Proteomic analysis of malignant B-cell derived Physiology and Biochemistry 2008;22:373–80.

microparticles reveals CD148 as a potentially useful [109] Mannu F, Arese P, Cappellini MD, Fiorelli G, Cappadoro M,

antigenic biomarker for mantle cell lymphoma diagnosis. Giribaldi G, et al. Role of hemichrome binding to

Journal of Proteome Research 2009;8:3346–54. erythrocyte membrane in the generation of band-3

[92] Banﬁ C, Brioschi M, Wait R, Begum S, Gianazza E, Pirillo A, alterations in beta-thalassemia intermedia erythrocytes.

et al. Proteome of endothelial cell-derived procoagulant Blood 1995;86:2014–20.

microparticles. Proteomics 2005;5:4443–55. [110] Arese P, Turrini F, Schwarzer E. Band

[93] Liu Y, Huang W, Zhang R, Wu J, Li L, Tang Y. Proteomic 3/complement-mediated recognition and removal of

analysis of TNF-alpha-activated endothelial cells and normally senescent and pathological human erythrocytes.

endothelial microparticles. Molecular Medicine Reports Cellular Physiology and Biochemistry 2005;16:133–46.

2012. [111] Stowell SR, Smith NH, Zimring JC, Fu X, Palmer AF, Fontes J,

[94] Lacroix R, Plawinski L, Robert S, Doeuvre L, Sabatier F, et al. Addition of ascorbic acid solution to stored murine

Martinez de Lizarrondo S, et al. Leukocyte- and red blood cells increases posttransfusion recovery and

endothelial-derived microparticles: a circulating source for decreases microparticles and alloimmunization.

ﬁbrinolysis. Haematologica 2012;97:1864–72. Transfusion 2013 [in press].

[95] Kabaso D, Shlomovitz R, Schloen K, Stradal T, Gov NS. [112] Dumaswala UJ, Wilson MJ, Wu YL, Wykle J, Zhuo L,

Theoretical model for cellular shapes driven by protrusive Douglass LM, et al. Glutathione loading prevents free

and adhesive forces. PLoS Computational Biology radical injury in red blood cells after storage. Free Radical

2011;7:e1001127. Research 2000;33:517–29.

[96] Gov NS, Safran SA. Red blood cell membrane ﬂuctuations [113] D’Alessandro A, Gevi F, Zolla L. Red blood cell metabolism

and shape controlled by ATP-induced cytoskeletal defects. under prolonged anaerobic storage. Molecular Biosystems

Biophysical Journal 2005;88:1859–74. 2013 [in press].

[97] Park Y, Best CA, Auth T, Gov NS, Safran SA, Popescu G, et al. [114] Marrocco C, Pallotta V, D’Alessandro A, Alves G, Zolla L. Red

Metabolic remodeling of the human red blood cell blood cell populations and membrane levels of

membrane. Proceedings of the National Academy of peroxiredoxin 2 as candidate biomarkers to reveal blood

Sciences of the United States of America 2010;107: doping. Blood Transfusion 2012;10(Suppl. 2):s71–7.

1289–94. [115] D’Amici GM, Mirasole C, D’Alessandro A, Yoshida T,

[98] Auth T, Safran SA, Gov NS. Fluctuations of coupled ﬂuid Dumont LJ, Zolla L. Red blood cell storage in SAGM and

and solid membranes with application to red blood cells. AS3: a comparison through the membrane

Physical Review E: Statistical, Nonlinear, and Soft Matter two-dimensional electrophoresis proteome. Blood

Physics 2007;76:051910. Transfusion 2012;10(Suppl. 2):s46–54.

[99] Burnier L, Fontana P, Kwak BR, Angelillo-Scherrer A. [116] D’Alessandro A, Giardina B, Gevi F, Timperio AM, Zolla L.

