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

Abstract

Plasma microparticles (MPs, <1.5 mum) 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.

Fig 1

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).

Fig 2

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).

Fig 3

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).

Fig 4

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.

Fig 5

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.