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. Author manuscript; available in PMC: 2010 Jun 16.
Published in final edited form as: Br J Haematol. 2008 Jun 5;142(4):627–637. doi: 10.1111/j.1365-2141.2008.07230.x

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Yara Banz 1, Guido Beldi 1, Yan Wu 1, Ben Atkinson 2, Anny Usheva 3, Simon C Robson 1
PMCID: PMC2886720  NIHMSID: NIHMS210675  PMID: 18537971

Summary

Plasma microparticles (MPs, <1.5 μm) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Keywords: microparticles, CD39/ecto-nucleoside triphosphate diphosphohydrolase1, endothelium, platelets, purinergic signalling

Cell-derived microparticles (MPs) in the blood plasma are small phospholipid vesicles ranging from nanometres to 1.5 μm in size that are released from platelets, circulating leucocytes and endothelial cells upon activation or in the setting of apoptosis (Wolf, 1967; Jimenez et al, 2003). MPs are thought to originate from lipid rafts and contain defined bioactive molecules (Leeuwenberg et al, 1992) that are potentially implicated in thrombogenesis and trans-cellular activation (Barry et al, 1997). Additionally, pro-inflamma-tory functions may be mediated by putative ligand-receptor interactions (Forlow et al, 2000), classical pathway complement activation (Gasser & Schifferli, 2005) and by triggering or modifying target cells and their functions (Mesri & Altieri, 1998). Furthermore, MPs represent a population of phosphatidylserine-exposing subcellular fragments (Satta et al, 1994), function as ‘transporters’ of circulating tissue factor (Falati et al, 2003), and may be valuable in maintaining normal haemostasis when platelet function is impaired or reduced, for instance in thrombocytopenic states (Keuren et al, 2007).

The release of tissue factor-expressing MP may be induced by stimulation of monocyte and dendritic cell types by extracellular nucleotides (Baroni et al, 2007). Nucleotides may be released from aggregating platelets, degranulating macrophages, stressed, injured or dead cells and relay signals via the P2-purinergic receptors (Dubyak & el-Moatassim, 1993). Within the vasculature, conditions of increased ATP flux, as occurs (for instance) during hypoxia, may mediate pro-inflammatory responses in the endothelium. CD39 is the prototype ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase1) that is highly expressed within specialized lipid raft domains (Koziak et al, 2000) in plasma membranes of endothelium, monocytes and populations of lymphocytes (Maliszewski et al, 1994; Marcus et al, 1997; Pulte et al, 2007). CD39 hydrolyzes extracellular nucleotides, preferentially adenosine tri- and diphosphate (ATP and ADP), converting these in concert with CD73, to adenosine.

The role of CD39 as an anti-inflammatory (Goepfert et al, 2000; Mizumoto et al, 2002) and thromboregulatory factor within the vasculature (Dwyer et al, 2004; Furukoji et al, 2005), has been confirmed in several experimental models using mutant mice and experimental gene therapeutics. Importantly, cellular activation initially results in loss of CD39 bioactivity from vascular cells that requires reconstitution by transcriptional upregulation of CD39 (Robson et al, 1997).

This study found functional CD39 expression within plasma MP and phenotypic characterization, implying shedding of the ectonucleotidase from vascular endothelial and other cells. Deletion of Entpd1 (Cd39) in mice impacts plasma MP numbers, characteristics and function in vitro. Absence of CD39 clearly augments the pro-inflammatory potential of MP. We propose that CD39 within MP might be an important regulator of their formation and function.

Materials and methods

Animals

Entpd1-null mice on the C57BL/6 background were housed and bred in a pathogen-free facility and have been characterized previously (Enjyoji et al, 1999). Wild type C57BL/6 control mice were obtained from Jackson Laboratories (Germantown, NY, USA). Animal housing was compliant with the requirements of animal care as specified by the United States Department of Agriculture and the Department of Health and Human Services. Animals were maintained on a 12-h light/ dark cycle and had unrestricted access to commercially available rodent chow and tap water. The Beth Israel Deacon-ess Medical Center Animal Care and Use Program approved all relevant experimental protocols.

