Keywords microvesicles - myocardial infarction - cardiovascular diseases - vascular homeostasis
Introduction
Extracellular vesicles (EVs) constitute a heterogeneous population ranging from 0.03
to 1 µm in size that are released from cells either by membrane budding (microvesicles)
or by exocytosis of intracellular multivesicular bodies (exosomes).[1 ]
[2 ] Exosomes can also be released by secretory autophagy with and without membrane fusion.[3 ]
[4 ]
[5 ] The release of EVs is induced by cellular activation, injury, or stress, and their
functions vary broadly depending on the source, the activation state of the parental
cell, and the generation process.[1 ]
[6 ] This results in EV populations with different content and surface antigens that
disseminate the functions of the parental cell and makes it possible to detect and
characterize them.[7 ]
[8 ] EVs are involved in many physiological processes such as coagulation, vascular homeostasis,
and intercellular transfer of biological messengers such as miRNA and lipids.[8 ]
[9 ]
[10 ]
[11 ] Elevated levels of EVs are found in many disorders, including cancer and immunological
and cardiovascular diseases, which renders them a high interest for clinical assessment.[1 ]
[6 ]
[9 ]
[10 ]
[11 ]
[12 ]
[13 ]
[14 ]
[15 ] The phospholipid membranes of the cell surface–derived EVs are also often rich in
negatively charged phosphatidylserine (PS) in the outer leaflet and provide a procoagulant
surface that contributes to the hypercoagulability observed in many diseases.[16 ]
[17 ]
Tissue factor (TF) is the main initiator of the coagulation cascade in vivo.[18 ]
[19 ] This 47-kDa cell surface–bound protein is expressed in tissues surrounding vessels
and initiates clotting after an injury by the binding and activation of FVII to FVIIa.
TF expression can also be induced in vascular cells, mainly monocytes, which contributes
to thrombus propagation in vivo.[16 ]
[20 ]
[21 ]
[22 ] Beyond initiation of coagulation, the formation of the TF/FVIIa complex initiates
intracellular signaling which contributes to the progression of cancer, diabetes,
and acute coronary syndromes by regulating inflammation, cell motility, cell survival,
and angiogenesis.[23 ]
[24 ]
[25 ] TF in plasma is mostly found on EVs,[26 ]
[27 ] and this pool of TF is also most likely to be procoagulant due to the presence of
negatively charged phospholipids providing a surface for activation of the coagulation
cascade.[28 ]
The most commonly used technique to study EVs in clinical samples is flow cytometry,
mainly due to the possibility to rapidly analyze a range of EV populations using multicolor
labeling. However, using this technique in particular small EVs with low protein levels
on the surface are difficult to measure accurately. They often fall under the detection
limit for forward and side scatter[12 ]
[29 ] and have weak fluorescent signals. The true events might also be confused by the
presence of antibody aggregates formed in the antibody reagents and centrifugation
of antibodies and buffer is required prior to staining to diminish this problem.[30 ] Moreover, protocols for analysis of EVs in plasma most often include washing procedures
that can lead to the loss of small EVs and the occurrence of centrifugation-induced
artifacts.[31 ]
[32 ] Whole blood and plasma analysis may on the other hand increase the background noise
that may complicate the analysis. Thus, the small size and the low protein levels
on EVs make measurement challenging and the lack of sensitive and standardized methods
may have slowed the implementation of EVs as clinical biomarkers. Newer instruments
and technical development have yielded better resolution for EVs and improvements
of flow cytometry and other techniques will be valuable for the understanding of EVs
in various diseases.[33 ]
[34 ]
[35 ]
The solid-phase proximity ligation assay (SP-PLA) that relies on at least triple recognition
of the target vesicle/protein and with real-time PCR quantification has been demonstrated
to have excellent sensitivity and specificity for protein detection in solutions.[36 ]
[37 ] Previously, it has been demonstrated that cholera toxin subunit-B (CT-B, binding
to ganglioside GM1)[38 ] and Annexin V (binding to PS) capture different populations of EVs in plasma.[39 ] In contrast to Annexin V, lactadherin is known to bind PS independently of Ca2+ .[40 ] We therefore established a SP-PLA method to specifically capture and detect PS-positive
populations of platelet-derived EVs in small amounts of plasma using lactadherin as
capture agent. In addition, we used CT-B to capture EVs with ganglioside GM1. These
populations were then analyzed with proximity ligation for CD41/CD61 or TF antigen.
We demonstrate here that platelet-derived EVs (CD41 + /CD61+ and PS + ) captured with
lactadherin, but not CT-B, are detected and quantified by SP-PLA, and that these measurements
correlate well with high-sensitivity flow cytometry. The SP-PLA also allowed detection
of EVs smaller than 0.2 µm. TF-positive EVs were detected using SP-PLA with CT-B capture,
but not lactadherin, from LPS and TRAP-stimulated whole blood from healthy individuals.
