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Sander, Tara L.*; Ou, Jing-Song*; Densmore, John C.*; Kaul, Sushma*; Matus, Isaac; Twigger, Simon; Halligan, Brian; Greene, Andrew S.; Pritchard, Kirkwood A. Jr*; Oldham, Keith T.*

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doi: 10.1097/SHK.0b013e3181454898



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Microparticles (MPs) are circulating fragments of the plasma membrane derived from a variety of cell types in the vascular system. They were first reported by Wolf (1) in 1967 and termed "cell dust.“ They have since been recognized and termed as cell membrane vesicles or particles in the vascular system that are shed from platelets (2-6), leukocytes (7, 8), lymphocytes (7, 9, 10), erythrocytes (7, 11, 12), and cells that compose the vessel wall, including endothelial cells (10, 13, 14) and vascular smooth muscle cells (10, 15). Microparticles are composed of lipids and cell surface proteins that are reflective of the cellular origin. For example, MPs derived from endothelium contain endothelial-type proteins such as tissue factor and CD31 (or platelet/endothelial cell adhesion molecule 1) (16). A common feature of MPs is their asymmetrical distribution of negatively charged phospholipids such as phosphatidyl serine on the cell surface. The exact mechanism of MP formation is poorly understood, but it is thought that plasma membrane shedding may be in response to cell injury, apoptosis, or activation via agonists such as TNFα, IL-1, and plasminogen activator inhibitor type 1 (PAI-1) (12, 16, 17). Microparticles can also mediate long-range signaling and alter downstream cell function (10). In particular, EMPs have been shown to initiate coagulation (8, 10, 18), increase monocyte adhesion (8, 19), activate neutrophils (20), regulate proliferation and angiogenesis (10, 21, 22), and impair vasodilation and vasorelaxation (13, 23). Therefore, EMPs are both markers and mediators of cellular dysfunction.

Under normal physiologic conditions, EMPs are present in the circulation at relatively low concentrations. However, in various pathologic states, their plasma concentration has been reported to be elevated (13-15). Ex vivo and in vitro observations indicate that patients with antiphospholipid syndrome (1, 19, 24) and systemic lupus erythematosus (25) as a result of autoimmune process involving antiphospholipid antibodies have significantly increased levels of EMPs. Studies have also shown a direct relationship between elevated plasma EMP levels and arterial dysfunction in end-stage renal failure (26), and increased EMP concentrations in diabetes (6), vasculitis (27, 28), cancer (29), and inflammatory disorders (8, 10) may be a direct result of endothelial dysfunction (6). Furthermore, Shet et al. (30) found a strong correlation between the severity of sickle cell disease and circulating plasma MP concentration. Altogether, an extensive amount of evidence suggests that elevated levels of MPs are directly associated with disease phenotype. However, it is not clear whether the increase in EMP concentration is the cause or effect of pathologic abnormality. Higher numbers of MPs have been observed in hemophiliacs, and the number further increases with acute bleeding. This suggests that MPs may act as antihemostatic agents (17) and can be a result of the pathologic abnormality. We have previously reported EMP-induced acute lung injury and cardiac valve endothelial dysfunction (21, 31). These data demonstrate that elevated levels of circulating EMPs independently cause widespread pathologic abnormality.

Various groups have examined the mechanism by which EMPs cause or contribute to disease. An in vitro study by Mezentsev et al. (32) on rat aortic rings demonstrated that high levels of EMPs impair endothelial function by diminishing vasorelaxation and NO production. In addition, high concentrations of EMPs inhibited angiogenesis by decreasing total capillary length, number of meshes, and branching points (22, 32). Recent work by our laboratory has further confirmed that EMPs propagate local vascular injury by direct inhibition of eNOS activity and NO production, leading to disruption of vasodilation (31). Furthermore, we have demonstrated that EMPs antagonize human mitral valve endothelial cell function in a dose-dependent manner by a mechanism that involves inhibition of vascular endothelial growth factor-induced proliferation and migration (21).

