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THROMBOSIS AND HEMOSTASIS
Laser-induced endothelial cell activation supports fibrin formation
Ben T. Atkinson,1,2 Reema Jasuja,1,2 Vivien M. Chen,1,2 Prathima Nandivada,1,2 Bruce Furie,1,2 and Barbara C. Furie1,2
1Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA; and 2Harvard Medical School, Boston, MA
Laser-induced vessel wall injury leads to
rapid thrombus formation in an animal
thrombosis model. The target of laser
injury is the endothelium. We monitored
calcium mobilization to assess activation
of the laser-targeted cells. Infusion of
Fluo-4 AM, a calcium-sensitive fluoro-
chrome, into the mouse circulation
resulted in dye uptake in the endothelium
and circulating hematopoietic cells.
Laser injury in mice treated with eptifi-
batide to inhibit platelet accumulation
resulted in rapid calcium mobilization
within the endothelium. Calcium mobiliza-
tion correlated with the secretion of
lysosomal-associated membrane protein
1, a marker of endothelium activation. In
the absence of eptifibatide, endothelium
activation preceded platelet accumu-
lation. Laser activation of human
umbilical vein endothelial cells loaded
with Fluo-4 resulted in a rapid increase in
calcium mobilization associated cell
fluorescence similar to that induced
by adenosine diphosphate (10M) or
thrombin (1 U/mL). Laser activation of
human umbilical vein endothelial cells
in the presence of corn trypsin inhibitor
treated human plasma devoid of platelets
and cell microparticles led to fibrin for-
mation that was inhibited by an inhibitory
monoclonal anti–tissue factor antibody.
Thus laser injury leads to rapid endothe-
lial cell activation. The laser activated
endothelial cells can support formation
of tenase and prothrombinase and may
be a source of activated tissue factor as
well. (Blood. 2010;116(22):4675-4683)
Introduction
The endothelium serves as a metabolically active interface between
the blood and underlying tissues. It maintains vascular tone,
regulates vessel permeability and inhibits thrombus formation.
The resting endothelium secretes 3 inhibitors of platelet activation,
nitric oxide,1prostacyclin,2,3 and the ectonucleotidase CD39,4
which together form a defense against platelet thrombus formation.
The resting endothelium also supports multiple anticoagulant
pathways, most importantly that of activated protein C, which is
both anticoagulant and cytoprotective.5Hemostasis and thrombus
formation are usually associated with exposure of the subendothe-
lial matrix rich in collagen and tissue factor that lead to accumula-
tion and activation of platelets and thrombin generation, respec-
tively, at the site of injury. While some animal models of
thrombosis mimic this exposure of the subendothelial matrix, in
our laser-induced injury model the endothelium remains intact and
the vessel wall is not denuded of endothelial cells.6In our
endothelial sparing model of laser-induced thrombus formation no
collagen is detected at the site of injury but platelet thrombus
formation and fibrin deposition both occur rapidly.7,8
We have examined thrombus formation after laser injury in
Par4⫺/⫺mice whose platelets lack the protease activated receptor
required for thrombin activation of mouse platelets.9Fibrin forma-
tion after laser injury in these mice is normal despite formation of a
very small platelet thrombus in which platelet activation is
significantly delayed. Fibrin formation is thrombin-dependent and
thrombin generation requires assembly of the tenase complex,
activated factor VIII and activated factor IX, and the prothrombi-
nase complex, activated factor V and activated factor X, on cell
surfaces with exposed phosphatidylserine.10 While it has been
generally accepted that activated platelets supply this critical
surface our results in Par4⫺/⫺mice indicate that either minute
quantities of activated platelets may be sufficient to support
thrombin generation or that other cell surfaces, such as those of
activated endothelial cells may provide the surface for enzyme
assembly. Therefore we investigated the hypothesis that endothelial
cells can be activated rapidly at a site of laser-induced injury and
can participate in thrombus formation.
Methods
Cells
Primary human umbilical vein endothelial cells (HUVECs), Medium
200, and low serum growth supplement were obtained from Cascade
Biologics. Human dermal microvascular endothelial cells (HDMECs),
human aortic endothelial cells (HAECs), and corresponding endothelial cell
medium were obtained from ScienCell Research Laboratories.
