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VASCULAR BIOLOGY
Platelets can enhance vascular permeability
Nathalie Cloutier,1 Alexandre Paré,2 Richard W. Farndale,3 H. Ralph Schumacher,4 Peter A. Nigrovic,5 Steve Lacroix,2 and
Eric Boilard1
1Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de
l’Université Laval, Québec, QC; 2Centre de Recherche du Centre Hospitalier Universitaire de Québec and Department of Molecular Medicine, Université Laval,
Québec, QC; 3Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom; 4University of Pennsylvania and Veterans Administration
Medical Center, Philadelphia, PA; and 5Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Platelets survey blood vessels, searching
for endothelial damage and preventing
loss of vascular integrity. However, there
are circumstances where vascular permeability increases, suggesting that platelets sometimes fail to fulfill their expected
function. Human inflammatory arthritis is
associated with tissue edema attributed
to enhanced permeability of the synovial
microvasculature. Murine studies have
suggested that such vascular leak facilitates entry of autoantibodies and may
thereby promote joint inflammation.
Whereas platelets typically help to promote microvascular integrity, we examined the role of platelets in synovial vascular permeability in murine experimental
arthritis. Using an in vivo model of autoimmune arthritis, we confirmed the presence of endothelial gaps in inflamed synovium. Surprisingly, permeability in the
inflamed joints was abrogated if the platelets were absent. This effect was mediated by platelet serotonin accumulated
via the serotonin transporter and could
be antagonized using serotonin-specific
reuptake inhibitor antidepressants. As opposed to the conventional role of platelets to microvascular leakage, this demonstration that platelets are capable of
amplifying and maintaining permeability
adds to the rapidly growing list of unexpected functions for platelets. (Blood.
2012;120(6):1334-1343)
Introduction
Platelets patrol blood vessels seeking vascular injuries. When
damage to the endothelium is detected, platelets promptly aggregate and release mediators to prevent blood loss and promote
wound repair.1 In addition to their role in primary hemostasis,
platelets also protect the vasculature against leakage.2 Indeed,
although basal levels of vascular permeability is necessary to allow
the passage of small molecules, such as nutrients, ions, and water,
the ultrastructural changes to the endothelium that accompany
thrombocytopenia prompt vasculature leakage and can lead to
edema or uncontrolled bleedings.2-6 Further, induction of thrombocytopenia engenders hemorrhages localized at the site of inflammation during Arthus reaction, stroke, sunburn, and endotoxininduced lung inflammation,7,8 pointing to a critical role of platelets
in maintenance of blood vessels in inflammatory conditions.
Rheumatoid arthritis (RA) is the most common autoimmune
inflammatory disorder that affects the joints.9 The presence of
microvascular injuries in joints of patients with RA has long been
appreciated.10,11 Transcellular fenestrations and interstices between
endothelial cells, called gaps, are frequently observed in RA
synovium vasculature and contribute to the enhanced permeability
of the diseased joint.10,12-16 Importantly, the permeability that
preferentially arises in joints during experimental arthritis has been
proposed to contribute to the organ preference of autoimmune
inflammatory arthritis.17,18
During murine autoimmune arthritis, platelets are activated
through collagen receptor glycoprotein VI (GPVI) and release
platelet microparticles (MPs), submicron vesicles shed from plate-
Submitted February 22, 2012; accepted April 18, 2012. Prepublished online as
Blood First Edition paper, April 27, 2012; DOI 10.1182/blood-2012-02-413047.
There is an Inside Blood commentary on this article in this issue.
The online version of this article contains a data supplement.
1334
let surface.19 These MPs are enriched in the inflammatory cytokine
IL-1 and can exacerbate inflammation by stimulating resident
fibroblast and potentially other cells. Notwithstanding the role of
platelets in inflammation, the permeability that prevails in the joint
vasculature during arthritis evidences the inability of platelets to
preserve the vessel wall integrity during this disease. Herein, we
aimed to specifically scrutinize the role of platelets in the maintenance of the endothelial barrier function in arthritis disease while
the inflammation is in place. We observed that, rather than
preserving vascular integrity during arthritis, platelets enhance the
formation of interendothelial cell gaps in joints via serotonin.
Methods
Reagents and antibodies
Fluorescent Sky Blue and Nile Red polystyrene microspheres were
obtained from Spherotech. Various sizes were used: 0.04 to 0.09 ␮m (mean,
0.09 ␮m), 0.1 to 0.3 ␮m (mean, 0.22 ␮m), 0.4 to 0.6 ␮m (mean, 0.45 ␮m),
0.7 to 0.9 ␮m (mean, 0.84 ␮m), 2.5 to 4.5 ␮m (mean, 3.2 ␮m), and 10.0 to
14.0 ␮m (mean, 10.2 ␮m). Antibody preparation for mouse platelet depletion (a mixture of purified rat monoclonal antibodies directed against mouse
GPIb [CD42b]) and polyclonal nonimmune rat immunoglobulins were
obtained from Emfret Analytics. Purified Rat Anti-Mouse Ly-6G and Ly-6C
and Purified Rat IgG2b (Isotype Control) were obtained from BD Biosciences. The cross-linked form of collagen-related peptide (CRP, Gly-CysHyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly) was generated as described.20
Serotonin hydrochloride was obtained from Sigma-Aldrich. Mouse recombinant
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by The American Society of Hematology
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
IL-1␤ was obtained from Cedarlane. Dextran-fluorescein 40 000 molecular
weight was obtained from Life Technologies.
Human SF
Human knee synovial fluids (SFs) were obtained as discarded material from
patients with various arthritides undergoing diagnostic or therapeutic
arthrocentesis. Arthritis diagnosis was ascertained by an American Board of
Internal Medicine–certified rheumatologist and/or by review of laboratory,
radiologic, and clinic notes and by applying ACR classification criteria. All
studies received Laval University Institutional Review Board approval.
PLATELETS CAN ENHANCE VASCULAR PERMEABILITY
1335
platelet-rich plasma was thereafter centrifuged for 5 minutes at 1300g to
pellet platelets. Platelets were lysed by addition of buffer (20mM Tris HCl,
pH 7.8, 1.25mM EDTA, 120mM NaCl, 0.5% NP40, 0.5% Triton; Complete
Protease inhibitor, Roche; PhosSTOP Phosphatase inhibitor, Roche; and
0.5mM PMSF). The serotonin content in platelet lysates and in mouse
whole blood was measured using serotonin ELISA Kit (Fitzgerald Industries International). The serotonin content in SF of human patients affected
by RA and osteoarthritis (OA) was measured using serotonin (ultrasensitive) ELISA Kit (Fitzgerald Industries International).