However, SP-PLA with lactadherin capture allows detection of TF-positive EVs spiked
into plasma with a similar sensitivity as a direct TF procoagulant activity (PCA)
assay.
Methods
Clinical Samples and Ethical Permission
Plasma samples from 17 patients with ST-elevation myocardial infarction from the Relevance
of Biomarkers for Future Risk of Thromboembolic Events in UnSelected Post-myocardial
Infarction patients (REBUS) trial collected 3 to 5 days after the index event and
at 1 year follow-up[41 ] were analyzed for platelet-derived EVs. The study was approved by the Uppsala University
local ethics committee (Dnr 2009/210). Plasma samples from healthy individuals were
drawn to study the generation of EVs by in vitro stimulation of whole blood and the
study was approved by the Uppsala University local ethics committee (Dnr 2013/116).
Blood Sampling and Preparation of Platelet-Free and EV-Free Plasma
Blood was drawn from healthy individuals using vacutainers with 3.2% citrate. Whole
blood was then stimulated with 20 µM adenosine diphosphate (ADP), 10 µM thrombin receptor
activating peptide (TRAP), or 20 ng/mL lipopolysaccharide (LPS) during 3 hours to
generate EVs. Samples were subsequently centrifuged at 2,500 × g for 15 minutes and the platelet-poor plasma (PPP) was transferred to new tubes. The
PPP was then centrifuged at 2,500 × g for 15 minutes again and the platelet-free plasma (PFP) was aliquoted and frozen
at −70°C. Repeated freeze–thaw cycles were avoided. The patient samples were centrifuged
at 2,000 × g for 15 minutes and aliquots were frozen at −70°C. After thawing, samples were centrifuged
again at 2,500 × g for 15 minutes.
To prepare EV-free plasma, PPP from 10 individuals was pooled and centrifuged at 100,000 × g for 18 hours at 4°C. After centrifugation, EV-free plasma was aliquoted and frozen
at −70°C.
Purification of Platelet-Derived EVs
Blood was drawn from healthy individuals using vacutainers with 3.2% citrate and was
centrifuged at 140 × g for 20 minutes and platelet-rich plasma (PRP) was transferred to new tubes. PRP was
stimulated using 20 μM ADP and 10 μM TRAP and incubated at 37°C for 2 hours with agitation
every half an hour. After incubation, PRP was centrifuged at 2,500 × g for 15 minutes twice to remove any residual platelets. The plasma was thereafter
centrifuged for 60 minutes at 20,000 × g and the plasma was subsequently removed. The pellet was dissolved using 200 µL HEPES-buffered
saline (HBS), and aliquots of 20 μL were frozen at −70°C.
Antibodies and Reagents
Antibodies used in SP-PLA were mouse antihuman CD41 (555465), mouse antihuman CD61
(555752), and mouse IgG1 (557273) from BD Pharmingen (San Jose, California, United
States). Goat anti-TF polyclonal antibody (AF2339) and goat IgG (AB-108-C) were from
R&D Systems (Minneapolis, Minnesota, United States). Antibodies for flow cytometry
were anti-CD41 (6607115) from Beckman Coulter (Fullerton, California, United States).
Lactadherin-FITC (Haematologic Technologies Inc., Essex Junction, Vermont, United
States) or Annexin V – Pacific blue (Biolegend, San Diego, California, United States)
was used to detect PS-positive EVs. Goat IgG for blocking buffer was purchased from
R&D Systems.
Biotinylation of Lactadherin
Bovine lactadherin was biotinylated using the EZ-Link Micro NHS-PEG4 -Biotinylation kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States)
according to instructions from the manufacturer. Fifty-fold excess NHS-PEG4 was added to lactadherin and the mixture was incubated for 30 minutes at room temperature.
Zeba spin desalting columns were used to remove unreacted biotin. Aliquots of 2 µM
biotinylated lactadherin were frozen at −70°C until use.
DNA Oligonucleotides, Primers, and Splint Sequences
DNA oligonucleotides[14 ] for preparation of PLA probes were modified with a thiol group at the 5′ or 3′ end
(5′ SH-CGCATCGCCCTTGGACTACGACTGACGAACCGCTTTGCCTGACTGATCGCTAAATCGTG 3′ OH) and (5′
P-TCGTGTCTAAAGTCCGTTACCTTGATTCCCCTAACCCTCTTGAAAAATTCGGCATCGGTGA-3′ SH), the latter
oligonucleotide having a 5′-phosphate group (Eurogentech). Forward (5′-CATCGCCCTTGGACTACGA-3′)
and reverse (5′-GGGAATCAAGGTAACGGACTTTAG-3′) primers for PCR and a connector oligonucleotide
(5′-TACTTAGACACGACACGATTTAGTTT-3′) to guide ligation of the two DNA oligonucleotides
were purchased from DNA technology (Risskov, Denmark).