Although important insights have been gained into the causal relationship between elevated numbers of EMPs and disease, the molecular and cellular basis of the EMP-mediated injury remains poorly understood. Plasminogen activator inhibitor type 1 (PAI-1), a known agonist of EMP production, is the major physiologic inhibitor of tissue-type plasminogen activator in plasma. Interestingly, the plasminogen activator system plays a central role in the pathogenesis of vascular injury, and PAI-1, in particular, is elevated in a variety of clinical situations associated with increased risk of vascular disease (33, 34). Thus, PAI-1-derived EMPs may be key components in the pathogenesis of disease. To gain an in-depth knowledge of the physiologic function of EMPs in disease, it is critical that their protein and lipid composition be defined. Therefore, to gain insight into the mechanisms by which EMPs are produced and function upon PAI-1 stimulation, we determined the protein composition of PAI-1-derived EMPs from human umbilical vein endothelial cells using two-dimensional (2D) gel electrophoresis followed by mass spectrometry.


EMP generation

Endothelial MPs were generated in vitro as previously described by Densmore et al. (31) and Brodsky et al. (35). Commercially available human umbilical vein endothelial cells (HUVECs; Clonetic, San Diego, Calif) at passage 5 were used for generating EMPs. Cells were grown on 15 T75 culture flasks (Fisher) coated with 1% gelatin (Sigma, St. Louis, Miss) and cultured in M-199 media (Invitrogen, Carlsbad, Calif) containing 20% (vol/vol) fetal bovine serum (Clonetic), 0.01% (wt/vol) heparin (Sigma), 1% (vol/vol) penicillin:streptomycin:glutamine (Invitrogen), and 0.05% (wt/vol) endothelial mitogen (BioMedical Research Associates, Akron, Ohio). At 100% confluence, media were aspirated, and the cells were incubated with endothelial basal medium 2 (Clonetic) base media without serum or growth factors for 2 h followed by 3-h incubation in fresh base media containing PAI-1 (10 ng/mL) (American Diagnostica, Stamford, Conn).

Conditioned media containing shed EMPs were transferred from each flask (total of 15) into 50-mL conical tubes (Fisher) and centrifuged at 100g for 4 min to separate MPs from cell debris. Supernatant from 50-mL conical tubes containing EMPs was transferred into a 90-mL bottle (Kendora) and ultracentrifuged for 1.5 h at 100,000g and 4°C in a Sorvall centrifuge. Supernatant was discarded, and the EMP pellet was resuspended in 300 μL Hanks Balanced Salt Solution (Invitrogen) (20 μL per T75 flask) and stored at 4°C until further use (EMP pellets were not stored for more than 3 days). Protein concentration in the sample was estimated using a BCA protein assay kit (Pierce, Rockford, Ill).

EMP lysis and protein solubilization

Aliquots of 300 μg protein were pelleted by centrifugation at 20,000g for 15 min followed by resuspension of the EMP pellet in 300 μL urea3-cholamidopropoyl-dimethylammonio-1-propane-sulfonate (CHAPS) lysis buffer (9 M urea [Sigma], 4% CHAPS [Sigma], 2 M thiourea [Sigma], and 0.002% bromophenol blue [Sigma]) as previously described (36-38). Immediately before use, 1× protease and phosphatase inhibitors (Sigma), 60 mM dithiothreitol (DTT) (Sigma), and 2% pharmalyte (GE Healthcare, Chalfont St. Giles, U.K.) were added to the EMP lysate. After 20 min incubation on ice, the sample underwent two freeze-thaw cycles of 20 min and was sonicated twice for 30 s using a sonic dismembrator (Fisher, Pittsburgh, Pa). Insoluble protein material was separated by centrifugation at 20,000g and 4°C for 15 min. The supernatant was incubated with ice-cold acetone (Fisher) three times its volume at −20°C for at least 2 h to remove any lipid contamination and precipitate out protein. Next, the sample was centrifuged for 20 min at 20,000g and 4°C. Supernatant was discarded. The protein pellet was partially dried to remove residual acetone and then resuspended in 220 μL rehydration buffer (8 M urea, 0.5% CHAPS [wt/vol], 0.5% pharmalyte [vol/vol], pH 4-7, 0.2% DTT [wt/vol], and 0.002% bromophenol blue). If necessary, samples were washed, filtered, and concentrated using Vivaspin ultrafiltration (Argos Technologies, Elgin, Ill) spin columns to obtain a cleaner preparation.

Isoelectric focusing

To overcome the precipitation of protein during isoelectric focusing (IEF) due to hydrophobicity, DTT was added to the rehydration buffer, and the immobilized pH gradient (IPG) strips were rehydrated for 12 h before IEF (38). Immobilized pH gradient strips (GE healthcare) 11 cm long, 4-7 pH, were used for protein separation based upon their isoelectric point. Ettan IPGphor II (GE Healthcare) was used for IEF. The strips were rehydrated overnight with the soluble protein sample (described above) in rehydration buffer (final volume, 200 μL). Isoelectric focusing strips were covered with silicon oil, and electrophoresis was performed at 20°C as previously recommended (39). Focusing was done in three steps of 500, 1,000, and finally at 16,000 Vhr (40).

Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Immobilized pH gradient strips were equilibrated in buffer containing 50 mM Tris-HCl (Sigma), 6 M urea, 30% glycerol (vol/vol) (Bio-Rad, Hercules, Calif), 2% sodium dodecyl sulfate (SDS) (wt/vol) (Sigma), 0.002% bromophenol blue, and 1% DTT (wt/vol) for 15 min followed by another 15-min equilibration with 2.5% iodoacetamide (wt/vol) (Sigma) instead of DTT. The strips were placed on top of a 12.5% Tris-HCl criterion gel (Bio-Rad) and electrophoresed at a constant current of 9 W. The gel was then washed three times for 5 min each in distilled water and incubated in colloidal Coomassie G250 stain (Bio-Rad). Coomassie staining was selected over silver stain because of poor reproducibility of the latter as reported by Gorg et al. (39). The gel was stained for 1 h followed by two washes with deionized water and stored in water at 4°C.

The protein spots were manually excised from the gel (within 72 h of Coomassie staining) using wide-bore pipet tips and placed in a 96-well plate (GE Healthcare) for tryptic digestion. Saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% acetonitrile/0.5% trifluroacetic acid was used as matrix on Ettan matrix-assisted laser desorption ionization (MALDI) target slides (GE Healthcare). An automated Ettan spot handling workstation from GE Healthcare was used for peptide extraction and spotting on MALDI target slide. The samples were analyzed by MALDI mass spectrometry (MS) Ettan MALDI-time of flight (TOF) Pro (GE Healthcare). Mode of operation for the instrument was set at reflector with positive polarity. Up to one missed cleavage was allowed. Trypsin (Promega, Madison, Wis) autolysis peaks were used for internal calibration.

Protein identification and data analysis

Proteins were identified by performing robust searches against the nonredundant National Center for Biotechnology Information database using Ettan MALDI-TOF Pro software (GE Healthcare) (40). The name description, the frequency observed among all six experiments, the percent coverage, and Swiss-Prot or Accession ID was retrieved and complied for each identified protein. Swiss-Prot IDs were identified at In some cases, the same Swiss-Prot ID was given for two different proteins, which likely indicates protein aliases. These include P30101 (ER-60; protein disulfide isomerase (PDI)-associated 3), P11021 (glucose-regulated protein; BiP), and P07237 (P4HB protein; Prolyl4-hydroxylase, β-subunit). Proteins were also reconfirmed by performing searches against the MASCOT database using the Voyager DE pro software (Applied Biosystems, Foster City, Calif).

Using the Gene Ontology (GO) database at, the Human GO Slim annotation file was searched for GO annotations assigned to each Swiss-Prot ID. Each GO term found was recorded, and the number of proteins annotated to that particular term was determined. If the protein had been annotated to that term more than once (from GO annotations with different evidence and/or different publication), the protein was counted once. Gene ontology term annotations for cellular component, molecular function, or biological process from human, rat, and mouse were downloaded for each Swiss-Prot ID. The percentage of proteins with a specific term was charted in the form of a bar graph. It should be noted that in some cases, one protein was annotated to more than one GO term (e.g. both cytoplasm and nucleus annotations). A complete list of proteins with their respective terms from each species is provided in supplementary data available at

Western blot analysis

Aliquots of 20 μg protein lysate were loaded on to a 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel (Bio-Rad) and separated at 100 V for 1.5 h, followed by transfer of proteins onto nitrocellulose membrane (Bio-Rad) and incubation with antihuman Annexin V (Vac-α) mouse monoclonal antibody (dilution, 1:1,000) (Bender MedSystems, Vienna, Austria), antihuman PDI rabbit polyclonal antibody (dilution, 1:5,000) (Calbiochem), antihuman Vimentin mouse monoclonal antibody (dilution, 1:1000) (Affinity BioReagents, Golden, Colo) or antihuman Prohibitin rabbit polyclonal antibody (1:1,000) (Affinity BioReagents). Protein bands on the membrane were detected by chemiluminescence using an ECL-Plus kit (GE Healthcare).