Fluorescence imaging
Mice
We used 6- to 9-week-old mice for all of our studies. Guidelines of the
Canadian Council on Animal Care were followed in a protocol approved by
the Animal Welfare Committee at Laval University. C57BL/6J, Slc6a4
(solute carrier family 6 member 4; also called serotonin transporter [SERT])
null mice (backcrossed to C57BL/6J ⬎ 10 times) and KitW-sh mutant mice
(87% C57BL/6J genetic background) and their age- and sex-matched
controls were obtained from The Jackson Laboratory. BALB/c mice were
obtained from Charles River. LysM-eGFP breeders were obtained from Dr
Gregory Dekaban (Robarts Institute, London, ON), with authorization from
Dr Thomas Graf (Barcelona, Spain). LysM-eGFP mice showed specific
green fluorescence in macrophages and neutrophil granulocytes.21 When
microspheres were used, 1 ␮g of the microspheres was injected intravenously (100 ␮L/mice). Platelet depletion was achieved by injecting 4 ␮g/g
of depleting antibody or matched isotype antibody to mice as described by
the manufacturer (Emfret). These antibodies were administrated one hour
before K/BxN injection and at day 3 for arthritis experiments or 18 hours
before K/BxN injection in flare experiments. When mouse recombinant
IL-1␤ was administrated, it was injected intraperitoneally in mice receiving
the platelet-depleting antibody 4 hours before K/BxN injection and on day 1
and 2 (2 ␮g per injection). Gr1 cell depletion was achieved by intraperitoneal injection of 100 ␮g isotype control or Ly-6G and Ly-6C antibody to
arthritic mice in combination with 2.5 ␮g IL-1␤ at day 7 after K/BxN
injection for arthritis experiments or 18 hours before K/BxN injection for
flare experiments. Efficient Gr1 cell depletion was confirmed by leukocyte
counts in experimental animals. For the GPVI stimulation in vivo, CRP-XL
(40 ␮g/kg) was injected intravenously 30 minutes before microsphere injection.
Serotonin was dissolved in normal sterile saline and was administrated at
20 mg/kg intravenously 5 minutes before microsphere injection. Dextranfluorescein was dissolved in normal saline and was administrated at 40 mg/kg
intravenously 2 minutes before the microsphere injection.
Serum transfer protocol and arthritis scoring
Arthritogenic K/BxN serum was transferred to recipient C57BL/6J, LysMeGFP, and BALB/c mice via intraperitoneal injection (125, 125, and 75 ␮L
K/BxN serum, respectively) on experimental day 0 and day 2 to induce
arthritis as described.19 Ankle thickness was measured at the malleoli with
the ankle in a fully flexed position, using a spring-loaded dial caliper (DGI
Supply). For flare experiments, the K/BxN serum was transferred to
recipient mice via intravenous injections (100 ␮L/mouse).
Pharmacologic inhibition of SERT
When fluoxetine was used in vivo, the drug was dissolved at 160 mg/L in
drinking water and changed once per week for 3 weeks before the
administration of K/BxN serum. When bolus administration of fluoxetine
was used, the drug was administrated by intraperitoneal injection (2.5 mg/
kg) in arthritic mice one hour before the microsphere injection.
Mice were anesthetized with 5% isoflurane in O2 and placed inside an in
vivo imaging system Xenogen IVIS200 (Xenogen) 2 to 45 minutes after the
injection of microspheres. The excitation filter was 615 to 665 nm, the
emission filter was 695 to 770 nm, and the acquisition time was 2 seconds.
The images were analyzed using Living Image Version 4.0 software. A
Region of Interest tool was used to measure the radiant efficiency from each
ankle and compared with control ankles in conditions where the highest
specific signal-to-background ratio was achieved. The radiant efficiency
was also examined in externalized harvested heart, kidney, liver, spleen, and
lung in all animal groups.
Electron microscopy analysis
RA patients underwent a Parker-Pearson needle biopsy of the synovium.
Specimens were immediately placed into half-strength Karnovsky
paraformaldehyde-glutaraldehyde fixative for electron microscopy. Specimens were postfixed in cold Palade osmium-veronal and embedded in
epoxy resin. Thick sections were obtained, stained with uranyl acetate and
lead citrate, and examined on a Zeiss EM 10 electron microscope under a
50-kV beam.
Multiphoton microscopy
Arthritic and nonarthritic control LysM-eGFP mice were anesthetized with
isoflurane and immobilized in the supine position, and a midline incision
was performed along the malleolus of the ankle. A 6-0 silk suture (Ethicon)
was used to gently spread the skin to maximize the imaging area. The whole
setup was placed on the stage underneath the objective, and a 0.9% saline
solution was applied over the region to be imaged. Dextran conjugated with
fluorescein and Nile Red polystyrene microspheres (size 0.4-0.6 ␮m) were
injected 5 minutes before the beginning of the imaging session to visualize
blood vessels and blood vessel permeability, respectively. Body temperature was recorded through a rectal probe connected to a temperature
controlling device (RWD Life Science Co), and the temperature was
maintained at 37°C during all procedures.
All images were acquired on an Olympus FV1000 MPE 2-photon
microscope dedicated to intravital imaging. The 2-photon Mai Tai DeepSee laser
(Spectra-Physics, Newport Corp) was tuned at 950 nm for all experiments.
Tissues were imaged using an Olympus Ultra 25x MPE water immersion
objective (1.05 NA), with filter set bandwidths optimized for CFP (460-500 nm),
YFP (520-560 nm), and Texas Red/DsRed (575-630 nm) imaging. Detector
sensitivity and gain were set to achieve the optimal dynamic range of
detection. Using Olympus Fluoview Version 3.0a software, images with a
resolution of 256 ⫻ 256 pixels were acquired at different zoom factors
(2-4⫻) at 2.5 frames per second with auto-HV option enabled, and exported
as 24-bit RGB TIF files, whereas metadata for subsequent automatic
setting-detection were exported in a TXT file. No Kalman filter was used to
avoid slowing down the acquisition speed.
