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PLATELETS AND THROMBOPOIESIS
Platelets and platelet-like particles mediate intercellular RNA transfer
Antonina Risitano,1 Lea M. Beaulieu,2 Olga Vitseva,2 and Jane E. Freedman2
1Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA; and 2Department of Medicine, University of Massachusetts Medical
School, Worcester, MA
The role of platelets in hemostasis and
thrombosis is clearly established; however, the mechanisms by which platelets
mediate inflammatory and immune pathways are less well understood. Platelets
interact and modulate the function of
blood and vascular cells by releasing
bioactive molecules. Although the platelet is anucleate, it contains transcripts
that may mirror disease. Platelet mRNA is
only associated with low-level protein
translation; however, platelets have a
unique membrane structure allowing for
the passage of small molecules, leading
to the possibility that its cytoplasmic RNA
may be passed to nucleated cells. To
examine this question, platelet-like particles with labeled RNA were cocultured
with vascular cells. Coculture of plateletlike particles with activated THP-1, monocytic, and endothelial cells led to visual
and functional RNA transfer. Posttransfer
microarray gene expression analysis of
THP-1 cells showed an increase in HBG1/
HBG2 and HBA1/HBA2 expression that
was directly related to the transfer. Infu-
sion of wild-type platelets into a TLR2deficient mouse model established in vivo
confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also
observed external RNA was functional in
the recipient cells. The observation that
platelets possess the capacity to transfer
cytosolic RNA suggests a new function
for platelets in the regulation of vascular
homeostasis. (Blood. 2012;119(26):
6288-6295)
Introduction
derived from the megakaryocyte. To test the hypothesis that
platelet-derived RNA is transferred, we have developed an in vitro
transfer model using MEG-01 cells, a human megakaryoblastic cell
line known to release platelet-like particles (PLPs) with characteristics similar to human platelets.24-26 These PLPs were then
examined for potential transfer of nucleic acids to recipient cells. In
this study, we show not only that PLPs are able to transfer RNA to
THP-1 cells, a monocytic cell line, and human umbilical vein
endothelial cells (HUVECs), but also that the transferred RNA is
functional.
Platelets are anucleate cells generated from megakaryocytes and
released in the bloodstream where they play an active role in
hemostasis, inflammation, and immune responses.1-4 They also are
involved in tumor progression and metastasis, with complex
molecular mechanisms that continue to be elucidated.5-9 Recent
studies have focused on analyzing platelet RNA content including
microRNA (miRNA) and messenger RNA (mRNA).10,11 Although
lacking nuclei, platelets retain megakaryocyte-derived cytoplasmatic RNA and may translate small amounts of mRNAs as well as
process miRNAs.12-14 Platelets express high levels of miRNAs, and
profiling studies have shown expression changes. Platelet transcript
variability also has been described in a large community-based
cohort study with inflammatory RNA significantly correlated with
specific cardiovascular risk factors, notably increased body mass
index.15 The platelet transcriptome mirrors the protein expression
profile, but only 69% of proteins have been found at the mRNA
level leaving open the question about the functionality of the
remaining RNA.16-18
Cell-cell communication can be achieved not only through
direct contact but also through exchange of soluble factors or
vesicles containing bioactive molecules. Recent studies have
shown that microparticles and exosomes, released from different
cell types, transfer not only proteins and lipids but also nucleic
acids to recipient cells.19,20 Activated platelets shed microparticles
that represent the most abundant microparticles in the bloodstream.21 Microparticles have been associated with hemostasis and
thrombosis; they are actively involved in cancer metastasis,
angiogenesis, and immune diseases.22,23
Taken together, these observations suggest that platelet transcript content may play a biologic role beyond being remnant RNA
PLPs were isolated from MEG-01 cultures treated with 100 ng/mL
recombinant human thrombopoietin (TPO; R&D Systems) for 48 hours.