Conjugation of Antibodies
Antibodies were conjugated according to previously published protocol.[36 ] Briefly, 20 µg antibodies were activated with 2 µL 4 mM sulfo-SMCC (Thermo Fisher
Scientific), diluted in DMSO, and incubated for 2 hours at room temperature. When
1 hour was left of the incubation time, the oligonucleotides (PLA probes) were reduced.
Three microliters (100 µM stock) for each antibody were reduced with 3 µL DTT (stock
100 mM freshly dissolved in phosphate-buffered saline [PBS]/5 mM EDTA). PLA probes
were incubated 1 hour at 37°C. Excess SMCC and DTT were removed by Zeba spin desalting
columns with 7K cutoff (Thermo Fisher scientific) according to instructions from the
manufacturer. The antibodies were then split into two aliquots and mixed with either
of the two oligo arms and incubated 30 minutes at room temperature. Finally, the conjugates
were dialyzed against PBS overnight at 4°C. Dialyzed antibodies were diluted to a
final concentration of 500 nM in PBS supplemented with 0.1% bovine serum albumin (BSA)
and 0.05% NaN3 and stored at 4°C.
Solid-Phase Proximity Ligation Assay for Measurements of EVs in Plasma
Five microliters of biotinylated CT-B at a concentration of 1 mg/mL (Thermo Fisher
Scientific) or 100 μL 2 μM in house biotinylated lactadherin was immobilized on 100
μL washed MyOneStreptavidin T1 dynabeads (Thermo Fisher Scientific) diluted to a final
volume of 200 μL with PBS/0.1% BSA. Beads were incubated for binding of capture agent
with rotation at room temperature for 1 hour. The beads were then washed twice with
500 µL PBS/0.05% Tween-20 and reconstituted in PBS with 0.1% BSA. All the washing
steps were done using the DynaMag-96 Side magnet (Thermo Fisher Scientific).
Dynabeads with lactadherin or CT-B were vortexed and 1 µL for each reaction was added
in a microcentrifuge tube, beads separated on magnet, and the storage buffer removed.
Then 5 µL PLA buffer (PBS, 0.1% BSA, 0.05% tween-20, 100 nM Goat IgG, 0.1 µg/µL salmon
sperm DNA, and 5 mM EDTA)/reaction was added to the beads and 5 µL bead suspension
distributed into each well of a 96-well PCR plate. Plasma samples were then diluted
1–10 × (TF measurement) or 100 × (CD41 CD61 measurement) in PLA buffer. Diluted plasma
samples (45 µL) were thereafter added and the plate was incubated with rotation for
1.5 hours at room temperature.
After incubation, samples were washed twice and 50-µL probe conjugated antibodies
(CD41 and CD61 antibodies conjugated to PLA probe 1 and PLA probe 2, respectively,
for platelet-EV detection; TF polyclonal antibody conjugated to either PLA probe 1
or PLA probe 2) were added at a final concentration of 500 pm . Samples were incubated 1.5 hours on rotation at room temperature and then washed
three times. To remove background, samples were moved to a new 96-well plate. PCR
and ligation mix was prepared by the addition of 0.01 U/µL T4 DNA ligase (Thermo Fisher
Scientific) and 0.08 mM ATP to the SYBR Select master mix (Applied Biosystems, Thermo
Fisher Scientific). Primers and splint were also added, at a final concentration of
0.1 µM each. The mix (50 µL) was added to each sample and the plate was incubated
for 5 minutes at room temperature before the start of the qPCR program with an initial
incubation step for 2 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C
and 1 minute at 60°C. Samples were analyzed in duplicates or triplicates. The cycle
threshold (CT) values were measured in the exponential phase of the amplification,
where amplification is most efficient and therefore quantification is least affected
by reaction-limiting conditions. For analysis, the CT values were linearized (2^-CT)
and related to background controls. To each plate and antibody combination, samples
with EV-free plasma and/or plasma-free samples were included for background control.