Determination of serotonin content
Mouse blood was drawn by cardiac puncture using acid citrate dextrose
anticoagulant (0.085M sodium citrate, 0.0702M citric acid, 0.111M dextrose, pH 4.5). Blood was diluted by addition of 300 ␮L Tyrode buffer, pH
6.5 (134mM NaCl, 2.9mM KCl, 0.34mM Na2HPO4, 12mM NaHCO3,
20mM HEPES, 1mM MgCl2, 5mM glucose, 0.5 mg/mL BSA) and centrifuged at 600g for 3 minutes. The platelet-rich plasma was further
centrifuged for 2 minutes at 400g to pellet contaminating RBCs. The
Statistical analysis
Results of mouse arthritis experiments are presented as mean plus or minus
SEM. The statistical significance for comparisons between groups was
determined using 2-way ANOVA, followed by Bonferroni correction.
Statistical significance for experiments using radiant efficiency and ELISA
was determined using unpaired Student t test. All statistical analysis was
done using Prism software package Version 4.00 (GraphPad Software).
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1336
CLOUTIER et al
Figure 1. Gaps are present between endothelial cells in the synovium vasculature from patients with RA. Synovium from RA patients was scrutinized by electron
microscopy. Black arrows indicate one gap between endothelial cells. E indicates
endothelial cells; PL, platelets; L, lymphocytes; and RBC, red blood cells. Original
magnification ⫻48 400.
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
platelets to the prevention of endothelial gap formation during
arthritis using thrombocytopenic mice. We found a complete
abrogation of gaps in the absence of platelets (Figure 3A). Given
that platelet MPs can provide IL-1 and so exacerbate arthritis,19 this
reduction in gap production could have resulted from an overall
diminution of inflammation (Figure 3B). However, in opposition to
the striking blockade of vascular leakage, the arthritis severity in
absence of platelets was only partially reduced, pointing to the
uncoupling of inflammation and permeability. To maintain inflammation despite platelet depletion, we thus injected recombinant
IL-1␤ concurrently to the K/BxN serum and monitored gap
formation.23,24 Surprisingly, we observed that the permeability in
joints remained suppressed in mice lacking platelets, even in
experimental conditions where severe inflammation is maintained
(Figure 3C-D).
Platelet-derived serotonin triggers gap formation
Results
Vascular permeability occurs in joints during arthritis
For this study, we aimed to examine platelets in a context where
increased vasculature permeability and inflammation are present.
Gaps are found between endothelial cells in the synovial vasculature from patients with RA,10 and our electron microscopic
analyses of synovial tissues from these patients also reveal platelets
in the vicinity of these gaps10 (Figure 1). Thus, autoimmune
inflammatory arthritis appeared to us as an ideal condition to
investigate the role of platelets in the context of enhanced
vasculature permeability.
We first confirmed the presence of vasculature leakage in joints
during active arthritis. We used the K/BxN serum transfer model of
inflammatory arthritis for our studies. In this model, arthritis can be
induced by passive transfer of autoantibody-containing serum from
K/BxN mice into wild-type mice.22 To visualize the formation of
interendothelial cell gaps and to obtain an estimate of their width
during arthritis in vivo, we injected fluorescent microspheres of
various diameters (0.09-10.2 ␮m) intravenously into control mice
and mice at the peak of arthritis disease (day 7 after K/BxN serum
injection) and monitored their localization using in vivo imaging.
Although none of the microspheres could reach the joints of control
nonarthritic mice, microspheres of diameter 0.84 ␮m, 0.45 ␮m
(Figure 2A), 0.22 ␮m and 0.09 ␮m (supplemental Figure 1,
available on the Blood Web site; see the Supplemental Materials
link at the top of the online article) promptly and preferentially
localized to arthritic joints (supplemental Figure 2A). Entry was
direct and not via attachment to migrating neutrophils (supplemental Figure 2B-C). Using intravital 2-photon microscopy, we further
validated the microsphere egress from blood vessels and their
concomitant accumulation within the collagen mesh where the
inflammation culminated (Figure 2B-C; full resolution video
available at http://bloodjournal.hematologylibrary.org/content/early/
2012/04/27/blood-2012-02-413047/suppl/DC1). Importantly, those
microspheres having diameters of 3.2 ␮m and 10.2 ␮m failed to
reach the arthritic joints (Figure 2A), showing that gaps in joint
vasculature during arthritis enable passive influx only of particles
of submicron dimension.
Platelets promote vascular permeability in inflamed
vasculature
Because one function of platelets is to maintain microvascular
integrity, platelet depletion would be expected to exacerbate
vascular permeability. We therefore evaluated the contribution of
Platelets play a crucial role in hemostasis by preserving the
integrity of the vasculature.2 Therefore, how could the platelets
contribute to the increased joint permeability during arthritis?
Given its abundance in platelets, serotonin (5-hydroxytryptamine)
was initially detected in blood,25 and studies have examined its
function(s) in vascular permeability26,27 and joint edema.17 A
pioneering electron microscopic investigation showed that local
injection of serotonin in the cremaster muscle induced the formation of gaps, 0.1 to 0.8 ␮m in width, between endothelial cells.26
Interestingly, the authors frequently observed the presence of
platelets, sometimes bridging these gap, sometimes plugging them,
and also noticed disintegrating platelets near these gaps.26 Given
the striking similarities between the gaps observed in RA synovium10 (Figure 1A) and those observed after serotonin injection,26
we hypothesized that platelets may promote the gap formation via
serotonin.
We thus sought to determine the presence of serotonin in joint
fluids where platelets are present. In contrast to the SF of patients
with OA, the SF of patients with RA contains platelet MPs,19 a
hallmark of platelet activation. Consistent with presence of MPs in
RA SF, we measured an average of 10.3 ng/mL serotonin in SF
from RA patients (Figure 4A). Interestingly, lower levels of
serotonin were measured in the SF of patients with OA (Figure 4A).
We next evaluated whether serotonin could promote the formation
of gaps in mouse joints. We found that a single systemic injection
of serotonin to nonarthritic mice sufficed to promote formation of
gaps in joint vasculature (Figure 4B), independently of neutrophils
(supplemental Figure 2D).