2D/MALDI-TOF/MS of EMPs generated from PAI-1-stimulated HUVECs

Endothelial microparticles were generated from PAI-1-stimulated HUVECs and subjected to 2D gel electrophoresis for subsequent proteomic analysis (41). Figure 1 shows protein distribution of 300-μg EMPs in a 2D polyacrylamide gel based upon their mass and isoelectric point. The results of six separate EMP isolations are shown. An IPG strip of pH 4 to 7 for IEF, in combination with a 12.5% polyacrylamide gel for separation of proteins by mass, yielded the best distribution of proteins in the sample. Protein spots were visualized by Coomassie stain.

Fig. 1:
Two-dimensional gel analysis of proteins isolated from PAI-1-generated EMPs. Three hundred micrograms of EMP proteins from six different experiments were separated using an IPG strip of pH 4 to 7 for IEF in combination with SDS-PAGE (12.5%) and stained by Coomassie blue. The six Coomassie-stained gels are shown; one representative gel is enlarged for highlighting spots that contained identified proteins. In the larger gel, 71 spots were picked for MALDI-TOF/MS analysis, and 21 proteins (indicated with an arrow) were identified. Proteins identified in each spot are (1) calreticulin, (2) Gp96, (3) prolyl 4-hydroxylase, (4) BiP, (5) myosin heavy polypeptide 9, (6) ACTB protein, (7) CAE45818.2, (8) HsP 70, (9) ER-60, (10) glucosidase, alpha; neutral AB protein, (11) DNAJB11, (12) BiP, (13) annexin A2, (14), protein disulfide isomerase, (15) laminin-binding protein, (16) PRDX4, (17) prohibitin, (18) ubiquinol-cytochrome c reductase, (19) glucosidase II β-subunit, (20) smooth muscle myosin alkali light chain, (21) BAB71275.1.

Upon separation of the proteins by 2D gel electrophoresis, all of the spots detected were picked from every gel and then identified by MS. The spots detected in the six gels shown in Figure 1 were manually excised and the proteins extracted by tryptic digestion for subsequent MALDI-TOF/MS analysis. Proteins contained within approximately 25% of the extracted gel spots were identified as significant matches. For example, in Figure 1, 71 spots were picked for MALDI-TOF/MS analysis, and 21 proteins (indicated with an arrow) were identified. The unmatched proteins from the other 50 spots may be due to low abundance or because the protein is unknown and not represented in the database. Proteins that were identified from six independent EMP isolations and showed an expectation (E) value less than 0.05 were collapsed into one data set. We identified 58 unique proteins. The complete list of proteins identified is shown in Table 1, in which the protein description, the percent (%) coverage, the frequency observed among all six experiments, and Accession ID are given. Most proteins were reconfirmed by performing robust searches against the MASCOT database using the Voyager DE pro software (Applied Biosystems).

Table 1:
Proteins identified in EMPs from PAI-1-stimulated HUVECs

Some proteins (e.g., 4 and 12 in Fig. 1) were present at multiple spots in the same gel, which is likely due to posttranslational modifications that changed the mass and/or pI of the protein and has been reported by others (42). Sixteen proteins were identified in more than 50% of the experiments, whereas the remaining 42 were identified less than 50% of the time. For example, PDI and BiP were identified in all six experiments, whereas others such as Wnt were only detected once. In addition, more than 50% of the proteins were identified at least two times from two separate EMP isolations. This suggests that our EMP populations were similar, consisted of a basic pool of proteins, and that there were no serious problems with our reproducibility of EMP populations. A well-known limitation of 2D gels is that the method is somewhat stochastic in that the frequency of identifying a protein will be proportional to protein abundance. As such, more abundant proteins will be detected at a higher frequency, whereas the identification of lower abundant proteins will be variable with lower frequency. Furthermore, if one spot contains more than one protein, there is no guarantee that every protein will be identified in the spot. In fact, when multiple proteins of similar abundance comigrate on the gel, identification is extremely difficult. Therefore, it is likely that the proteins identified at a lower frequency represent the relatively low sensitivity of the 2D methods employed.