Cell-derived microparticles in haemostasis and vascular Clinical metabolomics: the next stage of clinical

medicine. Thrombosis and Haemostasis 2009;101:439–51. biochemistry. Blood Transfusion 2012;10(Suppl. 2):

[100] Foller M, Huber SM, Lang F. Erythrocyte programmed cell s19–24.

death. IUBMB Life 2008;60:661–8. [117] Antonelou MH, Tzounakas VL, Velentzas AD, Stamoulis KE,

[101] Iida K, Whitlow MB, Nussenzweig V. Membrane Kriebardis AG, Papassideri IS. Effects of pre-storage

vesiculation protects erythrocytes from destruction by leukoreduction on stored red blood cells signaling: a

complement. Journal of Immunology 1991;147:2638–42. time-course evaluation from shape to proteome. Journal of

[102] Willekens FL, Werre JM, Kruijt JK, Proteomics 2012;76. Spec No. [220-38].

Roerdinkholder-Stoelwinder B, Groenen-Dopp YA, van den [118] Gevi F, D’Alessandro A, Rinalducci S, Zolla L. Alterations of

Bos AG, et al. Liver Kupffer cells rapidly remove red blood red blood cell metabolome during cold liquid storage of

cell-derived vesicles from the circulation by scavenger erythrocyte concentrates in CPD-SAGM. Journal of

receptors. Blood 2005;105:2141–5. Proteomics 2012;76. Spec No. [168-80].

[103] Willekens FL, Werre JM, Groenen-Dopp YA, [119] Felix CF, Oliveira VH, Moreira OC, Mignaco JA, Barrabin H,

Roerdinkholder-Stoelwinder B, de Pauw B, Bosman GJ. Scofano HM. Inhibition of plasma membrane Ca -ATPase

Erythrocyte vesiculation: a self-protective mechanism? by heparin is modulated by potassium. International

British Journal of Haematology 2008;141:549–56. Journal of Biochemistry and Cell Biology 2007;39:586–96.

[104] Stewart A, Urbaniak S, Turner M, Bessos H. The application [120] Pompeo G, Girasole M, Cricenti A, Boumis G, Bellelli A,

of a new quantitative assay for the monitoring of Amiconi S. Erythrocyte death in vitro induced by starvation

integrin-associated protein CD47 on red blood cells during in the absence of Ca(2+). Biochimica et Biophysica Acta

storage and comparison with the expression of CD47 and 2010;1798:1047–55.

t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52 51

[121] Bosman GJ. Erythrocyte aging in sickle cell disease. Cellular [141] Aatonen M, Gronholm M, Siljander PR. Platelet-derived

and Molecular Biology 2004;50:81–6. microvesicles: multitalented participants in intercellular

[122] van Beers EJ, Schaap MC, Berckmans RJ, Nieuwland R, Sturk communication. Seminars in Thrombosis and Hemostasis

A, van Doormaal FF, et al. Circulating erythrocyte-derived 2012;38:102–13.

microparticles are associated with coagulation activation [142] Suades R, Padro T, Vilahur G, Badimon L. Circulating and

in sickle cell disease. Haematologica 2009;94:1513–9. platelet-derived microparticles in human blood enhance

[123] Westerman M, Pizzey A, Hirschman J, Cerino M, thrombosis on atherosclerotic plaques. Thrombosis and

Weil-Weiner Y, Ramotar P, et al. Microvesicles in Haemostasis 2012;108:1208–19.

haemoglobinopathies offer insights into mechanisms of [143] Boilard E, Blanco P, Nigrovic PA, Platelets:. active players in

hypercoagulability, haemolysis and the effects of therapy. the pathogenesis of arthritis and SLE. Nature Reviews

British Journal of Haematology 2008;142:126–35. Rheumatology 2012;8:534–42.

[124] Tantawy AA, Adly AA, Ismail EA, Habeeb NM, Farouk A. [144] Varon D, Hayon Y, Dashevsky O, Shai E. Involvement of

Circulating platelet and erythrocyte microparticles in platelet derived microparticles in tumor metastasis and

young children and adolescents with sickle cell disease: tissue regeneration. Thrombosis Research 2012;130(Suppl

relation to cardiovascular complications. Platelets 2012. 1):S98–9.

[125] Setty BN, Rao AK, Stuart MJ. Thrombophilia in sickle cell [145] Mause SF. Platelet microparticles: reinforcing the

disease: the red cell connection. Blood 2001;98:3228–33. hegemony of platelets in atherothrombosis. Thrombosis

[126] Horne 3rd MK, Cullinane AM, Merryman PK, Hoddeson EK. and Haemostasis 2012:109.