Although the enterochromaffin cells in the gastrointestinal tract
are the main biosynthetic source of serotonin,28 the expression of
SERT enables platelets to store serotonin.29 We thus validated the
role of platelets in serotonin transport and used neutrophil-depleted
mice and mast cell-deficient mice (KitW-sh) for comparison. We find
that, in contrast to blood from mast cell-deficient mice and
neutrophil-depleted mice, blood from platelet-depleted mice contained no detectable serotonin (Figure 5A). Further, although the
production of serotonin by the intestine is intact in SERT deficient
mice (Slc6a4⫺/⫺),30 the platelets isolated from these mice contained
only minimal serotonin levels (Figure 5B). Thus, compatible with
the existing literature on platelet serotonin,29 our results confirm
that platelets require the expression of SERT to operate as
serotonin’s main vehicle in blood.
We therefore explored the contribution of SERT to gap formation during arthritis using Slc6a4 ⫺/⫺ mice. Interestingly, we found
that these mice exhibited a profound defect in gap formation and a
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
PLATELETS CAN ENHANCE VASCULAR PERMEABILITY
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Figure 2. Presence of vasculature permeability during autoimmune arthritis in vivo. (A) Nonarthritic control (left) and arthritic (right) mice (day 7 after K/BxN
serum injection) were injected intravenously with 0.45-,
0.84-, 3.2-, or 10.2-␮m-diameter microspheres and visualized 5 minutes later using a Xenogen IVIS in vivo
imaging system. Control and arthritic mice received the
same concentration of fluorescent microspheres. Radiant efficiency quantifications in the ankle joints for control
and arthritic mice for all microsphere sizes are presented
at right. (B-C) In vivo 2-photon imaging of the vasculature
permeability in the arthritic ankle joint. LysM-eGFP arthritic mice injected intravenously with dextran-fluorescein
and 0.45 ␮m Nile-Red-conjugated microspheres, and
ankle joints were imaged to visualize the microsphere
egress from the blood circulation and accumulation in the
subendothelial collagen-rich matrix. (B) Two-photon images from time-lapse recordings taken at 0, 120, and
444 seconds demonstrating a microsphere leaving the
blood circulation independently of transportation by a
neutrophil or a monocyte. (C) Representative image
evidencing the accumulation of microspheres outside the
vasculature in an arthritic joint. Red represents microspheres; green, blood vessels; cyan blue, leukocytes; and
Indigo blue, collagen (second-harmonic generation). Scale
bar represents 25 ␮m (panel B), and 50 ␮m (panel C).
consistent, yet modest, reduction in arthritis severity (Figure
5C-D). Serotonin modulates a diversity of neuropsychologic
processes, and there exist a variety of drugs used in psychiatry that
target its uptake.31 Mice pretreated with the antidepressant SERT
inhibitor fluoxetine exhibited a significant decrease in gap formation in the course of arthritis (Figure 5E), compatible with the
reduced serotonin levels in platelets from these mice (Figure 5F).
Consistent with platelet half-life (4-5 days) and their constant
storage of serotonin, a single administration of fluoxetine was not
sufficient to affect the production of gaps in arthritic mice and the
serotonin level in platelets (supplemental Figure 3A-B). Taken
together, these results point to a role of platelet serotonin in the
formation of gaps during arthritis.
GPVI is a platelet collagen receptor, and its stimulation is
reported to promote release of serotonin and MPs from platelets.19,32 We aimed to determine whether GPVI activation could
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1338
CLOUTIER et al
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
Figure 3. The presence of gaps in the inflamed arthritic joint vasculature depends on platelets. (A) The platelet-depleting antibody or the isotype control antibody was
injected to mice on day 0 and day 3. Arthritis was induced by injection of 75 ␮L of K/BxN serum on day 0 and day 2. On day 4, the 0.45-␮m microspheres were intravenously
injected in platelet (PLT)–depleted mice and control mice (with PLT), and the fluorescence was measured in ankle joints 5 minutes later. Nonarthritic mice were used as
controls. Representative results are presented. ***P ⬍ .0001. (B) The signs of arthritis were monitored daily and presented as mean ⫾ SEM. Arrow indicates parenteral
administration of platelet-depleting antibody or isotypic control; and arrowheads, K/BxN serum administration. (C) Antibody injection, arthritis induction, microsphere injection,
fluorescence measurements, controls, and results presentation were performed as in panel A. Mouse recombinant IL-1␤ was injected on days 0, 1, and 2 in mice receiving the
platelet-depleting antibody. ***P ⫽ .0002 (top). ***P ⫽ .0001 (bottom). (D) The signs of arthritis were monitored as in panel B. Arrow indicates parenteral administration of
platelet-depleting antibody or isotypic control; arrowheads, K/BxN serum administration; and double arrow, IL-1␤ administration.
promote vasculature leakage in mouse distal joints. We therefore
stimulated GPVI by intravenously injecting the specific GPVI
agonist, the CRP-XL,33 to mice and monitored the joint localization
of the microspheres. We found that this in vivo stimulation of
platelet GPVI sufficed to trigger the gap formation in joints (Figure
6). Importantly, no gaps were produced when the GPVI stimulation
was carried out in mice treated with fluoxetine (Figure 6),
demonstrating that platelet GPVI stimulation induces the formation
of gaps in joints via release of serotonin.
Discussion
Maintenance of vasculature integrity and prevention of fluid
leakage in interstitial tissue are a defined function of circulating
platelets. To our surprise, we revealed a counterintuitive role for
platelets in arthritis, namely, that platelets are the major cause of
joint vasculature permeability via serotonin release, potentially
serving to promote disease.17,18
Herein, we aimed to study the role of platelets in leakage of the
inflamed vasculature. Despite the absence of detectable vasculature
permeability in joints of IL-1␤–injected thrombocytopenic mice
and SERT-deficient mice, the arthritis persisted in these animals, as
measured via evaluation of the ankle thickness (an indication of
inflammation and tissue remodeling). This suggests that the
endothelial gaps in the joint vasculature may not be a prerequisite
for progression to inflammation where the concentrations of
inflammatory cytokines are excessively elevated. Indeed, neutrophils are capable of migrating through the endothelial barrier in the
absence of permeability,34 and many effector cells, such as synovial
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
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Figure 4. Serotonin is present in the SF of patients with RA
and can promote the gap formation in joint vasculature.