Flow Cytometry
Antibodies and lactadherin or annexin V were mixed to a volume of 50 µL with HBS (with
5 mM Ca2+ in case of annexin V inclusion) and centrifuged at 18,000 × g for 5 minutes to remove antibody aggregates. After centrifugation, the antibody solution
was transferred to new tubes and mixed with 30 µL plasma sample and incubated for
20 minutes at room temperature for staining. Samples were then diluted with 240 µL
HBS (with 2.5 mM Ca2+ if annexin V was included) and 30 µL of 3.17 µm AccuCount Blanc particles (Spherotec
Inc, Lake Forest, Illinois, United States) for volume control. Samples were then analyzed
within 30 minutes using a Navios or a CytoFLEX flow cytometer (Beckman Coulter). The
Navios flow cytometer was set to detect both large (0.5–1 µm) and small (0.3–0.5 µm)
EVs using Megamix-Plus FSC beads (Biocytex, Marseille, France).[29 ]
[42 ] The CytoFLEX was configured to detect EVs using the 405-nm violet laser. Gates for
EVs were set using polystyrene beads sized 0.1 to 0.9 μm in diameter (Megamix-Plus
FSC and Megamix-Plus SSC, Biocytex) and FSC/VioletSSC scatter plot according to instructions
from Beckman Coulter (Spittler, Set-up of the CytoFLEX for EV measurement, Beckman
Coulter).
TF Procoagulant Activity Assay
The PCA of TF on the surface of EVs was assessed using a direct activity assay that
measures the ability of EVs to convert FX to activated FXa in the presence of FVIIa
as described previously.[43 ] Plasma (50 µL) was incubated for 1 hour with 1 µL lactadherin–bound T1 beads (described
previously), and then plasma was separated from beads using a magnet and beads were
washed twice with 200 µL HBS with 0.1% bovine serum albumin (HBSA). The beads were
resuspended in 50 µL HBSA containing 10 nM FVIIa and 300 nM FX and incubated for 2
hours at 37°C. FXa generation was stopped by the addition of 25 µL 25 mM EDTA/HBSA,
and then 25 µL FXa substrate (S-2765, Chromogenix, Orangeburg, New York, United States)
was added and the samples incubated for 15 minutes at 37°C. The beads were removed
using a magnet and the supernatant was pipetted to a 96-well plate and absorbance
at 405 nm was measured using a spectrophotometer. The samples were measured in duplicates
and a standard curve between 0 and 100 pg/mL was created by using relipidated human
TF (Dade Innovin, Siemens, Erlangen, Germany).
Calibrated Automated Thrombogram
The calibrated automated thrombogram (CAT) assay was used to analyze thrombin generation
and TF activity in plasma samples as previously described.[31 ] Fluorescence was measured using a 96-well plate fluorometer (Fluoroscan Ascent,
Thermo Fisher Scientific). Briefly, 80 µL of plasma was mixed with 20 µL of HEPES
buffer supplemented with BSA (20 mM HEPES, 140 mM NaCl, 5 mg/mL BSA, pH 7.4) to measure
thrombin generation. In parallel with the test samples, thrombin calibrator (Thrombinoscope)
was run. The samples were run in duplicates and fluorometric measurements were performed
after automated addition of 20 µL FluCa-kit. No exogenous TF or phospholipids were
added and thrombin generation was followed for 120 minutes. The lag time before the
start of thrombin generation has previously been shown to depend on TF activity.[31 ]
[44 ]
Statistics
Statistical analyses were performed in GraphPad Prism and differences between experimental
groups were analyzed with paired or unpaired two-tailed Student's t -tests. Correlation between measurements was studied using Pearson's correlation.
Results were considered significant when p < 0.05.
Results
Evaluation of the SP-PLA Method for the Detection of Platelet-Derived EVs in Plasma
To detect different EV populations in plasma, we applied the SP-PLA as illustrated
in [Fig. 1 ]. To evaluate the method, a series of experiments were conducted on platelet-derived
EVs (CD41 + /CD61 + ). Lactadherin, but not CT-B, readily captured platelet-derived
EVs in plasma from both unstimulated and ADP-stimulated whole blood ([Fig. 2A ]). Flow cytometric analysis further confirmed that almost all of the detectable CD41-positive
EVs in plasma were indeed positive for PS ([Fig. 2B ]) and that CT-B and lactadherin seemed to stain different EV populations ([Fig. 2C ]). Exchanging the antibodies used for the detection with a mouse control IgG completely
removed the PLA signal demonstrating the requirement of specific antibodies for detection
of EVs ([Fig. 2D ]). Treating purified platelet EVs with 0.5% triton X-100 prior to analysis also depleted
the signal, showing that the assay is detecting vesicles ([Fig. 2E ]).
Fig. 1 SP-PLA method for detecting EVs. CT-B or lactadherin is bound to capture beads. Capture
beads with affinity reagent are then incubated with plasma diluted in PLA buffer to
capture extracellular vesicles. After capture, the beads are washed and incubated
with antibodies detecting CD41 or CD61 conjugated to two different oligonucleotides,
PLA probes 1 or 2, to detect the CD41/CD61 integrin which is present on the platelet
surface as well as on platelet-derived EVs. When the two antibodies are bound in proximity,
PLA probes can be ligated and the ligation product serves as a template for a PCR
reaction. Then detection can subsequently be done sensitively using quantitative PCR.