Preparations were centrifuged at 150g for 15 minutes, and sediments were
discarded and centrifuged again at 750g for 15 minutes. Supernatants thus
obtained were centrifuged at 1600g for 15 minutes, and sediments containing PLPs were washed and resuspended in culture medium.27 In selected
experiments, PLPs were treated with 1 U/mL RNase A/T128 (Invitrogen) or
RNase ONE (Promega) for 1 hour at 37°C. Then, the PLPs were then
Submitted December 2, 2011; accepted May 4, 2012. Prepublished online as
Blood First Edition paper, May 17, 2012; DOI 10.1182/blood-2011-12-396440.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2012 by The American Society of Hematology
6288
Methods
Cell cultures
MEG-01 cells and THP-1 cells (ATCC) were grown in suspension in RPMI
1640 (Invitrogen) supplemented with L-glutamine, 10% heat-inactivated
fetal bovine serum (Invitrogen), and 1% antibiotic/antimycotic (Invitrogen). HUVECs were obtained from Lonza Walkersville, cultured in
endothelial growth medium (Lonza Walkersville) and used between second
and fifth passages.
PLP isolation
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BLOOD, 28 JUNE 2012 䡠 VOLUME 119, NUMBER 26
inhibited with 10 U/mL SUPERase䡠In RNAse (Invitrogen) inhibitor and
0.2M potassium phosphate buffer, respectively. PLPs were washed in
culture medium after this treatment.
PLP-vascular cell coincubation
THP-1 cells treated with 1 ␮g/mL Pam3CSK4 (PAM; InvivoGen) were
coincubated for 24 hours with PLPs isolated from MEG-01 cells cultures at
a ratio 1:10. PLPs were stained with 2␮M PKH67 (Sigma-Aldrich)
according to the manufacturer’s directions or with CD41a, CD42b FITCconjugated antibodies (eBioscience). PKH67 is a fluorescent green dye
attached to long aliphatic tails that is incorporated into the cell membranes.
HUVECs were treated with 0.5 U/mL thrombin (Sigma-Aldrich) for
10 minutes in endothelial basal medium (Lonza Walkersville) without
phenol red, washed, and coincubated with PKH67-labeled PLPs for
30 minutes in endothelial basal medium containing 2mM CaCl2 (SigmaAldrich) at the same ratio. After coincubation, both cell types were washed
in phosphate-buffered saline (PBS; Invitrogen) and analyzed by flow
cytometry and confocal microscopy.
BrUTP transfection and transfer
MEG-01 cells were transfected with 0.1mM 5-bromouridine-5⬘-triphosphate
(BrUTP; Invitrogen) using Lipofectamine 2000 (Invitrogen) according to
the manufacturer’s directions. Cells were treated with 100 ng/mL TPO for
24 hours, and then PLPs were collected and cocultured with THP-1 cells, as
described in “PLP-vascular cell coincubation,” with time points 0, 3, 6, 12,
and 24 hours. HUVECs were coincubated with PLPs for 1 hour in the same
conditions described in “PLP-vascular cell coincubation.” HUVECs and
THP-1 cells were analyzed for BrUTP positivity by flow cytometry using a
BrdU Flow kit (BD Biosciences) according to the manufacturer’s protocol.
GFP transfection and transfer
MEG-01 cells were transfected with a plasmid encoding green fluorescent
protein (pmaxGFP; Lonza Walkersville) using a Nucleofector II device and
Nucleofector kit C (Lonza Walkersville) optimized for MEG-01 cells
according to the manufacturer’s suggestions. Cells were allowed to recover
after nucleofection and grow for 48 hours with 100 ng/mL TPO treatment.
PLP collection and coincubations were performed as described in the
preceding paragraphs. THP-1 cells were analyzed after 24 hours and then
washed to eliminate residual PLPs and cultured for additional 24 hours.
HUVECs were coincubated with PLPs for 1 hour and then washed and
allowed to grow for 24 hours. GFP fluorescence was recorded by flow
cytometry.
PLATELETS AND PLATELET-LIKE PARTICLES TRANSFER RNA
6289
after LPS injection. Whole blood samples were tested for the percentage of
heterotypic aggregates (platelet-monocyte aggregates) and TLR2-positive
monocytes by flow cytometry. Isolated platelets and PBMCs also were
processed for RNA isolation and real-time RT-PCR as described in
“Real-time RT-PCR.”