Isolation of circulating microparticles from platelet poor plasma

Blood was drawn from the inferior vena cava of anaesthetized mice into citrate-anticoagulated syringes. In two spin-down steps (400 g, 5 min, 20°C) to maximize retrieval of MPs, cells and cell debris were separated from the plasma fraction. Resulting platelet rich plasma (PRP) was spun down again twice using a swing-out centrifuge (200 g, 6 min, 20°C). Prostaglandin-1 was added to the PRP to stabilize the platelets before spinning down for 5 min at 1000 g, 20°C. Resulting platelet poor plasma (PPP) was collected and centrifuged at 16 000 g for 25 min, 20°C to pellet microparticles. Following washing with Apo buffer (10 mmol/l HEPES, 5 mmol/l KCl, 1 mmol/l MgCl2, 136 mmol/l NaCl, pH 7.4, 0.2 μm filtered) and re-pelleting, the microparticles were re-suspended in Apo buffer for staining and fluorescence-activated cell sorting (FACS) analysis or in appropriate sample buffer and frozen at –80 C for subsequent two dimensional (2D)-gel or western blot analysis.

2D-gel electrophoresis of microparticles and sample preparation for mass spectrometry

Retrieved MPs were denatured and solubilized in a buffer containing 8 mol/l urea, 2% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonic acid), 0.8% pH 4–7 and 6–10 2D-gel electrophoresis ampholytes and dithiothreitol as the reducing agent. Soluble proteins were hydrated into pH 4–7/pH 6–10 isoelectric focussing (IEF) strips (Invitrogen, Carlsbad, CA, USA) overnight and electrophoresed the following day. The IEF strips were equilibrated according to the manufacturer's instructions (Invitrogen), and each gel was further separated with the use of 4–12% gradient sodium dodecyl sulphate polyacryalmide gel electrophoresis (SDS–PAGE) (Invitrogen) and stained with Coomassie Blue. Two 2D gels were obtained for both wild type as well as Entpd1-null microparticles (pooled from six animals each). Five bands found to be differentially expressed were excised. Following in-gel trypsin digestion of the selected spots (Shevchenko et al, 1996), peptides were extracted from the gel pieces, reconstituted in high-performance liquid chromatography (HPLC) solvent A (2.5% acetonitrile, 0.1% formic acid) and loaded onto a nano-scale reverse-phase HPLC capillary column via a Famos auto sampler (LC Packings, San Francisco, CA, USA). A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). Each eluted peptide was subjected to electrospray ionization and entered into an LTQ linear ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Eluting peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein or translated nucleotide databases with the acquired fragmentation pattern by the software program Sequest (ThermoFinnigan).

Freshly isolated microparticles from wild type and Entpd1-null mice were subjected to PAGE and immunoblotting with appropriate antibodies as detailed subsequently for further analysis.

Western blot analysis of microparticles

Relative expression patterns of CD39 and differentially expressed proteins in MPs were determined using western blot analysis. Equal protein amounts were subjected to 4–15% gradient SDS–PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA, USA) by electroblotting. Membranes were probed with the primary antibodies overnight at 4°C. Following blocking with non-fat milk (2%) in Tris phosphate-buffered saline (TBS) – 0.05% Tween for 1 h at room temperature. Antibodies included the polyclonal rabbit anti-CD39 (antibody raised in rabbits by direct inoculation of the encoding cDNA in pcDNA 1:1000 dilution (Enjyoji et al, 1999), monoclonal anti-talin (Sigma, St. Louis, MO, USA; clone 8d4, 1:100 dilution) and monoclonal anti-ATP synthase β subunit (Invitrogen, 1:200 dilution). Protein bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Pierce, Rockford, IL, USA; 1:50 000 dilution) followed by chemiluminescent substrate (Pierce).