Mice
Wild-type C57BL/6J mice were obtained from The Jackson Laboratory.
The Beth Israel Deaconess Medical Center Institutional Animal Care and
Use Committee approved all animal care and experimental procedures.
Antibodies, dyes, and reagents
Rat anti–mouse CD41 antibody (clone MWReg30) was from Emfret and rat
anti–mouse lysosomal-associated membrane protein 1 (LAMP-1) antibody
(clone 1D4B; isotype immunoglobulin G [IgG]2a) was from eBioscience.
Mouse anti–human fibrin monoclonal antibody (clone 59D8 kindly sup-
plied by Professor Lawrence Brass, University of Pennsylvania School of
Medicine) was purified by affinity chromatography using Protein A/G.
Inhibitory tissue factor antibody cH36 was obtained from Altor Bioscience.
Rat IgG2a isotype control was obtained from Pharmingen/BD Biosciences.
Fab fragments of the anti-CD41 antibody were generated using the
Submitted May 5, 2010; accepted July 26, 2010. Prepublished online as Blood
First Edition paper, July 30, 2010; DOI 10.1182/blood-2010-05-283986.
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.
© 2010 by The American Society of Hematology
4675BLOOD, 25 NOVEMBER 2010 䡠VOLUME 116, NUMBER 22
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ImmunoPure Fab Preparation Kit from Pierce-ThermoScientific. Fab
fragments of anti-CD41 antibody and mouse anti-fibrin antibody and
anti–LAMP-1 antibody as well as rat IgG2a nonimmune IgG antibodies
were labeled with Alexa Fluor 488 or Alexa Fluor 647 according to the
manufacturer’s instructions (Invitrogen). The molar ratio of Alexa Fluor to
protein, determined spectrophotometrically, varied from 2.0 to 3.5.
Fluo-4 AM and DIOC6(3,3⬘-dihexyloxacarbocyanine iodide) were
obtained from Molecular Probes/Invitrogen, and prepared by solution at
3mM into dimethyl sulfoxide with 20% (wt/vol) Pluronic F-127 (Sigma-
Aldrich) for in vitro experiments and by solution at 6mM into Cremophore
EL (Sigma-Aldrich) for in vivo experiments.
The agonist adenosine diphosphate (ADP) was from Sigma-Aldrich,
and thrombin was from Haematologic Technologies Inc. Eptifibatide
(Integrilin) was purchased from Schering Plough.
Endothelial cell culture and stimulation
HUVECs were grown in Medium 200 containing low serum growth
supplement and cells of passage 2-3 were seeded on gelatin-coated
(Chemicon and Millipore) coverslips at a density of 1 ⫻105per coverslip.
The endothelial cells were cultured for 2-3 days under a 5% CO2/air
atmosphere at 37°C until confluent. For calcium imaging using Fluo-4 AM,
the cells were loaded as per the manufacturer’s instruction at a final
concentration of 3M for 30 minutes.
Images of cells in the basal state were recorded for 1 minute prior to
activation. For laser activation cells were subjected to a single pulse from a
dye tuned (410-nm wavelength) nitrogen laser with continuous imaging to
enable recording of early kinetic changes. For stimulation of endothelial
cells with ADP (10M), thrombin (1 U/mL) or histamine (10M) agonists
or vehicle control were added at a dilution of 1:10 to ensure rapid and
complete mixing. Agonists were prepared in cell media and were pre-
warmed to 37°C. For in vitro fibrin generation experiments, blood was
collected into 4% sodium citrate at a ratio of 1:9 and immediately
centrifuged at 330g. Platelets were removed from the platelet rich plasma
by further centrifugation at 2000 rpm. The platelet poor plasma was
centrifuged at 106 000gfor 1 hour at 4°C, aliquoted, and stored at ⫺80°C.