Fig. 2 Detection of platelet-derived EVs using SP-PLA. (A ) Detection of CD41/CD61+ EVs in plasma (diluted 1:100 in PLA buffer) using SP-PLA
after lactadherin capture (black bars) and CT-B capture (white bars). Levels relative
to the negative control with only PLA buffer (set to 1) are shown. Error bars represent
standard deviation (n = 2). (B ) Plasma from ADP-stimulated whole blood was analyzed by flow cytometry using a Navios
from Beckman Coulter with the gate set on 0.3–0.9 μm particles defined by polystyrene
beads. One representative flow cytometry analysis (dot plot) of lactadherin-FITC and
CD41-PC7–positive events is shown. (C ) Representative flow cytometry analysis (dot plot) of CT-B-FITC and lactadherin PCB.
(D ) Mouse control IgG or CD41 and CD61 antibodies were applied in SP-PLA and signals
from EVs in plasma (diluted 1:100) from two individuals are shown relative to negative
control with only PLA buffer (set to 1). Error bars represent standard deviation from
replicates (n = 2). (E ) Purified platelet EVs were lysed or not with 0.5% triton-X-100 for 5 minutes prior
to SP-PLA analysis. Signal in un-lysed and triton lysed samples are shown as levels
relative to negative control with only PLA buffer (set to 1). Error bars represent
standard deviation from duplicates. (F ) Whole blood was activated or not with 20 μM ADP for 2 hours and plasma was generated.
The plasma samples were filtered or not with 0.2 μm filters and the levels of platelet
EVs (CD41/CD61 positive) were measured with SP-PLA and high-sensitivity flow cytometry
using a Navios from Beckman Coulter, with the gate set by polystyrene beads on 0.3–0.9
μm particles. Levels relative to unstimulated control are shown. Error bars represent
standard deviation from three individual experiments (n = 3).
To compare high-sensitivity flow cytometry (Navios, Beckman Coulter) with the SP-PLA
method, plasma from unstimulated whole blood and plasma from whole blood stimulated
with 20 μM ADP were analyzed before and after 0.2-μm filtration. Levels of CD41 + /CD61+
EVs relative to the respective unstimulated control before and after filtration are
shown in [Fig. 2F ]. The plasma filtration almost completely depleted the signal from the flow cytometer,
whereas EVs were still detectable with the lactadherin SP-PLA method. The mean reduction
after filtration of plasma from ADP-stimulated whole blood was 79% with PLA and 96%
with flow cytometry (p = 0.048, n = 3).
In addition to treatment with ADP ([Figs. 2A ] and [3A ]), whole blood was also stimulated with TRAP and LPS as indicated in [Fig. 3A ]. ADP and TRAP increased the formation of platelet-derived plasma EVs, whereas LPS
did not. The ADP and TRAP treatments also reduced the lag time for thrombin generation
in the CAT assay, showing that the stimulated sample was procoagulant ([Fig. 3B ]).
Fig. 3 Platelet-derived EVs in plasma samples measured by SP-PLA and a thrombin generation
assay. Whole blood was stimulated or not with 20 μM ADP, 10 μM TRAP, or 20 ng/mL LPS
in 37°C for 3 hours to generate EVs. (A ) CD41/CD61+ EVs were detected with SP-PLA and the levels are shown as fold induction
from the mean value of plasma prepared from unstimulated whole blood (unstimulated,
set to 1). Error bars represent standard error of the mean (SEM). Differences between
groups were tested with unpaired Student's t -test, and a p -value <0.05 was considered significant. (B ) Thrombin generation was studied in the same samples using calibrated automated thrombogram
(CAT). Fold induction of the lag time in plasma from unstimulated whole blood is shown.
Error bars represent SEM from three individual experiments. Differences between groups
were tested with a paired, two-tailed Student's t -test. A p -value < 0.05 was considered significant.
Determination of Limit of detection Score and Assay Variability
To further demonstrate the performance of SP-PLA, we spiked washed platelet-derived
EVs into EV-free plasma or into buffer. The platelet-derived EVs were first counted
using a Navios flow cytometer (Beckman Coulter) with a polystyrene bead-defined gate
set on 0.3 to 0.9 μm. The platelet-derived EVs were then spiked at different concentrations
either in EV-free plasma (diluted the same way as we dilute plasma for SP-PLA analysis,
1:100) or directly in PLA buffer. The obtained signals after the SP-PLA were similar
for both spike-in in buffer and plasma, showing that detection of platelet-derived
EVs in plasma is plausible ([Fig. 4A ]).
Fig. 4 Detection of spiked platelet-derived EVs. (A ) Platelet EVs were counted by flow cytometry (Navios, Beckman Coulter) and spiked
at different concentrations either in PLA buffer or in EV-free plasma (diluted 1/100).