Flow cytometric analysis of MPs

Samples of isolated MPs were re-suspended in Apo buffer and subjected to flow cytometry using a Becton Dickinson FACSCalibur, and analysed using Cellquestpro software (Becton Dickinson, San Jose, CA, USA). Events falling in the predefined MP gate (i.e. <1 μm in size) that were stained positive for an amphiphilic cell linker dye (which integrates non-specifically into lipid bilayers, PKH67; Sigma) were subsequently analysed.

To determine the cellular origin of circulating MPs, aliquots of washed MPs were incubated with phycoerythrin (PE)-labelled monoclonal antibodies (all from eBioscience, San Diego, CA, USA) for 20 min at room temperature, protected from light: CD41 for platelet MPs, CD31 for endothelial MPs, Ly-6G (Gr-1) for neutrophil MPs, CD3 for T-lymphocytes MPs, CD45R for B-cell MPs. Following labelling, samples were washed with 1 ml Apo buffer, re-pelleted and re-suspended in 400 μl Apo buffer for analysis. For each sample, control labelling was performed in parallel using appropriate isotype control antibodies.

Flow cytometric analysis of CD39 on human MPs

Human MPs were stained for CD39 with fluorescein isothiocyanate (FITC)-labelled antibody (BU61; AnCell, Bayport, MN, USA) and control, irrelevant IgG1-FITC antibody (AnCell).

Assay for enzymatic activity of MPs

Isolated microparticles from platelet poor plasma were washed and re-suspended in phosphate-free incubation buffer (20 mmol/l HEPES, 10 mmol/l glucose, 5 mmol/l KCl, 120 mmol/l NaCl, 2 mmol/l CaCl2 and 5 mmol/l tetramisole – to inhibit alkaline phosphatase activity – pH 7.5, 100 μl per 96-well). Enzyme activity was determined at 37°C following incubation with 1 mmol/l substrate (ATP or ADP) for 20 min. Reactions were terminated by the addition of 100 μl of ice-cold 10 % tetrachloracetic acid. Released phosphate [Pi] was determined and validated as described previously using malachite green reagent (Baykov et al, 1988; Wu et al, 2006).

Preparation of liver sinusoidal endothelial cells (LSEC)

Liver sinusoidal endothelial cells (LSEC) were isolated from wild type and Entpd1-null mice by collagenase digestion, modified from previously published methods (LeCouter et al, 2003). Briefly, livers were perfused through the infrahepatic vena cava with Ca2+-free buffer (142 mmol/l NaCl, 6.7 mmol/l KCl, 10 mmol/l HEPES, pH 7.4), followed by 10 ml of 0.02% collagenase type IV in collagenase buffer (67 mmol/l NaCl, 6.7 mmol/l KCl, 100 mmol/l HEPES, 4.8 mmol/l% CaCl2 2H2O), for 5 min. Livers were excised and passed through a 100 μm nylon mesh and the filtrate centrifuged at 50 g for 10 min. The supernatant was collected and the pellet discarded. The non-parenchymal cell supernatant fraction was washed twice. Labelling with electromagnetic beads was performed according to the manufacturers’ protocol (positive selection for LSEC using CD146; Miltenyi Biotec Inc., Auburn, CA, USA). After separation, cells were plated at a density of 105 per cm2 on fibronectin-coated plates and were cultured overnight in Dulbecco's modified Eagle medium (DMEM)-F12 cell culture medium (Invitrogen) supplemented with 20% fetal bovine serum (Hyclone, South Logan, UT, USA), 100 U/ml penicillin and 100 lg/ml streptomycin (Invitrogen) at 37°C in a 5% CO2 incubator. The purity of preparations was tested using LDL uptake and was judged to be >95%.

Incubation of liver sinusoidal endothelial cells with microparticles

Equal concentrations of freshly isolated, washed MPs from wild type and Entpd1-null mice were re-suspended in full cell culture medium and added to the LSEC. Incubation of LSEC with medium alone or lipopolysaccharide (LPS) at 100 ng/ml served as negative and positive controls respectively. Cell supernatants as well as the LSEC were harvested at defined time points (2, 8 and 24 h) for further analysis.