Flow cytometry
THP-1 cells were stained with anti–human CD11b–PE-Cy7 and CD11bAPC, and HUVECs were stained with CD54–PE-Cy5. Anti–human CD41aFITC and CD42b-FITC (eBioscience) antibodies were used for PLPs, and
anti–human BrdU-FITC (BD Bioscience) was used to detect BrUTPlabeled RNA. Mouse platelets and monocytes were stained with anti–
mouse CD41-PE and CD11b-APC (eBioscience), respectively, whereas
CD282(TLR2)–FITC (eBioscience) was used for TLR2 detection. All the
stainings were performed along with the corresponding isotype controls.
The experiments were run on a FACScan analyzer (BD Biosciences) using
CellQuest Pro Version 5.2 software (BD Biosciences).
Confocal microscopy
Samples from the experiments described thus far were analyzed by confocal
microscopy. Imaging was performed on LSM 510 (Carl Zeiss) and TCS
SP2 (Leica Microsystems) confocal microscopes. Images were captured
using and MetaMorph Version 6.1 software (Molecular Devices) and Leica
Confocal Version 2.5 software.
Microarray gene expression analysis
Total RNA was isolated from Pam3CSK4-treated THP-1 cells cultured in
presence and absence of PLPs using the miRNeasy Mini kit (QIAGEN)
according to the manufacturer’s directions. Microarray gene expression
profile was determined using a Human Genome U133 2.0 Plus chip, human
transcriptome complete (Affymetrix). The Gene Expression Omnibus
accession number for the data is GSE38027.
Real-time RT-PCR
Total RNA was isolated as described in “Microarray gene expression
analysis.” RNA was converted to cDNA using the High-Capacity cDNA
Reverse Transcription kit (Invitrogen). Gene expression of human GAPDH
and HBG1/HBG2, mouse GAPDH and TLR2 (primers and probes from
Invitrogen), using the TaqMan Gene Expression Master Mix (Invitrogen),
was assessed with real-time PCR (Applied Biosystems 7900 HT Fast
Real-Time PCR System with SDS Version 2.2.2 software; Invitrogen).
SOD2 activity and ROS measurement
Animal models and in vivo platelet RNA transfer
C57BL/6J (WT) and B6.129-Tlr2tm1Kir/J (TLR2⫺/⫺) mice were purchased
from The Jackson Laboratory and housed in the animal facility at the
University of Massachusetts Medical School. All procedures were approved
by the Institute Animal Care and Use Committee at University of
Massachusetts Medical School.
Platelets were isolated from wild-type (WT) mice by centrifugation
following a previously described method.29 In brief, whole blood was
collected into syringes containing citrate-phosphate-dextrose solution
(15.6mM citric acid anhydrous, 89.4mM sodium citrate, 18.5mM sodium
phosphate, and 2.56% dextrose, pH 7.35). Samples were centrifuged at
450g for 7 minutes. The top layer of plasma was removed and diluted by
2.33 times the volume with platelet wash buffer (10mM sodium citrate,
150mM sodium chloride, 1mM EDTA, and 1% dextrose, pH 7.4). Samples
were centrifuged at 300g for 4 minutes. The resulting pellet contains
peripheral blood mononuclear cells (PBMCs). The supernatant was further
diluted with 3 times the volume with platelet wash buffer and centrifuged at
3500g for 10 minutes. The resulting pellet contains platelets. Approximately 5.5 ⫻ 108 platelets resuspended in citrate-phosphate-dextrose solution were injected into TLR2⫺/⫺ mice via tail-vein injection (total volume,
165 ␮L). After 1 hour, TLR2⫺/⫺ mice were injected with 5 ␮g/g lipopolysaccharide (LPS; InvivoGen) or saline intraperitoneally. Blood samples were
collected into citrate-phosphate-dextrose solution (1:5) at 3 and 6 hours
THP-1 cells were cocultured for 24 hours with PLPs as described in “PLPvascular cell coincubation” and then tested for superoxide dismutase 2 (SOD2)
activity using EpiQuik Superoxide Dismutase kit (Epigentek) according to the
manufacturer’s protocol. Reactive oxygen species (ROS) generation was
measured using 5-(and-6)-carboxy-2⬘,7⬘-difluorodihydrofluorescein diacetate (carboxy-H2DFFDA; Invitrogen) as described previously.30 In brief,
cells were washed in PBS, treated with 5␮M 5-(and-6)-carboxy-2⬘,7⬘difluorodihydrofluorescein diacetate for 30 minutes at 37°C, and washed
twice with PBS. The resulting fluorescence was analyzed by flow cytometry.