Fluo-4 AM–loaded cells cultured on photo-etched coverslips were incu-
bated with prewarmed plasma in the presence of 0.1 mg/mL corn trypsin
inhibitor and 10mM calcium. In addition, either the inhibitory tissue factor
antibody cH36 or an isotype-matched control human IgG was added to the
plasma. Selected cells were stimulated with the laser and cell activation was
monitored in real time by fluorescence microscopy. A single cell within
each of 3 noncontiguous fields of the grid on the photo-etched slide was
activated and the x,y coordinates of the sites of activation were noted using
the numbers on the underside of the slide. EDTA(ethylenediaminetetraace-
tic acid; 20mM) was added 15 minutes after addition of Ca2⫹to inhibit
further thrombin generation and cells on the photo-etched coverslips were
fixed with 4% paraformaldehyde. The fixed cells were immunostained with
Alexa 647–labeled fibrin specific antibody (clone 59D8) or Alexa 647–
labeled isotype-matched control antibody. The areas at and contiguous to
the sites of injury were examined by differential interference contrast and
fluorescent microscopy using a 60⫻oil lens (1.47 numeric aperture). Cells
and nuclei were visualized by actin staining with fluorescein isothiocyanate
phalloidin (40nM) and DAPI (4⬘,6-diamidino-2-phenylindole; 300nM),
respectively.
Intravital microscopy
Intravital videomicroscopy of the cremaster muscle microcirculation was
performed as previously described.11,12 Mice were preanesthetized with
intraperitoneal ketamine (125 mg/kg body weight; Abbott Laboratories),
xylazine (12.5 mg/kg body weight; Phoenix Pharmaceuticals), and atropine
(0.25 mg/kg body weight; American Pharmaceutical Partners). A tracheal
tube was inserted, and the mouse was maintained at 37°C on a thermo-
controlled rodent blanket. To maintain anesthesia, Nembutal (Abbott
Laboratories) was administered through a cannulus placed in the jugular
vein. After the scrotum was incised, the testicle and surrounding cremaster
muscle were exteriorized onto an intravital microscopy tray. The cremaster
preparation was superfused with thermo-controlled (36°C) and aerated
(95% N2,5%CO
2) bicarbonate-buffered saline throughout the experiment.
Microvessel data were obtained using an Olympus AX microscope with a
60⫻water-immersion objective (0.9 numeric aperture). The intravital
fluorescence microscopy system has previously been described in detail.7
Digital images were captured with a Cooke Sensicam charge-coupled
device camera in 640 ⫻480 format (2 ⫻2 binning).
The endothelium of the cremaster microcirculation was loaded with
Fluo-4 AM by systemic infusion of Fluo-4AM/Cremophore via the femoral
artery. A period of 20 minutes was allowed after infusion for uptake and
de-esterification of the dye within the endothelium. Concurrently aggrega-
tion was inhibited by infusion of the ␣IIb3antagonist eptifibatide.
Eptifibatide (10 g/g mouse) was infused immediately prior to intiation of
the first thrombus and was reinfused every 20 minutes for the duration of
the experiment. Eptifibatide does not interfere with binding to platelets of
the monoclonal anti-CD41 antibody, MWReg30, used for detection of
platelets in these experiments. After laser-induced vessel wall injury,
changes to endothelial Ca2⫹levels were observed by excitation at 488 nm,
and images were recorded over time.
Laser-induced injury
Vessel wall injury was induced with a Micropoint Laser System (Photonics
Instruments) focused through the microscope objective, parfocal with the
focal plane and aimed at the vessel wall.7Typically, 1 or 2 pulses were
required to induce vessel wall injury. Multiple thrombi were studied in a
single mouse, with new thrombi formed upstream of earlier thrombi to
avoid any contribution from thrombi generated earlier in the animal under
study. There were no characteristic trends in thrombus size or thrombus
composition in sequential thrombi generated in a single mouse during an
experiment. Image analysis was performed using Slidebook Version 4.2 or
higher (Intelligent Imaging Innovations). Fluorescence data were captured
digitally at up to 50 frames/s and analyzed as previously described.12
Typically, widefield fluorescence images were captured at exposure times
of 20 milliseconds, whereas brightfield images were captured with exposure
times of 10 milliseconds. Data were collected for 3-5 minutes after vessel
wall injury. The representative intravital color images are displayed either
as binarized images with the threshold chosen as the mean of the maximum
fluorescence values of a mask upstream of the thrombus taken throughout
the capture, or as an intensity map in which the intensity of the fluorescence
of each pixel is represented by a pseudocolor with blue being least intense
and red being most intense. The complete data sets of the representative and
multiple identical experiments are presented graphically, plotting the
integrated fluorescence intensity of all pixels in the image as a function of
time. Pixels considered are those above the previously defined threshold
and corrected for background, with the background being the mean of the
maximum fluorescence values in the upstream mask. The kinetics of
thrombus formation were analyzed by determining median fluorescence
values over time in approximately 20-30 thrombi.7
Statistical analysis
In vitro data are presented as means ⫾SEM and statistical significance was
calculated with Student ttest. For intravital experiments data were
considered nonparametric and presented as medians. Welch correction was
used for unpaired Student ttest in the in vitro fibrin generation assay.