ELISA for interleukin-6, tumour necrosis factor-alpha and von Willebrand factor

Interleukin (IL)-6, tumour necrosis factor-α (TNF-α) and von Willebrand Factor (VWF) were determined in MP-free cell culture supernatants by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (eBioscience and Stago Diagnostica, Parsippany, NJ, USA).

Flow cytometry of LSEC

Following incubation with MPs and retrieval of supernatants, LSEC were washed twice with ice-cold phosphate-buffered saline (PBS), pH 7.4 and were rapidly dissociated from the cell culture plate with trypsin-EDTA (Invitrogen), washed and re-suspended in PBS-2 % bovine serum albumin (Sigma) for staining of cellular activation markers intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1; phycoerythrin-labels, eBioscience) for analysis by flow cytometry.

Cytokine/MMP analysis of circulating microparticles

Pooled microparticles from wild type (n = 6) and Entpd1-null (n = 6) mice were isolated and washed as described previously and were re-suspended in 120 μl PBS-0.1% Nonidet-P40 (NP-40, Sigma). Samples were analysed for IL1-β, IL-4, IL-6, IL-10, IL-13, P-selectin, interferon-gamma, TNF-α, TNF-receptor I, and matrix metalloproteinase (MMP)-2 and -9 using the SearchLight Chemiluminescent Protein Array by Pierce (quantitative, plate-based antibody arrays based on traditional ELISA).

Statistical analysis

Experiments were performed at least in triplicate. All flow cytometry and western blot images shown are representative data from one experiment. Data are shown as mean ± standard deviation. Statistical analysis was conducted using two-tailed Student's t-test. P < 0.05 were considered to be statistically significant.

Results

Murine and human microparticles express CD39 and exhibit NTPDase activity

Circulating microparticles from PPP from wild type mice expressed CD39, as determined by western blot analysis (Fig 1A). As expected, MPs from Entpd1-null mice were negative for CD39 protein. These studies were validated further by FACS analyses (see below).

Fig 1.

Fig 1

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CD39 is expressed in murine microparticles from platelet poor plasma of wild type mice (wt) but not in Entpd1-null mice (ko, A). Western blot analysis was performed under non-reducing conditions on 4–15% gradient SDS–PAGE, with transfer to a PVDF membrane, and probing with rabbit polyclonal antibody to mouse Cd39. Micro-particles exhibit NTPDase activity in vitro (B, *P < 0.05 wt versus ko). ATP and ADP were used as substrates for NTPDase activity for intact microparticles in suspension. Data were mean ± standard deviation. CD39 is also expressed within human microparticles, as detected by FACS analysis (C) and biochemical analyses (not shown).

Microparticles from wild type mice consistently exhibited NTPDase ecto-enzymatic activity following the addition of the substrates ATP and ADP to the MP suspension (Fig 1B). In contrast, microparticles from Entpd1-null mice showed a 3.5-fold reduced ATPase activity and 6.1-fold reduced ADPase activity respectively.

Fluorescence-activated cell sorting analysis (Fig 1C) and NTPDase assays (not shown), as described above, also confirmed functional expression of CD39 in normal human microparticles.

Microparticle numbers and characteristics in Cd39-null mice

Microparticles from murine PPP were stained for cell surface markers and analysed by flow cytometry to determine the respective numbers and cellular origin (Fig 2A–C). A known number of Tetraspeck Microspheres (0.5, 1 and 4 μm beads; Invitrogen) were added to the sample before acquisition for MP quantification. Acquisition was terminated after 20 000 beads were counted. Entpd1-null mice had significantly higher total numbers of circulating MPs when compared to wild type mice (P < 0.05, Fig 2D). Of these MPs, the greatest proportion was derived from platelets (CD41+) and endothelial cells (CD31+) in both Entpd1-null as well as wild type mice. However, Entpd1-null mice had significantly more platelet and endothelial MPs when compared with wild type mice (P < 0.05). Results are summarized in Table I.

Fig 2.