The effect of red blood cells on thrombin generation. [146] Nollet KE, Saito S, Ono T, Ngoma A, Ohto H. Microparticle

British Journal of Haematology 2006;133:403–8. formation in apheresis platelets is not affected by three

[127] Van Der Meijden PE, Van Schilfgaarde M, Van Oerle R, Renne leukoreduction ﬁlters. Transfusion 2013.

T, ten Cate H, Spronk HM. Platelet- and erythrocyte-derived [147] Hayon Y, Shai E, Varon D, Leker RR. The role of platelets

microparticles trigger thrombin generation via factor XIIa. and their microparticles in rehabilitation of ischemic brain

Journal of Thrombosis and Haemostasis 2012;10:1355–62. tissue. CNS & Neurological Disorders Drug Targets

[128] Ninomiya H, Kawashima Y, Hasegawa Y, Nagasawa T. 2012;11:921–5.

Complement-induced procoagulant alteration of red blood [148] Angelillo-Scherrer A. Leukocyte-derived microparticles in

cell membranes with microvesicle formation in vascular homeostasis. Circulation Research

paroxysmal nocturnal haemoglobinuria (PNH): implication 2012;110:356–69.

for thrombogenesis in PNH. British Journal of Haematology [149] Eken C, Gasser O, Zenhaeusern G, Oehri I, Hess C, Schifferli

1999;106:224–31. JA. Polymorphonuclear neutrophil-derived ectosomes

[129] Whitlow M, Iida K, Marshall P, Silber R, Nussenzweig V. interfere with the maturation of monocyte-derived

Cells lacking glycan phosphatidylinositol-linked proteins dendritic cells. Journal of Immunology 2008;180:817–24.

have impaired ability to vesiculate. Blood 1993;81:510–6. [150] Eken C, Sadallah S, Martin PJ, Treves S, Schifferli JA.

[130] Kozuma Y, Sawahata Y, Takei Y, Chiba S, Ninomiya H. Ectosomes of polymorphonuclear neutrophils activate

Procoagulant properties of microparticles released from multiple signaling pathways in macrophages.

red blood cells in paroxysmal nocturnal haemoglobinuria. Immunobiology 2013;218:382–92.

British Journal of Haematology 2011;152:631–9. [151] Lacroix R, Dignat-George F, Microparticles:. new

[131] Rank A, Nieuwland R, Toth B, Pihusch V, Delker R, Hiller E, protagonists in pericellular and intravascular proteolysis.

et al. Microparticles for diagnosis of graft-versus-host Seminars in Thrombosis and Hemostasis 2013;39:

disease after allogeneic stem cell transplantation. 33–9.

Transplantation 2011;92:244–50. [152] Ghosh AK, Secreto CR, Knox TR, Ding W, Mukhopadhyay D,

[132] Reid VL, Webster NR. Role of microparticles in sepsis. Kay NE. Circulating microvesicles in B-cell chronic

British Journal of Anaesthesia 2012;109:503–13. lymphocytic leukemia can stimulate marrow stromal cells:

[133] Delabranche X, Berger A, Boisrame-Helms J, Meziani F. implications for disease progression. Blood

Microparticles and infectious diseases. Médecine et 2010;115:1755–64.

Maladies Infectieuses 2012;42:335–43. [153] Savasan S, Buyukavci M, Buck S, Ravindranath Y.

[134] Campos FM, Franklin BS, Teixeira-Carvalho A, Filho AL, de Leukaemia/lymphoma cell microparticles in childhood

Paula SC, Fontes CJ, et al. Augmented plasma mature B cell neoplasms. Journal of Clinical Pathology

microparticles during acute Plasmodium vivax infection. 2004;57:651–3.

Malaria Journal 2010;9:327. [154] Kwaan HC, Rego EM. Role of microparticles in the

[135] D’Alessandro A, D’Amici GM, Vaglio S, Zolla L. Time-course hemostatic dysfunction in acute promyelocytic leukemia.

investigation of SAGM-stored leukocyte-ﬁltered red bood Seminars in Thrombosis and Hemostasis 2010;36:

cell concentrates: from metabolism to proteomics. 917–24.