Results
Rapid activation of arteriolar endothelium in vivo
Endothelial responses to laser injury in vivo were investigated
using intravital widefield microscopy and the cell permeant cal-
cium sensitive dye Fluo-4 AM, the fluorescence intensity of which
increases more than 100-fold after binding with calcium
(Kd⫽345nM). Fluo-4 AM was introduced into the mouse circula-
tion via the femoral artery to maximize the delivery and uptake of
dye in the cremaster muscle microcirculation. Fluo-4 AM is
4676 ATKINSON et al BLOOD, 25 NOVEMBER 2010 䡠VOLUME 116, NUMBER 22
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nonspecific in its uptake among cell types and therefore labels all
hematopoietic cells as well as the endothelium. To focus on the
endothelium we used the ␣IIb3integrin antagonist eptifibatide to
inhibit platelet accumulation at the site of injury. Inhibition of
platelet accumulation was verified in an independent experiment
using anti–mouse CD41 Fab fragments conjugated to Alexa 647 to
label endogenous platelets prior to laser injury and imaging
(data not shown).
Changes in endothelial cell Ca2⫹levels in Fluo-4 AM– and
eptifibatide–treated mice were recorded in one channel and a
brightfield image in a second channel (Figure 1A). The response of
the endothelium to laser injury was very rapid and showed an
increase in Fluo-4 fluorescence, reflecting Ca2⫹elevation within
seconds of the laser pulse. Fluo-4 fluorescence remained elevated
for several minutes. Analysis of the increase in integrated
Fluo-4 fluorescence over time for multiple experiments yielded the
median curve for Ca2⫹flux shown in Figure 1B. We performed the
same experiment using the nonspecific membrane stain DIOC6in
place of Fluo-4 to exclude the possibility that the increased
Fluo-4 fluorescence after laser injury was a result of noncalcium
related accumulation of Fluo-4 labeled species at the site of injury
rather than a rise in Ca2⫹. DIOC6has an excitation maximum at a
similar wavelength to that of Fluo-4 and once infused labeled the
membranes of endothelial cells and all hematopoietic cells at a
comparable level to the baseline Fluo-4 fluorescence. After laser
injury, DIOC6fluorescence did not increase at the injury site,
confirming that the increased Fluo-4 fluorescence observed at the
site of laser-induced vessel injury was the result of Ca2⫹mobiliza-
tion in endothelial cells activated by the laser pulse (Figure 1B).
The area of endothelial activation visualized in the
Fluo-4–treated animals was limited to the field of view indicating
that activation was ⬍400 m along the vessel after laser injury.
The signal was apparent on both the side of the vessel targeted by
the laser and the opposite side of the vessel indicating circumferen-
tial propagation of activation from the initial injury site. The extent
of calcium elevation after injury was determined by measuring
Figure 1. Activation of arteriolar endothelium in vivo
by laser-induced injury.The endothelium of the cremas-
ter microcirculation was loaded with either Fluo-4 AM or
DIOC6by systemic infusion of Fluo-4 AM/Cremophore
or DIOC6via the femoral artery. A period of 20 minutes
was allowed after infusion for uptake and de-esterifica-
tion of Fluo-4 AM or uptake of DIOC6within the endothe-
lium. Concurrently platelet accumulation was inhibited
by infusion of eptifibatide. After laser-induced vessel wall
injury, changes in endothelial Ca2⫹levels were observed
by excitation at 488 nm. (A) Representative composite
fluorescence and brightfield images after vessel injury
show Ca2⫹elevation in the endothelium in the absence
of platelet accumulation. The fluorescence signal is
presented as a pseudocolor intensity map where blue
represents the least intense and red represents most
intense fluorescence signal. (B) Calcium elevation after
vessel injury as determined by the median integrated
fluorescence intensity (y-axis) from Fluo-4–loaded endo-
thelium (top black curve, 27 thrombi from 3 mice) in
comparison to the median integrated fluorescence of the
inert dye DiOC6(bottom gray curve, 15 thrombi from
3 mice). (C) Propagation of Ca2⫹elevation and endothe-
lial activation along the vessel after injury is presented
as a pseudocolor intensity map as in panel A. Image is
representative of peak endothelial activation, the site of
injury is marked (X) and one line demarking the vessel
wall on the same side as the injury (purple) and a second
demarking the opposing side (gray). (D) Representative
trace from a single experiment showing quantitation of
the Fluo-4 fluorescence signal longitudinally along each
vessel wall. A line was drawn along each wall and the
intensity of the pixels at each step along this line was
determined and plotted. The start of the line (0 pixels) is
bottom right and the end (⬃300 pixels) is top left.