Fig 2

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Flow cytometric analysis of circulating microparticles from wild type and Entpd1-null mice. Microspheres of 0.5 lm (gate R1), 1.0 lm (gate R2) and 4.0 μm (gate R3) were used as size references to gate the microparticle (MP) population (gate R4, <1.0 lm) and regulate acquisition (A). Events falling in the MP gate (R4) were gated for positivity for the lipophilic dye PKH67, in the FL1 fluorescein isothiocyanate (FITC) gate (B). Of this population, identified as microparticles, populations were stained for cell membrane markers using phycoerythrin (PE)-labelled antibodies (FL-2,PE gate) to determine cellular origin (C and also Table I for results). Inlay in (C) shows a SSC/FL-2 histogram depicting the staining pattern for microparticles (shaded grey) and calibration beads (black line, peak 1: 0.5 μmol/l, peak 2: 1.0 μm, peak 3: 4.0 lm beads) for comparison. All dot blots depict representative examples of actual blots used for subsequent analyses. PKH67-positive MPs (see B) were quantified from plasma samples from wild type and Entpd1-null mice. The total amount of circulating microparticles is significantly greater in Entpd1-null mice when compared with wild type mice (*P < 0.05, D).

Table I.

Analysis of cellular origin of microparticles by flow cytometry.

Microparticle origin Wild type (%) Entpd1-null (%)
Platelets (CD41+) 49·0 ± 5·14* 54·0 ± .2·4
Endothelium (CD31+) 28·5 ± 1·7* 30·7 ± 1·9
PMNs (Gr-1+) 10·7 ± 1·7 8·6 ± 1·4
T-cells (CD3+) 5·9 ± 0·5 5·3 ± 0·8
B-cells (CD45R+) 5·3 ± 0·7 4·6 ± 0·9
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Entpd1-null murine plasma contains significantly more microparticles of platelet (CD41+) and endothelial (CD31+) origin when compared with wild type mice (*P = 0·042 and *P = 0·048 respectively). Other populations investigated include circulating microparticles from polymorphonuclear cells (PMN, Gr-1+), T cells (CD3+) and B cells (CD45R+).

Circulating microparticles from wild type and Entpd1-null mice were subjected to 2D-gel electrophoresis and proteome analysis. Five gel spots revealed prominent differences between the two populations of microparticles (Coomassie Blue staining). These were analysed by mass spectrometry (MS) analysis and identified using the software program Sequest as described in Materials and methods. Talin (two spots) and ATP-synthase β subunit (one spot) appeared more abundant in wild type MPs, tropomyosin alpha-4 chain (one spot) and profilin-1 (one spot) appeared more abundant in Entpd1-null mice; all proteins previously shown to be associated with MP (Jin et al, 2005). Subsequent western blot analyses on MP fractions definitively confirmed the differential expression data for talin and the ATP-synthase β subunit (Fig 3A1: wild type, wt; Fig 3A2: Entpd1-null, KO, Fig 3B and C).

Fig 3.

Fig 3

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2D-gel electrophoresis and western blot analysis of microparticles from wild type (wt; A1) and Cd39-null (ko; A2) mice. Two spots (marked 1 and 2) in the 2D-gels (pH 4–7) revealed differing expression patterns between both mouse populations and were analysed by mass spectrometry (1 = talin, 2 = ATP synthase β, wt >> ko. Results confirmed by western blot showed significantly higher expression of talin (B, bands at 225 and 190 kDa) and ATP synthase β (C, bands at 56 kDa) in wild type when compared with Entpd1-null mice. Protein concentrations were determined by densitometric measurements of western blots. Samples were standardized to IgG for internal control and expressed as arbitrary units (*P < 0.05 wt versus KO).

Microparticles contain an array of cytokines and matrix metalloproteinases

Washed microparticles from wild type as well as Entpd1-null mice were re-suspended in PBS-0.1% NP-40 and analysed for cytokines, P-selectin and MMPs after pooling. Results are summarized in Table II. Of the investigated analytes, differences were particularly noticeable for IL-10 (11-fold higher levels in wild type versus Entpd1-null MPs), TNF receptor I (4.9-fold higher levels in wild type versus Entpd1-null MPs), as well as MMP-2 (7.2-fold higher levels in wild type versus Entpd1-null MPs).