(A) Serotonin concentrations were measured in SF of RA and OA
patients using an ELISA serotonin kit (n ⫽ 27). **P ⫽ .0012.
(B) Serotonin suffices to promote prompt formation of gaps in
joints. Diluent (PBS) or serotonin (20 mg/kg) was injected intravenously in mice before administration of 0.45-␮m-diameter fluorescent microspheres. The fluorescence was portrayed using an in
vivo imaging system 45 minutes after the microsphere injection
(left). The radiant efficiency quantifications in the ankle joints for
control (diluent) and serotonin injections are presented on the
right. ***P ⬍ .0001; *P ⫽ .024.
fibroblasts35 and mast cells,23,36 are already localized in the joints.
We thus exploited this conditional independence of inflammation
and vasculature permeability to delineate the role of platelets in
vascular leakage while severe inflammation is present. We revealed
that platelets could induce vascular leak via serotonin, a process
inhibited by serotonin-specific reuptake inhibitor antidepressants.
The critical role of platelets in vasculature leakage may not be
unique to inflammatory arthritis. In models of lung inflammation
induced by acid or abdominal sepsis, platelet depletion prevents
both inflammation and vascular leakage,37-39 pointing to a potential
role of platelets in vascular permeability during lung inflammation.
However, it remains unclear whether leakage occurring in lungs is
mediated directly by a platelet-derived mediator or via the overall
enhancement of inflammation by platelets. Importantly, we identified serotonin as a platelet-derived mediator able to initiate the
formation of gaps in the vasculature during inflammation. In
addition to its function in the joint vasculature, serotonin also
promotes leakage of endothelial cells in culture and of the
vasculature in vivo when locally injected in the cremaster muscle,
the footpad, or the cerebral ventricular system,26,40-45 suggesting
that serotonin may indeed initiate the formation of gaps between
endothelial cells elsewhere, not solely in the synovial vasculature
during arthritis.
How did platelets evolve to enhance vasculature permeability?
There is a growing appreciation of the immune functions of
platelets.46 In contrast to leukocytes, platelets cannot actively exit
the vasculature to fight an infection. Our work described herein
demonstrates the capability of platelets to open in the vasculature
gaps of dimensions compatible with the extravasation of platelet
MPs.47 Lacking any notable migratory activity, a platelet could
open the endothelial wall and infuse its mediators and MP cargo
through the gaps. However, the gaps formed in joint vasculature
during autoimmune arthritis may be detrimental to the joint. Like
the IL-1–rich platelet MPs,19 immune complexes, the etiologic
agent in RA, are also of submicron dimensions, with diameters
varying between 0.1 ␮m and approximately 1 ␮m.47 It is thus
plausible that the gaps produced by platelets contribute to joint
invasion by both immune complexes and MPs, supporting a dual
contribution to joint inflammation.
Thereby, our demonstration that serotonin uptake by platelets is
a prerequisite for the promotion of joint permeability implies that
medications used to treat depression by targeting SERT may also
target vasculature permeability in RA disease. A clinical observation supporting this notion in exists,48 although whether the drug
regimen followed by the arthritic, depressed patient suffices to
efficiently deplete serotonin from platelets and impact the severity
of arthritis remains to be explored.
An earlier report showed that the rapid microvasculature
permeability, also called flare, which occurs immediately (within
minutes) after K/BxN serum injection and goes on approximately
7 minutes, relies on neutrophils and mast cells.17,49 Our findings are
complementary to this report. Indeed, we found that this initial
event is independent of the presence of platelets and expression of
SERT (supplemental Figure 4A-B). Because mast cells also require
the expression of SERT to store serotonin,30 our observations that
SERT is dispensable during the flare rule out the contribution of
mast cell-derived serotonin to the initial gap production that occurs
rapidly after K/BxN serum injection. We also examined the role of
mast cells in gap formation in the course of arthritis disease (day
7 after K/BxN serum injection). We found that the mast celldeficient mice showed no defect in gap formation during arthritis
(supplemental Figure 5), precluding an obligate function for mast
cell SERT in gap production. These results indicate that platelets,
not mast cells, fulfill a role in the continuous vasculature permeability that occurs once the disease has been well established (Figure 7,
schematic representation).
The mechanism(s) by which platelet activation can either
prevent or promote permeability of the vasculature is intriguing:
(1) vasculature composition, (2) the different mediators present in
situ, and (3) the platelet receptor(s) engaged may govern whether
platelets play anti- or pro-permeability roles. Indeed, plateletneutrophil and platelet-endothelial cell interactions are suggested
to participate to leakage of vasculature in lungs and brain.37-39,50-52
In addition, the 13 different receptor subtypes for serotonin are
capable of mediating distinct functions.53 Thus, the cell combination present in the vasculature and a unique pattern of expression of
the various serotonin receptors might conceivably result in prevention versus promotion of vascular permeability. Further, the local
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1340
CLOUTIER et al
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
Figure 5. Mice deficient in platelet-derived serotonin have reduced gap formation. (A) Serotonin concentrations were measured in whole blood from mast cell-deficient
mice (KitW-sh), neutrophil-depleted mice, platelet-depleted mice, and their corresponding control mice using an ELISA serotonin kit. (B) Serotonin concentration was measured
in platelet isolated from WT and Slc6a4⫺/⫺ mice. (C) WT and Slc6a4⫺/⫺ arthritic mice were injected intravenously with 0.45-␮m-diameter fluorescent microsphere, and the
fluorescence was visualized 5 minutes later using an in vivo imaging system. Control mice (WT nonarthritic) and WT and Slc6a4⫺/⫺ arthritic mice all received the same
concentration of fluorescent microspheres. The radiant efficiency quantifications in the ankle joints for all mice are presented, and representative results are presented at right.
