Aging- and activation-induced platelet microparticles suppress apoptosis in monocytic cells and differentially signal to proinflammatory mediator release

Abstract

Background: Platelet microparticles (PM) are the most abundant cell-derived microparticles in the blood, and accumulate in thrombo-inflammatory diseases. Platelets produce PM upon aging via an apoptosis-like process and by activation with strong agonists. We previously showed that long-term treatment of monocytic cells with apoptosis-induced PM (PMap) promotes their differentiation into resident macrophages. Here we investigated shorter term effects of various types of PM on monocyte signalling and function.

Methods and results: Flow cytometry and scanning electron microscopy revealed that PM formed upon platelet aging (PMap) or ultra-sonication (PMsonic) expressed activated αIIbβ3 integrins and tended to assemble into aggregates. In contrast, PM formed upon platelet activation with thrombin (PMthr) or Ca(2+) ionophore (PMiono) had mostly non-activated αIIbβ3 and little aggregate formation, but had increased CD63 expression. PM from activated and sonicated platelets expressed phosphatidylserine at their surface, while only the latter were enriched in the receptors CD40L and CX3CR1. All PM types expressed P-selectin, interacted with monocytic cells via this receptor, and were internalised into these cells. The various PM types promoted actin cytoskeletal rearrangements and hydrogen peroxide production by monocytic cells. Markedly, both aging- and activation-induced PM types stimulated the phosphoinositide 3-kinase/Akt pathway, suppressing apoptosis induced by several agonists, in a P-selectin-dependent manner. On the other hand, the PM types differentially influenced monocyte signalling in eliciting Ca(2+) fluxes (particularly PMap) and in releasing secondary mediators (complement factor C5a with PMap, and pro-inflammatory tumour necrosis factor-α with PMthr).

Conclusions: In spite of their common anti-apoptotic potential via Akt activation, aging- and activation-induced PM cause different Ca(2+) signalling events and mediator release in monocytic cells. By implication, aging and activated platelets may modulate monocyte function in different way by the shedding of different PM types.

Keywords: Aging; apoptosis; microparticles; monocytes; platelet activation; tumour necrosis factor.

Figure 1

Formation of PM from stimulated and aging platelets. Suspensions of human platelets were unstimulated or stimulated with thrombin (thr, 1 U/ml) and/or collagen (col, 5 μg/ml), or A23187 (15 μM), as indicated. Platelets were also sonicated by ultrasound. After indicated times, formation of PM was determined by flow cytometry and staining with fluorescently-labelled anti-CD61 mAb. For calibration, fixed amounts of 1 and 6 μm beads were added to all samples. A: Representative dot plots of (activated) platelet suspensions after 1 hour. Gated regions represent PM. B: Quantification of PM formed after 1-24 hours of stimulation at 22°C, or 1 hour at 37°C. C: Quantification of PM formed in washed platelets or in PRP after indicated time points. Mean ± SEM (n = 3); *p < 0.05 vs. control at 37°C; ●p < 0.05 vs. control at 22°C.

Figure 2

Morphological characterisation of different PM types. A: Flow-cytometry dot plots of PM types, obtained after ultracentrifuge isolation and filtering. Gated regions indicate aggregated (AG) and non-aggregated microparticles. B: Quantitative analysis of the extent of PM aggregation, determined by flow cytometry. Mean ± SEM (n = 4-6); °p < 0.05 vs. PM_ap. C: Scanning electron microscopic images of preparations of different PM types. Centrifuged PM types were fixed and immobilised on polylysine for electron microscopy. Shown are representative electron micrographs. Arrows indicate PM aggregates (bars = 1 μm).

Figure 3

Surface characteristics of PM types and ability to bind to monocytic cells. A: Flow cytometric staining of PM types and platelets (plts) for different glycoprotein markers. Platelets were resting or stimulated with thrombin (thr, 1 U/ml). Data represent geometric means of fluorescence intensity, after correction for staining with control IgG. B: Flow cytometry detection of PM binding to monocytic THP-1 cells, treated with indicated PM for 30 minutes in the presence or absence of either: neutralising antibodies (20 μg/ml), EDTA (5 mM), eptifibatide (50 μg/ml), or CX3CR1 inhibitor F1 (0.5 μg/ml). PM complexes with THP-1 cells were detected as THP-1 gated events staining for FITC anti-CD61 mAb. Blank represents control with unstained cells. Means ± SEM (n = 3-5); *p < 0.05 vs. PM_ap; °p < 0.05 vs. Plts.