Data analysis
All values are expressed as the average ⫾ SD of at least 3 different
experiments. Statistical analysis was performed using Student t test and
significance of P less than or equal to .05. Prism Version 5 software
(GraphPad) was used for all analyses.
Results
PLPs coincubation with HUVECs and THP-1 cells
In culture, MEG-01 cells have the ability to spontaneously produce
PLPs with size, structures, and characteristics very similar to
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RISITANO et al
BLOOD, 28 JUNE 2012 䡠 VOLUME 119, NUMBER 26
Figure 1. PLPs coincubation with HUVECs and THP-1 cells. (A) THP-1 cells, treated with 1 ␮g/mL Pam3CSK4, were coincubated with PLPs for 24 hours and stained with
CD41a-FITC, CD42b-FITC (open curve; solid line) for flow cytometry analysis. Isotype IgG (gray filled curve), control (no PLPs; open curve, dotted line). (B) The same
experiment was carried out staining THP-1 cells for CD11b-APC, CD41a-FITC, and CD42b-FITC. Samples were then mounted on coverslips for confocal microscopy (PLP).
IgG control (isotype) and THP-1 alone (control). Scale bar denotes 10 ␮m; 100⫻ objective lens. (C) Confocal microscopy also was performed on PKH67-labeled PLPs (green
fluorescence) cocultured for 24 hours with THP-1 cells to demonstrate PLP internalization. Blue nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI).
Scale bar denotes 10 ␮m; 60⫻ objective. (D) The experiment was performed as described in panel A using CD11b-FITC. Untreated cells (control), activated cells (PAM), PLP
(THP-1 cells cocultured with PLPs). (E) HUVECs were treated for 10 minutes with 0.5 U/mL thrombin and then coincubated with PKH67-labeled PLPs for 30 minutes (gray
filled curve) and analyzed by flow cytometry. Control (open curve, dotted line), untreated HUVEC ⫹ PLPs (open curve, solid line). (F) Confocal microscopy of the experiment
described in panel E with additional DAPI staining. HUVECs incubated with PKH67-labeled PLPs (ii-iv) and unlabeled PLPs (i). Scale bar denotes 20 ␮m; 40⫻ objective.
human platelets.24,31 In coincubation experiments, THP-1 cells
were stimulated with Pam3CSK4, a synthetic lipoprotein and
TLR2 ligand, to induce interaction with PLPs. As a previous
study29 showed, the concentration used for all experiments (1 ␮g/
mL) does not induce any platelet aggregation. PAM-treated THP-1
cells, after 24 hour coincubation with PLPs, were stained with
CD41a and CD42b, specific markers for human platelets (Figure
1A). Flow cytometry analysis showed that ⬃ 20% of THP-1 cells
were positive for the double staining, confirming the PLP binding.