***P ⫽ .004. (D) Mice (17/group) sufficient and deficient in SERT expression were injected with 125 ␮L K/BxN serum at day 0 and day 2 and the signs of arthritis monitored
daily. The mouse arthritis experiment is presented as mean ⫾ SEM. P ⬍ .01. (E) SERT pharmacologic inhibition was performed in C57BL/6J mice by treating the mice with
fluoxetine for 21 days before administration of K/BxN serum. Arthritic (treated or not with fluoxetine) and control (nonarthritic) mice were injected intravenously with
0.45-␮m-diameter fluorescent microsphere, and the fluorescence was visualized 5 minutes later using an in vivo imaging system. The radiant efficiency quantifications in the
ankle joints for all mice are presented, and representative results are presented at right. **P ⫽ .0092. (F) Serotonin concentrations in platelets isolated from mice treated or not
with fluoxetine were measured by ELISA.
concentrations of mediators, such as platelet-activating factor,
sphingosine-1-phosphate, thromboxane A2, and prostacyclin, to
name only a few, could influence platelet function and vascular
leakage.37,50,54-61 Thromboxane A2 and prostacyclin in particular
are counterbalancing mediators, the former stimulating platelet
aggregation and the latter inhibiting it.58-60 Prostacyclin is present
in mouse and human arthritic joints,62,63 and we have demonstrated
that platelets produce prostacyclin with synoviocytes using a
transcellular mechanism during arthritis.19,62,64 The balance between such mediators may thus impact platelet functions and guide
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BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
PLATELETS CAN ENHANCE VASCULAR PERMEABILITY
1341
stimulus may sort particular granules, enriched with propermeability rather than anti-permeability factors. Indeed, the
growing appreciation of platelet’s capability to make available
certain type of granule’s mediators under different conditions is in
agreement with this hypothesis.65,66
In conclusion, we revealed an unpredicted role for platelets in
vasculature during inflammatory arthritis. Platelet serotonin coordinates the formation of gaps between endothelial cells in the joint
microvasculature, of dimensions compatible with those of platelet
MPs and immune complexes, and with their extrusion into the
synovial space. The permeability created by the platelet-derived
serotonin in the joint vasculature may thus contribute to the joint
inflammation induced by MPs and to the joint-preference of
arthritis disease mediated by immune complexes. In contrast to its
accepted role in the maintenance of vascular integrity, the capacity
of the platelet to contribute to permeability in vasculature, demonstrated here, adds to the rapidly growing list of unexpected
functions for platelets.
Acknowledgments
Figure 6. GPVI activation induces the formation of gaps in joints via release of
serotonin. Nonarthritic mice (treated or not with fluoxetine for 21 days) were injected
intravenously with CRP (40 ␮g/kg) or its diluent PBS. Thirty minutes after the CRP
injection, the 0.45-␮m fluorescent microspheres were injected. Two minutes after the
microsphere injections, the fluorescence in ankle joints was evaluated using an in
vivo imaging system. The radiant efficiency quantifications in the ankle joints for all
mice and representative results are presented. ***P ⬍ .0001; **P ⫽ .0019.
the fate of the vasculature: its leakage or its preservation. Finally,
arthritis is dependent on platelet GPVI activation and independent
of GPIb.19 Platelets in the inflamed joint stimulated by this unique
Figure 7. Proposed pathway for the formation of gaps
and amplification of the vasculature permeability by
platelets during arthritis. Top panel: Gaps between
endothelial cells in arthritic joints are formed. The GPVIexpressing platelets are activated by the newly exposed
subendothelial matrix rich in GPVI ligands, such as
collagen and laminin. Bottom panel: Stimulated platelets
produce copious amounts of IL-1–rich microparticles and
release serotonin. Platelet-derived serotonin promotes
the production of additional gaps; thus, more platelets
can be activated. Serotonin at the site of inflammation
promotes the vasculature leakage during disease. Note
that the precise anatomic location of platelet activation
and the route by which microparticles enter the joint
remain speculative. Πrepresents serotonin; and E,
platelet microparticle. Illustration courtesy of Steve Moskowitz, Advanced Medical Graphics.
The authors thank Dr Jasna Kriz (CRCHUQ, Université Laval) for
the generous access to the Xenogen in vivo imaging system and Dr
Denis Soulet (Assistant Professor, Département de Psychiatrie et
Neurosciences de l’Université Laval) for his assistance with the
cover image.
This work was supported by the Canadian Institutes of Health
Research, the Arthritis Society, the Canadian Arthritis Network,
and the Fonds de la recherche en santé du Québec (all E.B.), the
British Heart Foundation (R.W.F.), and the Cogan Family Foundation (P.A.N.).
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1342
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
CLOUTIER et al
Authorship
Contribution: N.C. conceived and designed the study, acquired,
analyzed, and interpreted data, performed statistical analyses, and
prepared the manuscript; A.P. and H.R.S. acquired, analyzed, and
interpreted data and prepared the manuscript; R.W.F. generated
critical reagents and prepared the manuscript; P.A.N. contributed
critical reagents, analyzed and interpreted data, and prepared the
manuscript; S.L. analyzed and interpreted data and prepared the
manuscript; and E.B. conceived and designed the study, acquired,
analyzed, and interpreted data, and prepared the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Eric Boilard, Centre de Recherche en Rhumatologie et Immunologie, Centre de recherche du Centre hospitalier
universitaire de Québec, 2705 Laurier Boulevard, Room T1-49,
Québec, QC, G1V 4G2, Canada; e-mail: [email protected].
References
1. George JN. Platelets. Lancet. 2000;355(9214):
1531-1539.
2. Ho-Tin-Noé B, Demers M, Wagner DD. How
platelets safeguard vascular integrity. J Thromb
Haemost. 2011;9(Suppl 1):56-65.
3. Kitchens CS, Pendergast JF. Human thrombocytopenia is associated with structural abnormalities of the endothelium that are ameliorated by
glucocorticosteroid administration. Blood. 1986;
67(1):203-206.
4. Kitchens CS, Weiss L. Ultrastructural changes of
endothelium associated with thrombocytopenia.
Blood. 1975;46(4):567-578.
5. Jackson DP, Sorensen DK, Cronkite EP, Bond VP,
Fliedner TM. Effectiveness of transfusions of fresh
and lyophilized platelets in controlling bleeding due to
thrombocytopenia. J Clin Invest. 1959;38:16891697.
6. Danielli JF. Capillary permeability and oedema in
the perfused frog. J Physiol. 1940;98(1):109-129.