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integrated fluorescence intensity along both sides of the vessel wall
in the vicinity of the laser injury. Median values for integrated
fluorescence intensity over time are presented in Figure 1C. The
level of endothelial Ca2⫹elevation on the targeted side of the vessel
was approximately 3-fold higher than on the opposite side of the
vessel. Maximal Ca2⫹elevation was observed at the approximate
site of laser injury and the parallel point on the opposite side of the
vessel and decayed both proximally and distally from those points
on both sides of the vessel (Figure 1D).
Endothelium activation precedes platelet accumulation and
thrombus formation after injury
We established the temporal relationship between endothelial cell
activation and platelet thrombus formation after laser-induced
vessel wall injury. Platelets were labeled by infusion of anti–mouse
CD41 Fab fragments coupled to Alexa 647, and Fluo-4 AM was
infused as described in “Rapid activation of arteriolar endothelium
in vivo.” Images monitoring thrombus formation indicated that the
increase in fluorescence in the vessel wall at the site of injury
occurred prior to the appearance of platelets at the site (Figure 2A).
As platelets accumulated and became activated, they contributed to
the observed increase in Fluo-4 fluorescence due to platelet Ca2⫹
mobilization. To determine the increase in integrated endothelial
cell Fluo-4 fluorescence, we subtracted any Fluo-4 fluorescence
that was colocalized with Alexa 647 fluorescence from the total
Fluo-4 fluorescence. This method overestimated the platelet–
Fluo-4 fluorescence contribution because there was no way of
parsing among pixels that contained platelet- and endothelial-
derived Fluo-4 fluorescence and those that contained only platelet-
derived Fluo-4 fluorescence. However, the stringent nature of this
analysis did not compromise the conclusion that endothelial cell
activation occurs prior to platelet accumulation because a substan-
tial Fluo-4 signal was observed prior to platelet appearance at the
injury site. Median curves for the endothelial cell-derived increase
in Fluo-4 fluorescence and the platelet-associated Alexa
647 fluorescence over time indicate that, after laser injury, there
was rapid calcium mobilization in the endothelium that preceded
platelet detection by up to 30 seconds. (Figure 2B).
Figure 2. In vivo imaging of platelet accumulation concomitantly with
endothelial calcium elevation after laser-induced injury. Platelets
were labeled with anti–mouse CD41 Fab fragments conjugated to Alexa
647 infused via the jugular vein, and the endothelium of the cremaster
microcirculation was loaded with Fluo-4 AM. After laser-induced vessel
wall injury activation of the endothelium and thrombus formation were
observed and recorded over time. (A) Composite fluorescence and
brightfield images after vessel injury show Ca2⫹elevation (green) in the
endothelium in conjunction with and preceding platelet accumulation (red)
or presence of both signals (yellow). The fluorescence signal is shown
binarized for ease of visual interpretation. (B) Kinetic curves displaying the
median integrated Fluo-4 fluorescence (gray curve on left y-axis) and
median integrated platelet fluorescence (black curve on right y-axis) for
34 thrombi in 3 wild-type mice. Fluo-4 fluorescence originating from
platelets and not the endothelium was eliminated by subtracting any
Fluo-4 fluorescence in pixels where Alexa 647 fluorescence was observed.