This result was further supported by confocal microscopy (Figure
1B-C). Untreated THP-1 cells did not significantly bind PLPs (data
not shown). As shown in Figure 1B (PLP), there is colocalization
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BLOOD, 28 JUNE 2012 䡠 VOLUME 119, NUMBER 26
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Figure 2. RNA transfer from MEG-01 to HUVECs and THP-1 cells. (A) Flow cytometry analysis of 1 ␮g/mL PAM-treated THP-1 cells cultured in presence of BrUTP-labeled
PLPs for 6, 12, and 24 hours. No fusion was observed at 1- and 3-hour time points (*P ⬍ .05 compared with PAM). (B) The same experiment was analyzed by confocal
microscopy. THP-1 cells demonstrated BrUTP labeling after 24-hour incubation with RNA-labeled PLPs (iii-vi; iv and vi are in bright field; 100⫻ objective). THP-1 control cells
stained with IgG (i) and BrdU-FITC (ii). Scale bar denotes 10 ␮m; 100⫻ objective. (C) HUVECs, treated with 0.5 U/mL thrombin, were coincubated for 1 hour with
BrUTP-labeled PLPs and analyzed by flow cytometry (open curve, solid line). Isotype IgG (gray filled curve), control (no PLPs; open curve, dotted line). (D) PAM
(1 ␮g/mL)–treated THP-1 cells cocultured for 24 hours with GFP-PLPs treated (RNase) or not with RNase (PLP). THP-1 ⫹ untreated-PLPs were washed, after 24 hours, to
eliminate residual PLPs and cultured for additional 24 hours (No PLP 48 h; *P ⬍ .05 vs untreated PLPs). (E) HUVECs showed GFP fluorescence, by flow cytometry, after
1-hour coincubation with PLPs containing GFP (open curve, dotted line), cells were then washed and allowed to grow for 24 hours (open curve, solid line). Control (gray filled curve).
between a THP-1 cell and a PLP followed by the PLP internalization, an observation confirmed using a complementary staining
method (Figure 1C). Changes in THP-1 CD11b staining also were
measured during coincubation experiments; as seen in Figure 1D,
the staining of the adhesion molecule CD11b, in presence of PLPs,
was decreased, suggesting its involvement in the binding process.
Similar results were obtained with HUVECs over a shorter period
of coincubation. HUVECs were activated with 0.5 U/mL thrombin
to promote the adhesion to PLPs, in fact, 19% of activated
HUVECs were able to bind and internalized PLPs, although less
than ⬃ 4% of resting cells were fluorescently positive (Figure 1E).
Binding also was seen with HUVECs (Figure 1Fii), as was the
progressive increase of cytosol staining from PLP internalization
(Figure 1Fiii-iv).
RNA transfer from MEG-01 to HUVECs and THP-1 cells
PLPs from transfected BrUTP MEG-01 cells were coincubated
with THP-1 cells. After 6 hours of coincubation, labeled THP-1
cells demonstrated RNA transfer that continued gradually up to
24 hours (Figure 2A). The percentage of BrUTP-positive THP-1
cells after 24 hours was 4% to 5%. Previous studies reported
MEG-01 as a difficult cell line to transfect; consequently, the
amount of labeled-RNA was limited.32 Confocal microscopy
showed that labeled RNA was localized outside and inside the
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RISITANO et al
Table 1. Coincubation: THP-1 ⴙ PLPs gene expression analysis
Up-regulated genes
Down-regulated genes
HBG1/HBG2
HBE1
MMP13
MMP2
HBA1 /HBA2
CALB1
NFYA
CD80
HPGD
ACTR2
KIF2A
STXBP5L
DNAJC12
GREB1
GPLD1
RARA
TRAJ17 /TRAV20
SOD2
RAB7A
TNFRSF11A
XDH
TNFRSF10D
MRPS15
FGA
recipient cells, indicating an ongoing transfer after 24-hour coincubation (Figure 2Biii-vi). RNA transfer also was observed using
HUVECs as recipient cells; ⬃ 20% of activated HUVECs were
positive for labeled RNA after 1-hour coculture (Figure 2C).
