Whole blood was stored in a polyvinyl chloride collection bag system (Shanghai Transfusion Technology Co. Ltd., Shanghai, China) containing an anticoagulant, citrate dextrose solution B (ACD-B) (Shanghai Transfusion Technology Co. Ltd.). After the blood was stored overnight, the plasma was separated via centrifugation at 3820 × g for 20 min and mannitol-adenine-phosphate (MAP) additive solution (Shanghai Transfusion Technology Co., Ltd., Shanghai, China) was then added to produce a final hematocrit level of 50–65% to maintain RBC vitality for 35 days. Samples were drawn from stored RBCs at regular intervals of seven days using a sterile tubing welder (Terumo BCT, Lakewood, CO, USA).

MPs were isolated using the method previously described by Rubin et al. and Jy et al., with some modifications.26,27 Briefly, samples of stored RBCs were centrifuged at room temperature at 1850 × g for 20 min and the supernatant was collected; this process was then repeated. The supernatant was centrifuged at 4 °C and 20,000 × g for one hour and the resulting pellets were resuspended in phosphate-buffered saline (PBS; pH 7.2, Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 0.3% citric acid (Sigma, St. Louis, MO, USA). After the MPs were washed twice, they were prepared as a 10-fold concentrated suspension in PBS buffer.

To obtain MP-depleted supernatants, RBC supernatants were processed either by filtration or by centrifugation. Filtration was performed with a 0.1-μm polyvinylidene Fluoride membrane filter which is supposed to remove 99.9% MPs from RBC supernatants (Millipore Ltd., Darmstadt, Germany). The pre- and post-filtration supernatants were used in the corresponding experiments. For processing by centrifugation, the supernatants were centrifuged at 4 °C and 20,000 × g for one hour and collected.

The distribution of MPs in the RBC supernatants was assayed by nanoparticle tracking analysis (NanoSight LM10, Malvern Panalytical, UK). Three types of MPs in stored RBCs were counted by flow cytometric analysis. 28 RBC MPs, platelet microparticles (PMPs) and leukocyte microparticles (LMPs) in the RBC supernatant were double-labeled with a phycoerythrin (PE)-conjugated mouse anti-human CD235a (IgG1, clone HIR2) monoclonal antibody (BD Biosciences, San Jose, CA, USA), an APC-conjugated mouse anti-human CD61 antibody (IgG1, clone VI-PL2) or an APC-conjugated mouse anti-human CD45 (IgG1, clone HI30) antibody, respectively, combined with 10 µM carboxyfluorescein succinimidyl ester (CFSE - Life Technologies Corporation, OR, USA). Before flow cytometric analysis (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA), 500 μL of the labeled sample was pipetted into a BD TruCOUNT tube (BD Biosciences) and gently vortexed. The events positive for fluorescence of the respective antibody and CFSE were gated and counted. In addition, 5000 bead events were acquired. The number of MPs was calculated according to the manufacturer's instructions for the BD TruCOUNT tube.

Thrombin generation tests (TGTs) were performed by a calibrated automated thrombogram assay. Tests were performed with a plate reader (Fluoroskan Ascent, Thermo Labsystems, Helsinki, Finland) and computer software (Thrombinoscope, Thrombinoscope BV, Maastricht, the Netherlands) as described by the manufacturers. Briefly, 20 μL of the sample or thrombin calibrator (Thrombinoscope BV), 60 μL of MP-depleted plasma and 20 μL of platelet-rich plasma (PRP) reagent (Thrombinoscope BV) containing 1 pmol/L TF and a minimal amount of phospholipids were added to a 96-well plate in duplicate. After incubation, 20 μL of fluorescent calcium reagent (Thrombinoscope BV) was added automatically. Thrombin generation was recorded by the computer software. The data are expressed as nanomoles of thrombin generated per unit time (ETP, nM/min), peak, lag time (min) and time to peak (min).

The partial thromboplastin time (PT) and aPTT were assayed with an ACL7000 hemostasis testing system (Instrumentation Laboratory, Bedford, MA 01730, USA) combined with a HemosIL RecombiPlasTin 2G Kit or a HemosIL SynthASil Kit (Instrumentation Laboratory, Bedford, MA 01730, USA) for PT and aPTT, respectively. Briefly, the tray was placed in the instrument, the intended assay component (PT-fibrinogen or aPTT) was selected, the required reagent was added to the reagent cup, and the sample or calibration plasma was added to the indicated cup on the sample tray. The result is reported in units of seconds or percent activity.