Table II.

Analysis of microparticle content by multiplexed sandwich ELISA.

pg/ml IL-1b IL-4 IL-6 IL-10 IL-13 P-selectin IFN-γ TNF-α TNFRI MMP-2 MMP-9
Wild type 232·1 165·6 378·7 13·4* 41·2 3476 844·6 123·7 9·3* 7354* 1924·5
Entpd1-null 276·4 174·4 439·2 1·2 26·3 4267 836·7 189·9 1·9 1020 1724·4
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Pooled MP-suspensions from wild type and Entpd1-null mice (n = 6 each) were lysed in PBS-0·1% NP-40 and then analysed for content of interleukins (IL), P-selectin, IFN-γ, interferon-gamma; TNF-α, tumour necrosis factor alpha; TNFRI, TNF receptor I; MMP, matrix metallo-proteinase.

*

Greater than fourfold differences between the two MP populations.

Preferential LSEC activation and cytokine release by Entpd1-null microparticles

Microparticles from wild type as well as Entpd1-null mice were co-incubated with quiescent wild type and Entpd1-null primary LSEC cultures to analyse pro-inflammatory potential. IL-6 as well as TNF-α was detected in supernatants from LSEC incubated with MPs from wild type as well as Entpd1-null mice (Fig 4A and B). When compared with incubation with medium alone, both wild type as well as Entpd1-null MPs significantly increased IL-6 and TNF-α release from LSEC (P < 0.05).

Fig 4.

Fig 4

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Microparticles induce pro-inflammatory responses in liver sinusoidal endothelial cells (LSEC) in vitro. LSEC release IL-6 (A) and TNF-alpha (B) and shed von Willebrand Factor (VWF, C), as measured in cell culture supernatant by enzyme-linked immunosorbent assay (*P < 0.05 wt versus KO MPs, #P < 0.05 ctrl versus MPs). LSEC incubated with medium (ctrl) and 100 ng/ml serve as controls. Data were mean ± standard deviations.

However, Entpd1-null MPs induced significantly more IL-6 (Entpd1-null LSEC) and TNF-α (wt and Entpd1-null LSEC), when compared with wild type MPs (P < 0.05). When compared with normalized and baseline values of VWF release, the incubation of LSEC with wild type or Entpd1-null microparticles significantly increased VWF levels in supernatants (P < 0.05, Fig 4C). In particular, Entpd1-null MPs induced significantly more VWF release from LSEC, when compared with wild type MPs (P < 0.05).

Microparticles augment upregulation of cell adhesion molecules in LSEC

As analysed by flow cytometry, incubation of LSEC with microparticles for 8 h, at least partly induced upregulation of intercellular adhesion molecule-1 (ICAM-1, Fig 5A) and vascular adhesion molecule-1 (VCAM-1, Fig 5B). Whereas a significant upregulation of ICAM-1 occurred in wild type LSEC following incubation with 100 ng/ml LPS (positive control), no induction was noted following incubation with Entpd1-null or wild type MPs. In contrast, Entpd1-null LSEC significantly upregulated ICAM-1 after incubation with Entpd1-null MPs (P < 0.05). Expression of VCAM-1 was significantly induced in both wild type as well as Entpd1-null LSEC mice with both wild type as well as Entpd1-null MPs (P < 0.05). The induction of IL-6 and TNF-α production, the release of VWF and induction of adhesion molecules on the LSEC following incubation with CD39 null MPs is most pronounced in the combination of Entpd1-null MPs and Cd39-null LSEC (P < 0.05 versus wild type MPs + wild type LSEC).

Fig 5.

Fig 5

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Microparticles from wild type and Entpd1-null mice induce upregulation of ICAM-1 and VCAM-1 on liver sinusoidal endothelial cells (LSEC) in vitro. Flow cytometry of LSEC for ICAM-1 (A) and VCAM-1 (B). Representative histograms are shown on the left (of Entpd1-null LSEC, isotype control: shaded grey; medium/ctrl: black; wild type/wt MPs: green; Entpd1-null /ko MPs: blue; LPS: red) (*P < 0.05 wt versus ko MPs, #P < 0.05 ctrl versus MPs). Data were mean ± standard deviation.