GFP transfer from MEG-01 to HUVECs and THP-1 cells
To test mRNA functionality in the recipient cells, we transfected
MEG-01 cells with a GFP vector by nucleofection. By flow
cytometry, we observed that GFP was expressed in MEG-01 cells
and almost 25% of PLPs collected from cultures expressed mature
GFP, carried mature GFP, or both. During a coincubation experiment, both mRNA and proteins can be transferred; therefore, we
examined THP-1 cells after 24-hour fusion with PLPs treated or not
with RNase. To verify that PLP RNA was effectively degraded by
the RNase treatment, we compared the RNA content of RNasetreated PLPs with untreated PLPs; RNase-treated PLPs contained
⬃ 48% less RNA. Interestingly, more than 6% of THP-1 cells
without RNase treatment were found positive for GFP, a number
reduced to 3% in presence of RNase. This suggests that THP-1
cells received GFP both as a mature protein and mRNA that was
then translated. When THP-1 cells cocultured with untreated PLPs
were washed after 24 hours, to eliminate PLPs, and cultured for
additional 24 hours, fluorescence from GFP was still detectable as a
result of 2 ongoing processes: GFP-mRNA expression and transferred GFP mature protein degradation (Figure 2D). Six percent of
HUVECs were fluorescent after 1-hour coincubation due to the
GFP protein transfer. After 24 hours in absence of PLPs, 16.5% of cells
expressed the GFP messenger, a figure assumed to include the percentage of positive cells (6%) containing the mature protein (Figure 2E).
THP-1 cell microarray gene expression analysis
Although transfer of RNA is important, it is of greater interest if
found to be functional. To investigate the possible effect of the
newly transferred RNAs in the recipient cells, we carried out a gene
expression analysis of THP-1 cells in presence and absence of
PLPs. Microarray analysis showed that the most up-regulated
genes were ␥ globins (HGB1/HGB2), up to 2-fold, followed by ␣
globins (HBA1/HBA2) and ⑀ globin (HBE1). Two mitochondrial
genes (SOD2, MRPS15) and genes involved in transport and
Figure 3. THP-1 HBG1/HBG2 expression after PLP RNase treatment. Cells
cocultured for 24 hours with RNase-treated PLPs expressed less HBG1/HBG2
(RNaseA/T1 and RNase ONE) compared with the untreated PLP coculture (PLP).
HBG1/HBG2, normalized against GAPDH, was absent in the control, THP-1 cells
treated with 1 ␮g/mL PAM and 100 ng/mL TPO.
transfer mechanisms also were up-regulated. There were a small
number of modestly down-regulated genes (⬍ 1-fold) associated
with exocytosis pathways (STXBP5L, RAB7A, GPLD1), 2 types of
matrix metallopeptidase (Table 1). The most up- and downregulated genes in the control cells (PAM-activated THP-1), noted
in Table 2, also were not altered by the presence of the imported
RNA. Using the ␥ globins (the most expressed genes), the
microarray results were validated by real-time PCR verifying
whether the up-regulation was due to RNA transfer. PLPs were
treated with 2 different types of RNases before a coincubation and
RT-PCR confirmed that the increased RNA was from outside the
cells (Figure 3). The small percentage of residual RNA after RNase
treatment depends on the length of treatment: a complete degradation can be achieved with longer treatments but this would have
affected PLP vitality and integrity. As a negative control TPO/
Pam3CSK4-treated THP-1 cells were examined for HGB1/HGB2
expression to exclude any TPO carryover effect from the previous
culture. CD11B did not seem to be down-regulated, confirming that
the adhesion molecule was directly involved in the PLP binding,
and for this reason not accessible for antibody staining (Figure 1D).
Platelet TLR2 RNA transfer in vivo
Mouse studies also were performed to test whether a specific
mRNA could be transferred by platelets to monocytes in vivo.
Platelets were isolated from WT mice (TLR2 RNA present) and
Table 2. Pam3CSK4-treated THP-1 gene expression analysis
Up-regulated genes*
Down-regulated genes†
ICAM1
EPB41L3
THBS1
GFBP3
NEXN
RBM35A
VCAM1
ADAM28
RTN1
IFI44L
ANKRD55
SYNPO2
CCL2
EBI3
CCL4
S100A12
WDR49
SERPINI2
CXCL14
RASGRP1
CCL8
PRR16
CEACAM6
CA3
BCL2A1
TRPM1
TNFAIP6
BIRC3
PLA2G7
SLAMF7
CLEC7A
TLR8
LUM
TRPM1
EPYC
*Fold change of 2.