Validation of 2D MALDI/TOF data by Western blot analysis

Western blot analysis was used as a complimentary approach to validate our 2D MALDI/TOF findings. Because confirmation of all 58 identified proteins was not possible due to unavailability of certain antibodies, four EMP proteins identified above were selected for further analysis. In Figure 2, we show protein expression for prohibitin, annexin 5, PDI, and vimentin in whole cell HUVEC lysates unstimulated or stimulated with PAI-1. The EMP protein lysate generated from these stimulated parent cells is shown alongside. As expected, EMPs from PAI-1-stimulated HUVECs contain prohibitin, annexin 5, PDI, and vimentin, which validates data obtained from 2D MALDI/TOF. Differences in protein expression observed between the EMP and HUVEC lysates are likely because the EMP lysate is a concentrated sample of vesicle-contained proteins compared with HUVEC lysate, which is a composition of all cellular proteins.

Fig. 2:
Western blot analysis of EMPs generated by PAI-1. Whole cell extract from unstimulated HUVEC (lane 1), whole cell extract from PAI-1-stimulated HUVEC (lane 2), and EMP extract from PAI-1-stimulated HUVEC (lane 3). Equal amounts of protein were loaded and separated by SDS-PAGE electrophoresis on 12.5% gel, transferred to nitrocellulose membrane, and immunoblotted with antibodies specific to prohibitin, annexin 5, PDI, or vimentin. Proteins were detected by chemiluminescence and autoradiography.

Analysis of PAI-1-generated EMP protein composition

To gain insight into potential mechanisms contributing to EMP production and function, we used the GO database ( to annotate and analyze proteins identified in PAI-1-generated EMPs. The GO project provides a defined vocabulary that facilitates high-quality functional gene annotation (43). The GO terms assigned to each Swiss-Prot ID, with respect to cellular component, molecular function, or biological process, were identified in human, rat, and mouse species. The GO annotation terms were charted in the form of a bar graph shown in Figure 3. These data illustrate the number of proteins in EMPs that are localized to specific cell organelles, exhibit a particular molecular function, and are associated with a certain biological process. The complete list of proteins with their respective annotation term from human, rat, and mouse can be found in supplementary data available at

Fig. 3:
Protein analysis of PAI-1-derived EMPs. The cellular component (A), molecular function (B), and biological process (C) were annotated for each protein identified on PAI-1-derived EMPs. The GO database was searched for GO annotations using the Swiss-Prot ID assigned to each protein (Table 1). Each GO term found was recorded, and the number of proteins annotated to that particular term was determined. The GO term assigned to each protein that fell under the category of cellular component, function, or process was graphed in the form of a bar chart using Microsoft Excel software. In some cases, one protein was annotated to more than one GO term, so the number of annotations in a particular aspect do not represent the total number of proteins identified in EMPs (see supplementary data available at for a complete list of annotations assigned to each Swiss-Prot ID).


Multiple studies have demonstrated the existence of EMPs in plasma; however, the mechanism of EMP generation and their downstream function remains unclear (20). Because MPs are known to mediate downstream cellular responses, they might serve as dynamic signaling-type molecules that exchange information between cells (15). As such, the protein composition of EMPs will probably play a direct role in mediating specific cellular responses. The phenotype of MPs seems to be based upon the nature of the stimulant used (10, 44), which indicates that the microenvironment might be a predictor of MP protein composition. For example, Huber et al. reported that EMPs (or membrane vesicles) composed of proinflammatory oxidized phospholipids elicit distinct responses in endothelial cells leading to the adhesion of monocytes, unlike their native counterpart (19). Furthermore, oxidized phospholipids are present on MPs when oxidative stress contributes to particle formation (32), and MPs from patients with diabetes show high procoagulant activity (6). Because EMP numbers are elevated in diseased states, future studies are needed to determine to what degree increased EMP levels advance disease. There is evidence that certain therapies partially exert their effect by blocking the formation of MPs, suggesting that MPs may contribute to the pathologic abnormality (44). Others have reported that the number of MPs increases as the pathological condition progresses from diseased to crisis stage (30). In either case, EMPs may be a very useful diagnostic marker for disease states (28, 45). As such, identifying the protein composition of MPs provides key information for potential diagnostic and therapeutic applications in treating diseases associated with EMPs such as acute lung injury and autoimmune disorders.