Discussion

CD39 has been shown to comprise a component of circulating plasma microparticles and to maintain active ecto-enzymatic functions within these blood components. It is somewhat unclear why no CD39 was reported in previous MP proteome analyses (Jin et al, 2005). However, the apyrase conserved region of CD39 shows a certain homology to ATP-binding sites of actin (Robson et al, 2006). Specific detection may therefore have been missed as multiple actin fragments and associated proteins were detected in large amounts in the 2D analyses, possibly masking the actual CD39 protein.

We have shown that MPs maintain low levels of functional NTPDase activity. ADPase activity, more specifically associated with CD39, was noted to be substantially decreased in Entpd1-null MPs. Although other NTPDases have not been shown to be expressed within MPs to date, it cannot be excluded that ‘residual’ ATPase activity in Entpd1-null MPs may be secondary to other members of the CD39 family, such as NTPDase2 (CD39L1), a preferential ATPase (Sevigny et al, 2002). Curiously, alkaline phosphatases can be associated with trophoblast-derived MPs during gestation (Goswami et al, 2006); this ecto-enzyme was specifically inhibited in our assays and not considered to contribute to residual ATPase activity in Cd39 null MPs as we studied non-gravid mice.

Our current study showed CD39 to be a novel regulator of cellular activation that could be translocated within the vasculature by circulating MP. In the absence of Cd39, heightened levels of MPs were noted in plasma. Whilst absolute platelet numbers in Entpd1-null mice may be around 20% lower when compared with wild type animals (Enjyoji et al, 1999), proportionately more platelet-derived MPs were found in circulation in vivo, indirectly possibly indicating some degree of increased platelet turnover brought about by altered extracellular nucleotide fluxes.

Our analyses of plasma MP further suggest roles and functions as vectors for cytokines and other factors, such as MMPs. These data are in keeping with other previous studies that suggest in vitro generated neutrophil MPs carry MMP-9, elastase and myeloperoxidase (Gasser et al, 2003) and that platelet MPs roll on the endothelium to facilitate delivery of RANTES to the target cells (Mause et al, 2005).

Qualitative differences between the wild type and Entpd1-null MP populations, which may partly explain their differing pro-inflammatory potential, were noted for IL-10, TNFRI and MMP-2 contents; all were substantially decreased in null MPs. IL-10 mainly functions as an anti-inflammatory cytokine (Fiorentino et al, 1989). If indeed the MP-associated IL-10 downregulates intravascular inflammation, this may, at least in part, explain why MPs from Entpd1-null animals exhibited heightened pro-inflammatory properties in vitro. Any direct relationship to TNFRI changes remains speculative, but diminished levels could theoretically exacerbate inflammation because of a decreased capacity to scavenge TNF.

Both platelet- and endothelial microparticles as well as MMPs have been implicated in angiogenesis (Kim et al, 2004; van Hinsbergh et al, 2006). As platelets are known to contain MMPs, which may be released upon activation, and the majority of circulating MPs are platelet-derived, it is feasible that MP-derived MMP-2 and -9 might also play a direct role in angiogenesis. This new finding may also be of interest in explaining aspects of the pathological phenotype previously observed in Entpd1-null mice with respect to inhibition of angiogenesis (Jackson et al, 2007).

Another prominent difference between the MP population from wild type and Entpd1-null mice lies in the decreased amounts of talin and ATP-synthase β subunit detected in the null populations. Detection of these proteins within wt MPs is in accordance with previous proteomic analyses of plasma MPs in general and platelet MPs in particular (Garcia et al, 2005; Jin et al, 2005). The regulated binding of talin to integrin β tails is a final common element of cellular signalling cascades that control integrin activation (Tadokoro et al, 2003). As Entpd1-null mice show platelet hypofunction associated with purinergic type P2Y1 receptor desensitization and glycoprotein IIbIIIa integrin dysfunction, this might bring about decreased opportunity for talin binding to β integrins and association within the platelet-derived MP.