The PLA signals relative to background (only buffer) after lactadherin capture and
CD41/CD61 detection from a representative experiment (n = 1) is shown. (B ) To determine the LOD score, washed platelet-derived EVs were counted by flow cytometry
(Navios). The EVs were then spiked in PLA buffer to create a dilution series (1:10)
and detected with SP-PLA CD41/CD61. Error bars represent standard deviation (n = 3).
Limit of detection (LoD) is the lowest analyte concentration likely to be reliably
distinguished from the background and at which detection is feasible. To determine
the LoD for SP-PLA, platelet EVs were spiked in a 10-fold dilution series in EV-free
plasma diluted 1:100 with PLA buffer and detected with SP-PLA. The concentration of
CD41+ PS+ EVs was plotted against the SP-PLA signal ([Fig. 4B ]) and the LoD (CT value: 2 standard deviations above the background) corresponded
to a measured concentration of 0.1 EVs/μL. The intra-assay variability (mean %CV calculated
for technical triplicates at all five measured concentrations) was 16% and the inter-assay
variability was 17.7% for 1,500 CD41+ PS+ EVs/μL and 18.3% for EV-free plasma (estimated
by measuring samples in triplicates in 10 independent experiments and calculating
the %CV of the means).
SP-PLA Correlates Well with a High-Sensitivity Flow Cytometric Analysis
Next, to compare the SP-PLA signal for platelet-derived EVs with a high-sensitivity
flow cytometry assay, the CytoFLEX system (Beckman Coulter) was used according to
the guidelines for EV detection provided by the manufacturer (Spittler, Set-up of
the CytoFLEX for EV measurement, Beckman Coulter). The gates for EVs were set using
FSC/SSC scatter plot and polystyrene Mega mix beads with the diameters 0.1, 0.16,
0.2, 0.24, 0.3, 0.5, and 0.9 μm. All bead sizes could be resolved from one another
using the violet side scatter. Plasma samples from 17 patients with myocardial infarction
included in the REBUS study (at inclusion and at 1-year follow-up) and 3 controls
(in total 37 samples) were used and a significant correlation between the SP-PLA signal
and flow cytometric measurements of platelet-derived EVs was found (Pearson's correlation
[R ] = 0.63, p < 0.0001; [Fig. 5 ]).
Fig. 5 Correlation between SP-PLA and high-sensitivity flow cytometric measurements of platelet-derived
EVs. SP-PLA measurements of platelet-derived EVs in plasma from REBUS patients (relative
to EV-free plasma that was set to 1) were plotted against CD41 + AnnV+ EVs counted
by high-sensitivity flow cytometry using the CytoFLEX (Beckman Coulter) with the gate
set by polystyrene beads on 0.1–0.9 μm particles .Correlation was determined using
the Pearson product-moment correlation coefficient (R = 0.65, p < 0.0001, n = 34).
Assay for CT-B-Captured TF+ EVs Is Specific and Detects Extracellular Vesicles
To test if the method would work on a less abundant EV population, we applied the
SP-PLA method on TF+ EVs. For this purpose, we first captured the EVs with CT-B and
used a TF polyclonal antibody conjugated to PLA probes for detection. The TF+ EVs
were captured by CT-B in 5 µL plasma, and the total levels of EV TF (that can reflect
EV numbers and/or TF expression on EVs) were increased after stimulation of whole
blood with TRAP, LPS ([Fig. 6A ]), and ADP (data not shown). Treatment with 0.5% Triton X-100 for 10 minutes prior
to the SP-PLA ([Fig. 6A ]) and centrifugation of LPS plasma at 20,000 × g for 30 minutes ([Fig. 6B ]) both resulted in lower levels of TF+ CT-B+ EVs, indicating that the assay detected
EVs. To control for the specificity of antibodies, one of the detection antibodies
was exchanged for a mouse control IgG antibody. As demonstrated in [Fig. 6C ], the signal disappeared with this antibody pair which means that a specific detection
was recorded by our TF antibody pair. A 10-fold dilution series of plasma with buffer
furthermore decreased the SP-PLA signal proportionally ([Fig. 6D ]).
Fig. 6 Detection of TF+ EVs using SP-PLA and CT-B capture. (A ) The levels of TF+ EVs was measured with SP-PLA by using CT-B capture in unstimulated
plasma or in plasma from TRAP or LPS-stimulated whole blood with or without triton-X100
treatment. Levels relative to EV-free plasma are shown. (B ) The levels of TF+ EVs were measured with SP-PLA in plasma samples from unstimulated
and stimulated whole blood from two individuals before (white bars) and after (black
bars) centrifugation at 20.000 × g for 60 minutes. Levels relative to buffer alone are shown. Error bars represent standard
deviation from technical replicates (n = 2). (C ) Levels of TF+ EVs were measured by SP-PLA using the TF polyclonal goat antibody
conjugated to both PLA probes as usual (black bars) or by using the TF polyclonal
goat IgG (conjugated to probe 1) together with a control goat IgG (conjugated to probe
2) (gray bars). The levels relative to signal in buffer alone is shown. (D ) Levels of TF+ EVs were measured with SP-PLA using CT-B capture in plasma from LPS-stimulated
whole blood, diluted 1:2 in a dilution series. The plasma concentration is plotted
against linearized CT values. Error bars represent standard deviation of technical
replicates (n = 3).