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Calcium mobilization in the endothelium correlates with
granule secretion
To confirm that the fluorescence intensity increase in the endothe-
lium associated with calcium mobilization is an event associated
with endothelial cell activation and to determine whether laser
stimulation of endothelial cells leads to later stages of cell
activation, we examined the surface expression of LAMP 113,14
after laser-induced injury. LAMP 1, a lysosomal membrane protein
found in many cells including endothelial cells, is translocated to
the cell membrane upon cell activation and granule secretion.
Alexa 488–labeled anti–LAMP-1 and Alexa 647–labeled anti-
CD41 Fab fragments were infused and laser-induced thrombus
formation monitored. LAMP-1 was visible at the site of laser injury
on the vessel wall at a time prior to the accumulation of platelets
(Figure 3A). As platelets accumulated at the injury site and became
activated, they contributed to LAMP-1 fluorescence because
activated platelets also express LAMP-1 on their surface.15 These
results indicate that endothelial cell activation can be monitored in
vivo by both calcium mobilization and LAMP-1 secretion.
The increase in integrated endothelial cell–associated
LAMP-1–associated fluorescence was measured after correcting
for the LAMP-1 signal contributed by platelets. Laser-induced
vessel injury led to rapid accumulation of LAMP-1 antigen on the
surface of the surrounding endothelium and was detected before
platelet accumulation (Figure 3B). Similar to observations of
endothelial-associated Ca2⫹elevation, endothelial cell-associated
LAMP-1 accumulation after laser-induced injury was observed to
spread from the site of injury around the vessel and, in some cases,
to the opposite wall. Thus, calcium mobilization and granule
secretion in the endothelium precede platelet accumulation in vivo
during laser-induced vessel wall injury and thrombus formation.
Rapid activation of endothelial cells by laser injury in vitro
To determine that the endothelial cell activation and its sequelae
observed after laser injury in vivo are a direct consequence of the
laser pulse and not secondary factors we examined the calcium
response of cultured HUVECs to a laser pulse using Fluo-4 AM.
Fluo-4–loaded HUVECs were subjected to a single laser pulse of
approximately 300 J, and subsequent changes in Fluo-4 fluores-
cence recorded (Figure 4A). The laser pulse was focused to a
diffraction-limited spot of approximately 1.8-m diameter, a target
area well below the size of the targeted endothelial cell. The images
show a representative resting endothelial cell prior to laser injury
(0 seconds) and the increase in fluorescence as a result of increased
intracellular Ca2⫹after laser injury. At 0.5 seconds after the laser
pulse Ca2⫹elevation was observed within the cell body covering an
area of approximately 13 m in diameter centered at the laser
target. Within several seconds an increased Ca2⫹level was
observed throughout the cell. Later time points showed a gradual
reduction in fluorescence over approximately 3 minutes. No
fluorescence was observed when the laser was aimed at a cell-free
area of the cover-slip. The fluorescence signal from laser-activated
cells was quantitated by defining the cell perimeter from the
differential interference contrast image and calculating the mean
pixel intensity from the 488-nm fluorescence excitation channel
within the region of interest for each time point (Figure 4B). Plots
of fluorescence versus time for 1 representative cell and the mean
of 41 cells show peak Ca2⫹elevation occurred in less than
10 seconds after the laser pulse and then declined. In similar
experiments performed in Ca2⫹-free media approximately 25% of
the calcium mobilization was preserved, indicating that the
observed calcium flux was due to cell activation rather than
disruption of the plasma membrane (not shown).
Activation of Fluo-4–loaded HUVECs by ADP (10M), throm-
bin (1 U/mL), or histamine (10M), physiologic cell agonists, was
compared with laser activation. Calcium mobilization was moni-
tored by fluorescence, and all 3 agonists induced Ca2⫹elevation
within seconds of their addition to the cells (Figure 4C). Thrombin
induced the highest peak Ca2⫹elevation followed by ADP and
histamine. A comparison of these modes of activation revealed
similar kinetics for both agonist and laser stimulation. Decay of the
Ca2⫹signal was most rapid in thrombin-activated cells and slowest
in laser-activated cells. The peak Ca2⫹responses elicited by
thrombin, ADP, and laser pulse were significantly larger than
control (P⬍.05) and the thrombin and laser responses were
equivalent (P⬎.5). Similar results were obtained using cultured
HAECs and HDMECs.