†Fold change of 1.5.
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Figure 4. Quantitative RT-PCR of TLR2 in transfused mice. TLR2 fold changes
(expressed as 2⫺⌬⌬Ct) in platelets and PBMCs. All samples were normalized against
GAPDH. In each case we used 3 mice per group; the transfer of TLR2 mRNA to
PBMC was considered significant (*P ⫽ .05).
PLATELETS AND PLATELET-LIKE PARTICLES TRANSFER RNA
6293
platelet transcriptome.11,12,37 In this study, we explored the possibility that platelets influence peripheral blood and vascular cells by
transferring RNA to these cells. RNA horizontal transfer was
investigated by in vitro and in vivo models, shedding new light on
the relationship between platelets and other vascular cells.
This study demonstrates that there is successful transfer of RNA
from MEG-01 cells to HUVECs and THP-1 cells using PLPs as
mediators of the transfer (Figure 6). Vascular cells bound and
internalized PLPs with mechanisms similar to those already
described for platelets and microvesicles.38,39 For example, MEG-01
cells stimulated with TPO produced PLPs carrying mainly fetal
hemoglobin transcripts that were then transferred to the recipient
cells having almost no baseline expression of these genes. As
demonstrated in different cell lines, human TPO induces predominantly fetal and adult hemoglobin synthesis and, for the purpose of
this study, were an excellent marker to validate an RNA transfer.40-42
Interestingly, these data also suggest that RNA was not randomly
transferred because MEG-01 cells may have distributed transcripts
in the newly formed PLPs. This observation is supported by a
recent study describing how megakayocytes selectively transfer
matrix metalloproteases and their tissue inhibitors into platelets.43
PLP RNase treatments abrogated up to 75% of HGB1/HGB2
transfer after 24-hour coincubation, confirming that the mRNA
infused into TLR2-deficient animals. Complementary to the coincubation experiments, we induced the formation of heterotypic
aggregates using LPS, a bacteria LPS and TLR4 agonist. Flow
cytometry results from platelet transfusion experiments performed
on TLR2 knockout mice showed the presence of platelet-monocyte
heterotypic aggregates 3 hours after transfusion with an increase in
TLR2-positive monocytes in presence of LPS (83.60% ⫾ 2.24%)
compared with saline treatment (33.6% ⫾ 0.33%). LPS treatment
produced a modest increase in platelet-positive monocytes
(12.76% ⫾ 1.46) compared with the relative saline treatment
(9.56% ⫾ 3.46). Because coculture experiments showed RNA
transfer 6 hours after coincubation, we collected platelets and
PBMCs 6 hours after platelet transfusion and analyzed RNA to
verify whether TLR2 mRNA was transferred to the recipient cells.
A 6-fold increase in TLR2 mRNA clearly demonstrated that during
the stage of heterotypic aggregates formation platelet-monocyte
RNA transfer had occurred (Figure 4). This was particularly
important because it established that the process occurs in vivo and
with platelets as well as PLPs.
Relationship between SOD2 activity and ROS production in
THP-1 cells
Microarray results showed a modest increase in SOD2 expression
that was confirmed by assaying directly the activity in the cells
(Figure 5A); there was a 13% increase in SOD2 activity in presence
of PLPs compared with the control (PAM). ROS production also
was increased by the presence of PLPs. PAM-activated THP-1 cells
produced ⬃ 30% more ROS compared with resting cells; however,
in presences of PLPs, there was a further 12% increase that was
consistent with the amount of SOD2 activity recorded (Figure 5B).