Detection of PS+ TF+ EVs in Plasma
In contrast to CT-B, we were unable to capture TF+ EVs with lactadherin in 5 µL plasma
in the stimulated whole blood from healthy individuals. However, a spike-in of relipidated
TF could be detected in our EV-free plasma with the same efficiency as in buffer ([Fig. 7A ]). Upon TF spike-in, the SP-PLA for TF+ EVs, captured by lactadherin, had a detection
range of at least 4 logs and a LoD score of 0.5 pg/mL ([Fig. 7B ]). The intra-assay variability (mean %CV of technical triplicates at all the measured
concentrations) was 11%. Interassay variability (% CV of mean value) measured for
10 and 100 pg/mL in five independent experiments was 7 and 17%, respectively.
Fig. 7 Detection of PS+ TF+ EVs using SP-PLA and lactadherin capture. (A ) Relipidated TF was spiked into buffer or EV-free plasma and detected with SP-PLA
after lactadherin capture. Levels relative to control with EV-free plasma (set to
1) are shown. (B ) Relipidated TF was spiked at different concentrations in PLA buffer and TF+ EVs
were detected with SP-PLA after lactadherin capture. Error bars represent standard
deviation from technical replicates (n = 2). (C ) TF PCA of relipidated TF was measured using a direct activity assay after capture
directly in the buffer or after lactadherin capture.
TF PCA of Lactadherin-Captured TF+ EVs
The PCA of TF on the surface of EVs was determined using a direct activity assay measuring
the ability of EVs to convert FX to FXa in the presence of FVIIa.[43 ] The measured levels of relipidated TF were similar after capture with 1 μL lactadherin
T1 beads as if measured directly in the buffer ([Fig. 7B ]).
Discussion
In this report, we show that EVs can be accurately measured in only a few microliters
of plasma using a qPCR machine, standard equipment in most laboratories which is cheap
and easy to handle as compared with a flow cytometer. With PCR, very few templates
can be amplified and detected quantitatively using a real-time quantitative approach,
resulting in high sensitivity. Based on the SP-PLA technique, we have developed an
assay that specifically and sensitively detect platelet-derived EVs (CD41 + /CD61+
and PS + ) as well as TF+ EVs in small amounts of plasma. Treatment with detergent
or centrifugation of the samples depleted the SP-PLA signal, showing that the method
detects vesicular structures. In addition, by filtering the plasma with a 0.2-μm filter,
we completely removed the flow cytometry count using the Navios instrument from Beckman
Coulter, whereas the CD41/CD61 EVs were still detectable by SP-PLA. This indicates
that our method can detect vesicles smaller than 0.2 μm.
We also compared SP-PLA platelet EV detection using only 0.5 µL of plasma with the
new CytoFLEX system (Beckman Coulter), which can detect polystyrene beads as small
as 0.1 μm in diameter. We found that there was a significant correlation between the
two analysis methods (R = 0.6475; p ≤ 0.0001). The values tended to be higher with SP-PLA detection in some samples,
which may be explained by the fact that CytoFLEX measures EVs per microliter, whereas
SP-PLA measures total amount of antigen on all EVs. Samples with a higher degree of
large EVs with high expression of CD41/CD61 might thus increase the SP-PLA signal
without affecting the count in the CytoFLEX. As stated, with the SP-PLA also EVs below
even the detection limit of the CytoFLEX may be measured, which also could contribute
to the discrepancies in the measurements.