The studies described here provide the first definition of the proteome from PAI-1-derived endothelium-derived MPs using 2D gel electrophoresis. An interesting observation is that the proteins found on PAI-1-derived EMPs are quite diverse in that they originate from different cellular organelles, have a variety of functions, and are involved in a range of biological processes. For example, EMP proteins derive from the cytoplasm (e.g., annexin 5 and vimentin), nucleus (e.g., polymerase (DNA directed), ETA), and membrane (e.g., adenosine triphosphate synthase), and are involved in different biological processes such as angiogenesis, protein transport, cell motility, and cell adhesion. Most of the proteins on PAI-1-derived EMPs are from the endoplasmic reticulum and are involved in protein folding such as PDI. It was reported that PDI expression is increased in endothelial cells under hypoxic stress, suggesting that PDI may contribute to the ability of endothelial cells to tolerate hypoxia (46). It is interesting to speculate that under severe stress, endothelial cells might deliberately release EMPs that contain PDI protein as a form of a downstream signaling response. Interestingly, a fraction of the EMP proteins are unnamed or "hypothetical“ gene products that do not have an assigned GO annotation term associated with cellular component, molecular function, or biological process. This suggests that the biological activity for a significant portion of the EMP proteome is not known and warrants further investigation to fully understand the potential role of each protein in regulating EMP function. It should also be noted that CD31 and E-cadherin, which are known surface markers for endothelial cells, were not identified by 2D/MS analysis. This is probably because it is difficult to obtain soluble cell surface proteins, which has been reported by Blanchard et al. (9). In addition, a known limitation of 2D analysis is that less abundant proteins are difficult to detect (38). The fact that we previously detected CD31 and E-cadherin in EMP populations by Western blot and fluorescence-activated cell sorter analysis (31) further suggests that limitations in methodology prevented us from detecting these proteins here.

It has been reported that EMPs can elicit different effects depending on the generating cell type and the agonist or stimulus mediating EMP production (1, 13). In a recent study by Banfi et al. (16), the proteome of TNF-α-derived endothelial MPs was characterized. They found that TNF-α-generated EMPs consist of various proteins, including metabolic enzymes, adhesion molecules, and cytoskeletal proteins, of which some might be directly involved in causing increased endothelial cell procoagulant activity (16). However, it is not known whether the phenotype of the released EMP is dependent on the agonist or stimulus used to activate the parent cell. Therefore, it is possible that TNF-α-derived EMPs have a different protein composition and physiologic function compared with EMPs derived from different stimuli such as PAI-1.

When the proteins identified in PAI-1-derived EMPs from this study are compared with the proteins identified in TNF-α-generated EMPs in the report of Banfi et al. (16), there seems to be overlapping but distinct differences between the two proteomes. Proteins that were similar include proteins from the cytoskeleton such as actin, tubulin, vimentin, and prohibitin. It is known that MPs are formed in response to cell activation, which leads to increased cytosolic calcium and disruption of the cytoskeleton (41), so this could reflect a common process involving blebbing of these MPs, regardless of the stimulus. Additional proteins shared in common between the two stimuli are chaperones and endoplasmic reticulum-related products involved in protein folding such as PDI-associated 3, endoplasmic reticulum protein 29, glucose-regulated protein and heat shock protein 70. However, there are also distinct differences between the proteomes. For example, PAI-1-generated EMPs contain tropomysin 4, Wnt inhibitory factor 1, hypoxia up-regulated 1, and a zinc finger protein transcription factor, ZNF364, whereas TNF-α-stimulated EMPs do not. In addition, we show in Figure 2 that the proteome of PA1-1-derived EMPs contains annexin 5. The proteome of TNF-α-derived EMPs was reported to contain annexin 1 and 2 (16), but not annexin 5. Furthermore, other proteins were identified on TNF-α-induced EMPs that were not found in our study, including components of the nucleosome and various enzymes such as superoxide dismutase and cytochrome c oxidase (16). Altogether, these differences suggest that EMP production and their physiologic function might be tightly controlled by the upstream stimulus, and that the proteome of EMPs may reflect the original state of the cell in response to a particular environment (i.e., inflammation, shear stress, injury, apoptosis). However, one must leave open the possibility that there are technical explanations for the variability observed between the two studies because the approaches used to characterize the EMP proteomes were quite different. Nevertheless, more studies will need to be conducted to compare the protein composition of MPs generated by different stimulants and microenvironments to understand the nature of EMPs and their consequence in various pathologic conditions. Identifying the potentially unique protein composition of EMPs from different stimuli would provide fundamental insight into the mechanisms regulating the production of these particles and their physiologic role in different diseases. The use of methods such as liquid chromatography/MS are necessary to comprehensively compare protein composition of EMPs from different stimuli and are current lines of investigation in our laboratory.


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EMP; microparticles; endothelial cells; proteome; PAI-1

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