Haematologica 2012;97:107–15. [155] Van Aalderen MC, Trappenburg MC, Van Schilfgaarde M,

[136] Thon JN, Italiano Jr JE. Does size matter in platelet Molenaar PJ, Ten Cate H, Terpstra WE, et al. Procoagulant

production? Blood 2012;120:1552–61. myeloblast-derived microparticles in AML patients:

[137] Chen K, Rondina MT, Weyrich AS. A sticky story for STAT3 changes in numbers and thrombin generation potential

in platelets. Circulation 2013;127:421–3. during chemotherapy. Journal of Thrombosis and

[138] Siljander PR. Platelet-derived microparticles – an updated Haemostasis 2011;9:223–6.

perspective. Thrombosis Research 2011;127(Suppl. 2):S30–3. [156] Ma G, Liu F, Lv L, Gao Y, Su Y. Increased

[139] Varon D, Shai E. Role of platelet-derived microparticles in promyelocytic-derived microparticles: a novel potential

angiogenesis and tumor progression. Discovery Medicine factor for coagulopathy in acute promyelocytic leukemia.

2009;8:237–41. Annals of Hematology 2013;92:645–52.

[140] Mullier F, Bailly N, Cornet Y, Dubuc E, Robert S, Osselaer JC, [157] Sabatier F, Camoin-Jau L, Anfosso F, Sampol J,

et al. Contribution of platelet microparticles generation Dignat-George F. Circulating endothelial cells,

assay to the diagnosis of type II heparin-induced microparticles and progenitors: key players towards the

thrombocytopenia. Thrombosis and Haemostasis deﬁnition of vascular competence. Journal of Cellular and

2010;103:1277–81. Molecular Medicine 2009;13:454–71.

52 t r a n s l a t i o n a l p r o t e o m i c s 1 ( 2 0 1 3 ) 38–52

[158] Yong PJ, Koh CH, Shim W. Endothelial microparticles: [163] Pericleous C, Clarke LA, Brogan PA, Latchman DS, Isenberg

missing link in endothelial dysfunction? European Journal DA, Ioannou Y, et al. Endothelial microparticle release is

of Preventive Cardiology 2013;20:496–512. stimulated in vitro by puriﬁed IgG from patients with the

[159] Burger D, Kwart DG, Montezano AC, Read NC, Kennedy CR, antiphospholipid syndrome. Thrombosis and Haemostasis

Thompson CS, et al. Microparticles induce cell cycle arrest 2013;109:72–8.

through redox-sensitive processes in endothelial cells: [164] Tan Z, Yuan Y, Chen S, Chen Y, Chen TX. Elevated plasma

implications in vascular senescence. Journal of the endothelial microparticles together with TNF-alpha

American Heart Association 2012;1:e001842. and IL-6 in Kawasaki disease. Indian Pediatrics 2013

[160] Petrozella L, Mahendroo M, Timmons B, Roberts S, McIntire [in press].

D, Alexander JM. Endothelial microparticles and the [165] Fleitas T, Martinez-Sales V, Vila V, Reganon E, Mesado D,

antiangiogenic state in preeclampsia and the postpartum Martin M, et al. Circulating endothelial cells and

period. American Journal of Obstetrics and Gynecology microparticles as prognostic markers in advanced

2012;207(140):e20–6. non-small cell lung cancer. PLoS ONE 2012;7:

[161] Amabile N, Guignabert C, Montani D, Yeghiazarians Y, e47365.

Boulanger CM, Humbert M. Cellular microparticles in the [166] Ci HB, Ou ZJ, Chang FJ, Liu DH, He GW, Xu Z, et al.

pathogenesis of pulmonary hypertension. European Endothelial microparticles increase in mitral valve disease

Respiratory Journal 2013 [in press]. and impair mitral valve endothelial function. American

[162] Wu Q, Chen H, Fang J, Xie W, Hong M, Xia L. Elevated Journal of Physiology – Endocrinology and Metabolism

Fas/FasL system and endothelial cell microparticles are 2013;304:e695–702.

involved in endothelial damage in acute graft-versus-host [167] Alkhatatbeh MJ, Enjeti AK, Acharya S, Thorne RF, Lincz LF.

disease: a clinical analysis. Leukemia Research The origin of circulating CD36 in type 2 diabetes. Nutrition

2012;36:275–80. & Diabetes 2013;3:e59.