7. Goerge T, Ho-Tin-Noe B, Carbo C, et al. Inflammation induces hemorrhage in thrombocytopenia.
Blood. 2008;111(10):4958-4964.
8. Carbo C, del Conde I, Duerschmied D. Petechial
bleeding after sunburn in a patient with mild
thrombocytopenia. Am J Hematol. 2009;84(8):
523.
9. Firestein GS. Evolving concepts of rheumatoid
arthritis. Nature. 2003;423(6937):356-361.
10. Schumacher HR Jr. Synovial membrane and fluid
morphologic alterations in early rheumatoid arthritis: microvascular injury and virus-like particles.
Ann N Y Acad Sci. 1975;256:39-64.
11. Kulka JP, Bocking D, Ropes MW, Bauer W. Early
joint lesions of rheumatoid arthritis: report of eight
cases, with knee biopsies of lesions of less than
one year’s duration. AMA Arch Pathol. 1955;
59(2):129-150.
12. Schumacher HR. The microvasculature of the
synovial membrane of the monkey: ultrastructural
studies. Arthritis Rheum. 1969;12(4):387-404.
13. Brown DL, Cooper AG, Bluestone R. Exchange of
IgM and albumin between plasma and synovial
fluid in rheumatoid arthritis. Ann Rheum Dis.
1969;28(6):644-651.
14. Schumacher HR. Fate of particulate material arriving at the synovium via the circulation: an ultrastructural study. Ann Rheum Dis. 1973;32(3):212218.
15. Levick JR. Permeability of rheumatoid and normal human synovium to specific plasma proteins.
Arthritis Rheum. 1981;24(12):1550-1560.
16. Simkin PA, Bassett JE. Pathways of microvascular permeability in the synovium of normal and
diseased human knees. J Rheumatol. 2011;
38(12):2635-2642.
17. Binstadt BA, Patel PR, Alencar H, et al. Particularities of the vasculature can promote the organ
specificity of autoimmune attack. Nat Immunol.
2006;7(3):284-292.
18. Wipke BT, Wang Z, Kim J, McCarthy TJ, Allen PM.
Dynamic visualization of a joint-specific autoimmune
response through positron emission tomography.
Nat Immunol. 2002;3(4):366-372.
19. Boilard E, Nigrovic PA, Larabee K, et al. Platelets
amplify inflammation in arthritis via collagendependent microparticle production. Science.
2010;327(5965):580-583.
20. Knight CG, Morton LF, Onley DJ, et al. Collagenplatelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen. Cardiovasc Res. 1999;41(2):
450-457.
21. Faust N, Varas F, Kelly LM, Heck S, Graf T. Insertion of enhanced green fluorescent protein into
the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood.
2000;96(2):719-726.
35. Lee DM, Kiener HP, Agarwal SK, et al.
Cadherin-11 in synovial lining formation and pathology in arthritis. Science. 2007;315(5814):
1006-1010.
36. Lee DM, Friend DS, Gurish MF, Benoist C, Mathis D,
Brenner MB. Mast cells: a cellular link between autoantibodies and inflammatory arthritis. Science. 2002;
297(5587):1689-1692.
37. Zarbock A, Singbartl K, Ley K. Complete reversal
of acid-induced acute lung injury by blocking of
platelet-neutrophil aggregation. J Clin Invest.
2006;116(12):3211-3219.
22. Kyburz D, Corr M. The KRN mouse model of inflammatory arthritis. Springer Semin Immunopathol. 2003;25(1):79-90.
38. Asaduzzaman M, Lavasani S, Rahman M, et al.
Platelets support pulmonary recruitment of neutrophils in abdominal sepsis. Crit Care Med.
2009;37(4):1389-1396.
23. Nigrovic PA, Binstadt BA, Monach PA, et al. Mast
cells contribute to initiation of autoantibodymediated arthritis via IL-1. Proc Natl Acad Sci
U S A. 2007;104(7):2325-2330.
39. Zarbock A, Polanowska-Grabowska RK, Ley K.
Platelet-neutrophil-interactions: linking hemostasis and inflammation. Blood Rev. 2007;21(2):99111.
24. Chou RC, Kim ND, Sadik CD, et al. Lipidcytokine-chemokine cascade drives neutrophil
recruitment in a murine model of inflammatory
arthritis. Immunity. 2010;33(2):266-278.
40. Lee HZ, Lin WC, Yeh FT, Wu CH. 2-Phenyl-4quinolone prevents serotonin-induced increases
in endothelial permeability to albumin. Eur J Pharmacol. 1998;354(2):205-213.
25. Rand M, Reid G. Source of ‘serotonin’ in serum.
Nature. 1951;168(4270):385.
41. Lee HZ, Lin WC, Yeh FT, Lin CN, Wu CH. Decreased protein kinase C activation mediates inhibitory effect of norathyriol on serotonin-mediated
endothelial permeability. Eur J Pharmacol. 1998;
353(2):303-313.
26. Majno G, Palade GE. Studies on inflammation: 1.
The effect of histamine and serotonin on vascular
permeability: an electron microscopic study.
J Biophys Biochem Cytol. 1961;11:571-605.
27. Hilton BP, Cumings JN. An assessment of platelet
aggregation induced by 5-hydroxytryptamine.
J Clin Pathol. 1971;24(3):250-258.
28. Gershon MD. Serotonin receptors and transporters: roles in normal and abnormal gastrointestinal
motility. Aliment Pharmacol Ther. 2004;20(Suppl
7):3-14.
29. Mercado CP, Kilic F. Molecular mechanisms of
SERT in platelets: regulation of plasma serotonin
levels. Mol Interv. 2010;10(4):231-241.
42. Zhao D, Qin L, Bourbon PM, James L, Dvorak HF,
Zeng H. Orphan nuclear transcription factor TR3/
Nur77 regulates microvessel permeability by targeting endothelial nitric oxide synthase and destabilizing
endothelial junctions. Proc Natl Acad Sci U S A.
2011;108(29):12066-12071.
43. Westergaard E. The effect of serotonin on the
blood-brain barrier to proteins. J Neural Transm
Suppl. 1978(14):9-15.
44. Schwartz A, Askenase PW, Gershon RK. The effect of locally injected vasoactive amines on the
elicitation of delayed-type hypersensitivity. J Immunol. 1977;118(1):159-165.