Measuring the total antigen levels on the EV surface (as we do with SP-PLA) and counting
EVs (as with flow cytometry) are two different approaches which provide different
information about the EVs and the methods could complement each other. Flow cytometry
can separate the subsets of EVs based on scattering properties and fluorophores counting
the number of EVs. The SP-PLA method measures the total EV surface antigen levels
in a sensitive manner and provides additional information to the amount of EVs found
by flow cytometry. Since the surface levels of adhesion molecules or tissue factor
are relevant for functionality such as activation of coagulation, the total antigen
levels might be clinically relevant for certain EV populations. For example, platelet-derived
EVs with high levels of adhesion molecules adhere to sites of thrombotic activity,
whereas erythrocyte-derived EVs, which lack adhesion receptors, are found in the plasma.[45 ] In this article, we have focused on the platelet-derived EVs as well as those carrying
tissue factor. However, there are populations of EVs from other cell types found in
plasma with different functions and the SP-PLA method can be further developed to
detect other EV populations, in particular those with small size and/or low antigen
levels that may be difficult to detect with flow cytometry. In addition to solving
the problem with measuring EVs in the full-size range, SP-PLA and other proximity-based
technologies such as 4 PLA, ExoPLA, and multiplex extension assays[14 ]
[33 ]
[46 ] offer several more advantages over traditional flow cytometry. As exemplified by
our plasma SP-PLA method, these new techniques offer higher specificity as three affinity
reagents are necessary for detection. PLA has also been shown to give higher sensitivity
than ELISA.[47 ] These are clear advantages for the analysis of small EVs and/or EVs with low levels
of surface protein. Multiplex analysis is also possible[46 ] enabling parallel analysis of several EV populations in a sample.
To capture EVs, we used two capture agents, lactadherin and CT-B. CD41 + /CD61+ EVs
(platelet-derived EVs) were found in the EV population that binds to lactadherin,
whereas TF+ EVs were primarily found in the CT-B-bound population in plasma from in
vitro stimulated blood donated from healthy individuals. Flow cytometry measurement
confirmed that CD41+ EVs are PS positive, and that CT-B and lactadherin, at least
to a large extent, measure different vesicle populations. This is in agreement with
a recent report investigating biomarkers for preeclampsia. The authors of this report
found that distinct vesicles were captured by annexin V or CT-B.[39 ] CT-B binds to ganglioside GM1 in the plasma membrane. GM1 is enriched in lipid rafts
and caveolae[48 ] which, among other things, appears to favor membrane blebbing and EV formation.[49 ] Release of TF+ EVs has also been associated with lipid rafts[50 ] and we found that TF+ ganglioside GM1+ EVs are released upon stimulation with ADP,
TRAP, and LPS. The TF+ EVs were also depleted by triton-X-100 treatment or centrifugation
and were detected only in 5 µL plasma using the SP-PLA method. LPS stimulation of
whole blood has previously been shown to upregulate TF in monocytes and TF PCA has
been detected on EVs in centrifuged plasma derived from LPS-stimulated whole blood.[44 ]
[51 ]
[52 ]
[53 ]
PS+ TF+ EVs are present at very low concentrations in plasma and initially we did
not find PS+ TF+ EVs using the SP-PLA analysis with lactadherin capture. However,
spike-in of relipidated TF was captured with lactadherin and detected with SP-PLA
generating a similar signal to background ratio in our EV-free plasma pool as in PLA
buffer, showing that the assay works well also for PS+ EVs. PS+ TF+ EVs were detected
down to concentrations of below 1 pg/mL, which is similar to a direct TF PCA assay.[43 ] By using a previously described direct TF PCA assay,[43 ] we also found that lactadherin-captured relipidated TF spike-in generated a similar
activity level as if activity was measured directly in the buffer, showing that the
capture of PS+ EVs is efficient.
The levels of TF+ EVs in plasma are usually low and flow cytometric analysis and ELISA
have been shown to correlate poorly with activity.[51 ] A possible reason for a discrepancy between levels of TF+ EVs and TF PCA might be
that TF exists both in an active and an encrypted conformation and only the former
is procoagulant.[54 ] Functional assays can be used to detect TF+ EVs, such as a direct TF PCA assay or
CAT assay.[43 ]
[44 ] Then only the active/procoagulant TF, which might be more clinically interesting,
is accounted for. However, the presence of PS− TF+ EVs has been reported previously[55 ]
[56 ] and monocytic EVs have been shown to fuse with activated platelets to initiate coagulation,[50 ] implying that EVs lacking PS can still become procoagulant under certain circumstances.
The measurement of TF PCA in plasma often includes a high-speed centrifugation step
or capture with annexin V (affinity which is Ca2+ dependent) to separate the EVs from plasma.[43 ]
[51 ]
[52 ] Using SP-PLA with lactadherin capture, we can detect the TF+ PS+ procoagulant EVs
sensitively and without a need for centrifugation or Ca2+ .
In summary, our novel SP-PLA method with lactadherin and CT-B as capture reagents,
using only 0.5 to 45 µL sample, can detect different types of EVs with high specificity
and sensitivity, providing information of the total antigen level and has the potential
to be an attractive complement method to flow cytometric analysis of preclinical and
clinical samples. With this SP-PLA, the smallest EVs can also be analyzed. Techniques
for measuring EVs have rapidly improved during the last years, and this development
provides hope that it will be possible to study EVs more efficiently and accurately
in the future, with the purpose to identify populations of EVs that might be valuable
for clinical assessment of various diseases.