HMVECs (Pricell, Wu Han Biomedical & Technical Ltd., China) were grown to ≥90% confluence in 96-well plates. After the incubation of endothelial cells with MPs for 2, 6, 12 or 24 h, the cells were detached with 0.05% trypsin-EDTA (Gibco). To examine the expression of ICAM-1 on HMVECs, 1 × 106 cells were incubated with 10 μL of mouse anti-human CD54-PE antibody (BD, Biosciences, San Jose, CA, USA) in 100 μL of PBS for 30 min at room temperature. The cells were diluted to 600 μL with PBS before flow cytometric analysis. The mean fluorescence intensity of the HMVECs was recorded.

After incubation of HMVECs with MPs for 2, 6, 12 or 24 h, the medium was collected, centrifuged at 12,000 × g for five minutes to remove dead cells or debris, and stored at −80 °C. The levels of IL-6 and IL-8 were simultaneously determined using a human cytokine cytometric bead array (CBA Kit, BD Pharmingen) according to the instructions provided by the manufacturer. Cytokine bead staining was analyzed by flow cytometry, and the data were compiled with BD Biosciences CBA software.

HMVEC damage was induced as described in our previous study.29 Briefly, HMVECs were grown to ≥90% confluence in 96-well plates. The cells were treated with 200 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich) for six hours. PMNs were added to endothelial cells at an effector cell:target cell ratio of 10:1. After settling, the PMNs were exposed to MPs or buffer for 30 min. The numbers of viable cells were counted over a 1-mm2 surface area by fluorescence microscopy after staining with a LIVE/DEAD Cell Imaging Kit (Life Technologies Corporation, OR, USA). The PMN respiratory burst was inhibited by incubating PMNs with 300–1200 μM apocynin for 15 min at 37 °C before adding them to the HMVECs.

For the different experimental groups, the data are presented as means ± standard deviations (SDs) or standard errors of the mean (SEMs). To compare the differences among multiple groups, the 95% confidence intervals (95% CI) are included. One-way ANOVA was performed, followed by Tukey's multiple comparison test. A paired t-test was used to calculate differences between two groups. A p-value <0.05 was defined as statistically significant.

The results of the nanoparticle tracking analysis indicated that the size of the majority of the MPs in the RBC supernatant was approximately 100–300 nm. MPs were depleted significantly by filtration through a 0.1 µm filter (Figure 1A, B). Because non-leukoreduced RBCs were used in this study, three types of MPs in stored RBCs were counted by flow cytometric analysis. The results show that RBC MPs were the major MPs present (approximately 94.5%; Figure 2A) and that the RBC MP count increased significantly after RBCs were stored for 28 days (Figure 2B).

Figure 1.

Nanoparticle tracking analysis of microparticles (MPs) in supernatant obtained from red blood cells (RBCs) centrifuged at 1850 × g for 20 min. (A) Microparticles in supernatant of RBCs stored for 35 days. (B) Microparticles in supernatant of RBCs stored for 35 days after filtration with a 0.1 μm filter.

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Figure 2.

Flow cytometric analysis of microparticles (MPs) in red blood cell (RBC) supernatant. (A) RBC MPs, platelet MPs and leukocyte MPs in RBC supernatant were double-labeled with mouse anti-human CD235a, mouse anti-human CD61 and mouse anti-human CD45 respectively, combined with 10 µM of carboxyfluorescein succinimidyl ester (CFSE). The amount of MPs in pre- and post-filtration of RBC supernatant (0.1 μm filter) stored for 35 days were counted with a BD TruCOUNT tube by flow cytometric analysis (n = 3). (B) RBC MPs accumulated in RBCs during storage for 35 days were double-labeled with mouse anti-human CD235a combined with 10 µM of CFSE and counted by the same method (n = 5). * p-value <0.05.

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Concentrated MPs from RBCs stored for 35 days were serially diluted in MP-depleted fresh plasma. With increasing amounts of RBC MPs, the ETP and peak values increased, while the lag time and time to peak values decreased (Figure 3). These results indicated that the RBC MPs promoted thrombin generation in a dose-dependent manner from an RBC MP concentration of 500 to 5000 per microliter; no additional increase was observed with higher RBC MP concentrations. Depletion of MPs from the supernatant by either filtration or centrifugation reduced the amount of MPs and significantly decreased the potential of MPs to promote thrombin generation (p-value <0.05; Figure 4).