Discussion
Intercellular nucleic acid transfer is known to be an important
mechanism regulating metastasis and stem cell communication
networks.33-35 Platelets are modestly active in translating their own
mRNA,36 but few studies have examined the function of the
Figure 5. SOD2 activity and intracellular ROS in THP-1 cells. (A) SOD2 activity
assessed in THP-1 control cells, 1 ␮g/mL PAM-treated and in presence of PLPs
(PLP). (B) Cells coincubated with (PLP-T) or without PLPs (PAM-T) were treated with
5␮M 5-(and-6)-carboxy-2⬘,7⬘-difluorodihydrofluorescein diacetate for 30 minutes and
then evaluated by flow cytometry versus controls with (PLP) and without PLPs
(*P ⬍ .05 compared with PLP and **P ⬍ .001 compared with NT [NT, no treatment]).
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Figure 6. PLPs interaction with HUVECs and THP-1 cells.
recovered in THP-1 cells was from an outside source. The
hemoglobin transfer was detectable after 12-hour coincubation
(result not shown) in accordance with the slow process observed in
THP-1 cells with the bromine-labeled RNA transfer. Many of the
other gene expression changes were related to exo- and endocytosis.
In addition, the functionality of the PLP transcripts in the
recipient cells was demonstrated by the GFP transfer; both the
mature protein and mRNA presence were highlighted. PLPs
actively interacted and transferred their RNA and GFP messenger
to activated HUVECs as well; the process was faster and more
efficient compared with THP-1 cells. After the fusion in THP-1
cells, we also observed an increase in ROS content that may be the
result of the modest SOD2 overexpression, metabolic activity
increase, or both.
Results obtained from in vitro experiments were clearly supported by the first mouse model showing that platelets can transfer
TLR2 mRNA to PBMCs after an inflammatory stimulus. Considering the significant level of transcript detected 6 hours after transfusion in leukocytes, we conclude that RNA transfer in vivo is
relatively fast and more efficient compared with the in vitro model.
However, there are limitations to this experiment as transfer of only
1 transcript was studied.
Taken together, our observations suggest that platelets may
have an interesting additional role in intercellular communication
by acting as a source of transcripts. The transfer mechanism seems
to require the direct contact between the cells, although the specific
mechanism is not yet known. Cocultured cells have been shown to
transfer short interfering RNAs with a mechanism that involves
gap junctions.44 mRNAs instead were mainly identified in microparticles released from cells,18,45 indicating that size and structure
prevent larger nucleic acids from being transferred through membrane channels. Our findings are unique in showing PLP internalization and GFP mRNA transfer, suggesting that platelets have a more
complex regulatory role compared with microparticles.
Human platelets cannot be produced in vitro or cultured but
PLPs, except for the lack of thrombotic activity, are very similar to
human platelets. The RNA transfer model developed for this study
suggests an intriguing in vivo scenario. The study shows that RNA
was transferred and expressed in the recipient cells, and these
results add a new role for the platelet transcriptome. Thus, platelet
expression profiles analyzed in cancer and cardiovascular
diseases46-48 reveal transcript variations that may affect not only
platelet function but also influence platelet-interacting cells and
vascular homeostasis by direct transfer of genetic information.
Acknowledgments
The authors thank Dr Milka Koupenova for helpful assistance with
the animal studies.
This work was supported by National Institutes of Health grants
P01 A1078894 (J.E.F.) and T32 HL007224 (A.R. and L.M.B.).
Authorship
Contribution: A.R. designed and performed experiments, analyzed
data, and edited the manuscript; L.M.B. designed and performed
animal studies, analyzed related data, and edited the manuscript;
O.V. performed confocal microscopy shown in Figures 1B and 2B;
and J.E.F. designed experiments and edited the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Jane E. Freedman, Department of Medicine,
University of Massachusetts Medical School, 381 Plantation St,
Biotech V, Suite 200, Worcester, MA 01605-4319; e-mail:
[email protected].
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 28 JUNE 2012 䡠 VOLUME 119, NUMBER 26
PLATELETS AND PLATELET-LIKE PARTICLES TRANSFER RNA
6295
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2012 119: 6288-6295
doi:10.1182/blood-2011-12-396440 originally published
online May 17, 2012
Platelets and platelet-like particles mediate intercellular RNA transfer
Antonina Risitano, Lea M. Beaulieu, Olga Vitseva and Jane E. Freedman
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