30. Chen JJ, Li Z, Pan H, et al. Maintenance of serotonin in the intestinal mucosa and ganglia of mice
that lack the high-affinity serotonin transporter:
abnormal intestinal motility and the expression of
cation transporters. J Neurosci. 2001;21(16):
6348-6361.
45. Gershon RK, Askenase PW, Gershon MD. Requirement for vasoactive amines for production of
delayed-type hypersensitivity skin reactions.
J Exp Med. 1975;142(3):732-747.
31. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu Rev Med. 2009;60:355366.
46. Semple JW, Italiano JE, Freedman J. Platelets
and the immune continuum. Nat Rev Immunol.
2011;11(4):264-274.
32. Asselin J, Knight CG, Farndale RW, Barnes MJ,
Watson SP. Monomeric (glycine-prolinehydroxyproline)10 repeat sequence is a partial
agonist of the platelet collagen receptor glycoprotein VI. Biochem J. 1999;339(2):413-418.
47. György B, Modos K, Pallinger E, et al. Detection
and isolation of cell-derived microparticles are
compromised by protein complexes resulting
from shared biophysical parameters. Blood. 2011;
117(4):e39-e48.
33. Kehrel B, Wierwille S, Clemetson KJ, et al. Glycoprotein VI is a major collagen receptor for platelet
activation: it recognizes the platelet-activating
quaternary structure of collagen, whereas CD36,
glycoprotein IIb/IIIa, and von Willebrand factor do
not. Blood. 1998;91(2):491-499.
48. Krishnadas R, Cavanagh J. Sustained remission
of rheumatoid arthritis with a specific serotonin
reuptake inhibitor antidepressant: a case report
and review of the literature. J Med Case Reports.
2011;5:112.
34. Kim MH, Curry FR, Simon SI. Dynamics of neutrophil extravasation and vascular permeability
are uncoupled during aseptic cutaneous wounding. Am J Physiol Cell Physiol. 2009;296(4):
C848-C856.
49. Nigrovic PA, Malbec O, Lu B, et al. C5a receptor
enables participation of mast cells in immune
complex arthritis independently of Fcgamma receptor modulation. Arthritis Rheum. 2010;62(11):
3322-3333.
50. Roviezzo F, Brancaleone V, De Gruttola L, et al.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 9 AUGUST 2012 䡠 VOLUME 120, NUMBER 6
Sphingosine-1-phosphate modulates vascular
permeability and cell recruitment in acute inflammation in vivo. J Pharmacol Exp Ther. 2011;
337(3):830-837.
51. Asaduzzaman M, Rahman M, Jeppsson B,
Thorlacius H. P-selectin glycoprotein-ligand-1
regulates pulmonary recruitment of neutrophils in
a platelet-independent manner in abdominal sepsis. Br J Pharmacol. 2009;156(2):307-315.
52. Sun G, Chang WL, Li J, Berney SM, Kimpel D,
van der Heyde HC. Inhibition of platelet adherence to brain microvasculature protects against
severe Plasmodium berghei malaria. Infect Immun. 2003;71(11):6553-6561.
53. Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology.
1999;38(8):1083-1152.
54. Bossi F, Frossi B, Radillo O, et al. Mast cells are
critically involved in serum-mediated vascular
leakage in chronic urticaria beyond high-affinity
IgE receptor stimulation. Allergy. 2011;66(12):
1538-1545.
55. Camerer E, Regard JB, Cornelissen I, et al.
Sphingosine-1-phosphate in the plasma compart-
PLATELETS CAN ENHANCE VASCULAR PERMEABILITY
56.
57.
58.
59.
60.
61.
ment regulates basal and inflammation-induced
vascular leak in mice. J Clin Invest. 2009;119(7):
1871-1879.
Obinata H, Hla T. Sphingosine 1-phosphate in
coagulation and inflammation. Semin Immunopathol. 2012;34(1):73-91.
Lucke S, Levkau B. Endothelial functions of
sphingosine-1-phosphate. Cell Physiol Biochem.
2010;26(1):87-96.
Cheng Y, Austin SC, Rocca B, et al. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science. 2002;296(5567):539-541.
Kobayashi T, Tahara Y, Matsumoto M, et al. Roles
of thromboxane A(2) and prostacyclin in the development of atherosclerosis in apoE-deficient
mice. J Clin Invest. 2004;114(6):784-794.
James MJ, Walsh JA. Inter-relationships between
vascular thromboxane and prostacyclin synthesis. Prostaglandins Leukot Essent Fatty Acids.
1988;31(2):91-95.
Takahashi Y, Tokuoka S, Masuda T, et al. Augmentation of allergic inflammation in prostanoid
IP receptor deficient mice. Br J Pharmacol. 2002;
137(3):315-322.
1343
62. Chen M, Boilard E, Nigrovic PA, et al. Predominance of cyclooxygenase 1 over cyclooxygenase
2 in the generation of proinflammatory prostaglandins in autoantibody-driven K/BxN serumtransfer arthritis. Arthritis Rheum. 2008;58(5):
1354-1365.
63. Brodie MJ, Hensby CN, Parke A, Gordon D. Is
prostacyclin in the major pro-inflammatory prostanoid in joint fluid? Life Sci. 1980;27(7):603-608.
64. Boilard E, Larabee K, Shnayder R, et al. Platelets
participate in synovitis via Cox-1-dependent synthesis of prostacyclin independently of microparticle generation. J Immunol. 2011;186(7):43614366.
65. Italiano JE Jr, Richardson JL, Patel-Hett S, et al.
Angiogenesis is regulated by a novel mechanism:
pro- and antiangiogenic proteins are organized
into separate platelet alpha granules and differentially released. Blood. 2008;111(3):1227-1233.
66. Cognasse F, Hamzeh-Cognasse H, Lafarge S,
et al. Toll-like receptor 4 ligand can differentially
modulate the release of cytokines by human
platelets. Br J Haematol. 2008;141(1):84-91.
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2012 120: 1334-1343
doi:10.1182/blood-2012-02-413047 originally published
online April 27, 2012
Platelets can enhance vascular permeability
Nathalie Cloutier, Alexandre Paré, Richard W. Farndale, H. Ralph Schumacher, Peter A. Nigrovic,
Steve Lacroix and Eric Boilard
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