Figure 3.

Calibrated automated thrombogram (CAT) assay showing that serially diluted red blood cell (RBC) microparticles (MPs) promote thrombin generation in plasma (n = 5). RBC supernatant was centrifuged at 4 °C and 20,000 × g for one hour and washed with phosphate-buffered saline (PBS) containing 0.3% citric acid. They were prepared as a 10-fold concentrated suspension in PBS buffer before dilution. Thrombin generation was recorded by computer software. The data are expressed as nanomoles of thrombin generated per unit time (ETP, nM/min), lag time (min), peak of the thrombin concentration (nM) and time to peak (min). * p-value <0.05,**p-value<0.01,***p<0.001,****p<0.0001; compared with the group of 500 RMP counts/μl.

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Figure 4.

Effect of filtration or centrifugation on the generation of thrombin by microparticles from the supernatant of red blood cells stored for 35 days (*p-value <0.05,****p-value<0.0001; n = 5).

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Although the PT was not affected, the aPTT decreased on adding concentrated MPs to MP-depleted plasma, and the effects exhibited dose dependency for RBC MP concentrations of 1000–20,000 per microliter. There was no difference in the aPTT between the filtered supernatant and the control supernatant (Figure 5).

Figure 5.

Effects of microparticles (MPs) on partial thromboplastin time/activated partial thromboplastin time (PT/aPTT). The PT and aPTT were assayed with an ACL7000 hemostasis testing system. Samples were prepared by serially diluting red blood cell (RBC) MPs with platelet poor plasma. (A) Result of aPTT and PT (n = 7). (B) aPTT analysis for the pre- and post-filtration (0.1 μm filter) supernatant from RBCs stored for 35 days (n = 7). .

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In this experiment, endothelial cells were incubated with MPs for 24 h. The expression of ICAM-1 on HMVECs was increased significantly after six hours and increased continuously until 24 h of incubation (Figure 6A). Furthermore, MPs in stored RBCs induced the release of the inflammatory cytokines IL-6 and IL-8 by HMVECs after 12 h of incubation (Figure 6B & C).

Figure 6.

Flow cytometric analysis of human pulmonary microvascular endothelial cells (HMVECs) during 24 h of incubation. (A) ICAM-1 expression on HMVECs (n = 3). After the incubation of endothelial cells with microparticles (MPs) for 2, 6, 12 or 24 h, the cells were detached and labeled with mouse anti-human CD54 before flow cytometric analysis. Interleukin-6 (IL-6) (B) and interleukin-8 (IL-8) (C) released from HMVECs (n = 3). After incubation of HMVECs with MPs for 2, 6, 12 or 24 h, the medium was collected, centrifuged at 12,000 × g for five minutes to remove dead cells or debris. The levels of IL-6 and IL-8 were simultaneously determined using a human cytokine cytometric bead array (CBA Kit, BD Pharmingen) according to the instructions provided by the manufacturer. * p-value <0.05,**p-value<0.01,***p-value<0.001,****p-value<0.0001; compared with the contol group at same time point.

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The PMN-mediated HMVEC damage indicated that RBC MPs could induce damage to LPS-treated HMVECs, but this damage was prevented by inhibiting the PMN respiratory burst with apocynin (Figure 7).

Figure 7.

Red blood cell (RBC) microparticles (MPs) induced lipopolysaccharide (LPS)-activated human pulmonary microvascular endothelial cell (HMVEC) damage. The cells were treated with 200 ng/mL LPS for six hours. Polymorphonuclear neutrophils (PMNs) were added to endothelial cells at an effector cell:target cell ratio of 10:1. After settling, the PMNs were exposed to MPs or buffer for 30 min. The numbers of viable cells were counted over a 1-mm2 surface area by fluorescence microscopy after staining with a LIVE/DEAD Cell Imaging Kit. The PMN respiratory burst was inhibited by incubating PMNs with 300–1200 μM apocynin for 15 min at 37 °C before adding them to HMVECs. (A) Effect of serially diluted RBC MPs on HMVEC damage (n = 5). (B) Effect of treating PMNs with apocynin on RBC MP-induced HMVEC (3 × 104/μL) damage (n = 5). * p-value <0.05,**p-value<0.01,***p-value<0.001,****p-value<0.0001.

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