Figure 4

Binding and uptake of PM types by monocytic cells. A: Flow cytometric detection of FITC-labelled carboxylated beads coated with PM_ap or PM_thr. Beads were incubated with 3 × 10⁷ PM/ml, or otherwise as indicated. PM coating was verified by co-labelling with APC anti-CD61 mAb. B: Interaction of PM-coated beads with THP-1 cells, determined by flow cytometry. Cells were incubated with beads for 40 minutes at 22°C. Note abolished binding in the presence of EDTA (5 mM), but not with annexin A5 (1 μg/ml). C: Numbers of bound and internalised PM-coated beads per THP-1 cell, determined by confocal microscopy. D: Confocal microscopy of PM-coated beads (FITC label, green) bound and internalised into TPH-1 cells (APC anti-CD45 mAb, red). Shown are representative 3-dimensional reconstructions of recorded z-stacks (bars, 10 μm). Means ± SEM (n = 3-4); *p < 0.05 vs. control; °p < 0.05 vs. no inhibitor.

Figure 5

Effects of PM types on peroxide secretion and actin cytoskeleton rearrangement in monocytic cells. THP-1 cells (2.5 × 10⁶/ml) were treated with vehicle (ctrl), PM_ap, PM_thr, PM_iono or PM_sonic (2.5 × 10⁷/ml) for 2-24 hours. A: Hydrogen peroxide secretion measured after indicated treatment times. B: Filamentous actin (F-actin) formation after 24 hours, as assessed by staining with FITC-phalloidin and flow cytometry. Means ± SEM (n = 3-5); *p < 0.05 vs. control.

Figure 6

Anti-apoptotic effect of PM types on monocytic cells. THP-1 cells (1.25 × 10⁶/ml) were treated with vehicle (control), resting platelets or PM types (each 1.25 × 10⁷/ml) for 1-6 hours. The Akt inhibitor, AKT124005 (10 μM), was present as indicated. Cells were then stimulated with PMA (0.5 μg/ml) or ABT737 (10 μM) for 1 hour to start apoptosis. A: PM effect on PMA- or ABT737-induced caspase-3 activation. Caspase-3 activity is expressed as increased fluorescence compared to non-stimulated control cells. B, C: PM effect on Akt phosphorylation. B: Representative western blots of phosphorylated (P)-Akt or total Akt from cells incubated with PM_ap or PM_thr for 1-6 hours. C: Densitometric analysis of P-Akt bands, normalised to Akt staining. D: Effect of Akt inhibitor on PM-regulated caspase-3 activity. Means ± SEM (n = 3-7); *p < 0.05 vs. vehicle control; °p < 0.05 vs. PMA or ABT737.

Figure 7

Different effects of PM types on cytokine release by monocytic cells and monocytes. THP-1 cells (3 × 10⁵/ml) or monocytes (1 × 10⁶/ml) were treated with vehicle solution (ctrl), indicated PM types, or platelets for 24-44 hours at 37°C. A: Levels of complement factor C5a and TNFα, measured in supernatants after 24-hours treatment of THP-1 cells with PM (3 × 10⁶/ml). B: Dose effect of PM types (0.6-30 × 10⁶/ml) on C5a and TNFα release by THP-1 cells. C: Cytokine levels in supernatants after treatment of monocytes (44 hours) with PM types (1 × 10⁷/ml) or resting platelets (1 × 10⁷/ml). Means ± SEM (n = 3-4); *p < 0.05 vs. control; °p < 0.05 vs. PM_ap.

Figure 8

Different effects of PM types on Ca²⁺ responses in monocytic cells. Fluo-4-loaded CD14-positive monocytes on coverslips were left untreated (control) or treated with indicated types of PM or resting platelets (each 2 × 10⁸/ml). The agonist ATP (10 μM) was used as a positive control. Rises in Ca²⁺ in single cells were monitored by high-frequency fluorescence image recording. A: Time traces of pseudo-ratio Ca²⁺ responses (F/F_o) from 3 representative cells per condition. B: Average Ca²⁺ responses from > 25 cells. Means ± SEM; *p < 0.05 vs. control.