The mitochondrial ATP synthase β subunit, shown to be enriched in platelet MPs as compared to endothelial MPs (Smalley et al, 2007), is also decreased in the mutant, CD39 null MP population. Presently the significance of this finding is unclear and possible relevance to the proposed platelet dysfunction in Entpd1-null mice remains to be evaluated.

In vitro generated platelet MPs have been shown to induce upregulation of cell adhesion molecules on umbilical vein endothelial cells, stimulating adhesion of monocytes (Barry et al, 1998). Furthermore, MPs induce cytokine release and promote endothelial dysfunction by impairing the endothelial nitric oxide pathway (Mesri & Altieri, 1999). In our system, incubation of MPs with liver sinusoidal endothelial cells (LSEC) revealed two interesting findings in vitro. On the one hand, the absence of CD39 on MPs resulted in changes that exacerbated the pro-inflammatory response in LSEC, documented by increased cytokine and VWF release as well as upregulation of adhesion molecules, particularly VCAM-1. On the other hand, absence of CD39 on the endothelium itself further heightened its susceptibility to stimulation with MPs.

This cell-based effect was not entirely unexpected, as the ectonucleotidase function of CD39 is known to confer anti-inflammatory properties to endothelial and other cells (Robson et al, 2005). We did consider that the increased pro-inflammatory phenotype of the Entpd1-null MPs could, at least in part, directly relate to intrinsic lack of ectonucleotidase activity. Currently available inhibitors that could be used to test this by blocking wild type MP NTPDase activity, such as POM-1, a polyoxometalate (Muller et al, 2006), do not have absolute specificity for CD39. ARL 67156 is likewise not fully specific and is moreover ineffective when tested in presence of high levels of enzyme substrates (Levesque et al, 2007). In addition, relatively low levels of NTPDase activity in microparticles would be swamped by endogenous CD39 ectonucleotidase activity on wild type LSEC (Robson et al, 1997). Despite this, addition of Cd39 null versus wild type MPs have clearly different outcomes on causing IL-6, TNF and VWF release from wild type LSEC in the absence of any exogenous ATP. Therefore, the intrinsic effects of altered NTPDase activity in Cd39 null MPs are probably less relevant than differential cellular origins and the secondarily altered contents of cytokines e.g. IL-10 and TNFR1.

Another factor accounting for increased pro-inflammatory potential of Entpd1-null MPs may lie in the fact that significantly more MPs were found to be platelet- as well as endothelial-derived, when compared with wild type mice. A number of disease states, including sepsis and acute coronary syndromes, amongst others, have been shown to be associated with elevated levels of pro-coagulant, pro-inflammatory platelet and endothelial MPs (Nieuwland et al, 2000).

In summary, we have shown that ecto-enzymatically active CD39 is present on circulating MPs and that substantive quantitative and qualitative differences exist between wild type and Entpd1-null MP populations. Our data suggest that MPs may function as vectors for cytokines and MMPs. Pro-inflammatory properties of the Entpd1-null MPs on LSEC were also clearly noted in vitro. Whether these changes are a direct result of the intrinsic lack of CD39 or indirectly related to changes in content of cytokines and other factors in mutant MPs remains to be determined fully. Further in vivo studies will also be needed to investigate how CD39 expression by the vasculature and derived circulating MPs impacts localized or generalized states of inflammation and thrombosis.

Acknowledgements

SCR acknowledges grant support from NIH HL57307, HL63972 and HL076540. YB and GB thank the Swiss National Research Foundation for grant support (PBBEB-112760; PASMA-115700 and PBBEB-112764). The authors wish to thank Steven Gygi and his team from the Taplin Biological Mass Spectrometry Facility, Harvard Medical School, for MS analysis and Dr Bruce Furie at the Center for Hemostasis and Thrombosis Research BIDMC, Harvard Medical School, for insights, technical and scientific advice and encouragement. The authors declare they have no potential conflict of interest and no competing financial interests.

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