Figure 3. Exposure of activation and secretion marker LAMP-1 in comparison
with platelet accumulation in vivo after laser injury. Anti–mouse CD41 Fab
fragments conjugated to Alexa 647 and anti–mouse LAMP-1 antibodies conjugated
to Alexa 488 were infused to label platelets and LAMP-1, respectively. Injuries
were induced by laser pulses to cremaster arteriole vessel walls and subsequent
LAMP-1 accumulation and thrombus formation recorded over time. (A) Images from a
representative experiment showing fluorescence over time overlaying brightfield data
before and after vessel injury. LAMP-1 accumulated rapidly at the vessel wall (green)
followed by platelet accumulation (red) or presence of both signals (yellow). The
fluorescence signal is shown binarized for ease of visual interpretation. (B) Kinetic
curves displaying the median integrated platelet fluorescence (black curve on right
y-axis) and median integrated LAMP-1 fluorescence (gray curve on left y-axis) for
18 thrombi in 3 wild-type mice. LAMP-1 fluorescence originating from platelets and
not the endothelium was eliminated by subtracting any LAMP-1 fluorescence in pixels
where Alexa 647 fluorescence was observed.
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Propagation of endothelial cell activation to surrounding cells
after laser stimulation
In vivo endothelial cells do not exist as independent units but
rather as part of a confluent monolayer lining the lumen of the
vasculature. We therefore investigated the effect of laser stimu-
lation on a confluent cell population. A confluent monolayer of
HUVECs was loaded with Fluo-4 and a single cell within the
monolayer subjected to laser stimulation. Figure 5A shows
representative images of the response of a cell monolayer to a
single laser pulse fired at the point indicated (X). Calcium
mobilization, as monitored by fluorescence, begins in the target
cell 1 second after the laser pulse. Subsequent activation of the
surrounding cells was visible, nearest first and spreading
outward. Propagation of the calcium wave from one cell to
another was not inhibited by either 18-␣-glycyrrhetinic acid, a
gap junction inhibitor, or the ADP scavenger apyrase. Similarly,
after laser stimulation of a single cell we observed propagation
of the calcium wave among cells seeded at a low density with no
cytoplasmic bridges between them (data not shown).
We followed the spread of the wave of activation by quantifying
the rise and peak in Ca2⫹elevation in cells at varying distance from
the point of laser injury (Figure 5B). The indicated cells showed a
staggered rise in fluorescence indicating a time delayed propaga-
tion of cell-associated Ca2⫹elevation and activation as the distance
from the laser-injured cell increased. The spread of Ca2⫹mobiliza-
tion among the population diminished with distance from the point
of injury and was not observed beyond 3 to 4 fields-of-view,
equivalent to approximately 400-500 m. The mean speed of
propagation of the activation wave front was 15 ⫾2m/s. Trans-
lated to an in vivo setting this rate of propagation would
cross a 50 m diameter arteriole from one side to the other in 5.2
seconds. Laser-stimulation of endothelial cells in vitro results in
Figure 4. Rapid endothelial cell activation in vitro follows targeted
laser pulse. HUVECs were loaded with Fluo-4 AM (3M) and
observed using fluorescence microscopy. (A) Representative images of a
cell before and after a direct laser pulse to the point the indicated
(X). An increase in Fluo-4 fluorescence (green) reflects a rise in
intracellular Ca2⫹.(B) Quantification of this signal is plotted against time
showing 1 representative trace (solid line) and the mean trace of
laser-induced activation of 41 cells (dotted line). (C) Similarly prepared
HUVECs loaded with Fluo-4 AM were stimulated with ADP (10M),
thrombin (1 U/mL), or histamine (10M) as a comparison to the laser-
induced activation. Agonists or vehicle were added after 10 seconds of
image acquisition. The graph shows median curves as a comparison of
the kinetics of these modes of activation. ADP, solid line; thrombin,–––;
histamine, -.-.; laser, -- -; vehicle ...
. (D) The peak cell activation was
extracted from the kinetic data for each agonist and the laser. The mean
⫾SEM is plotted for each group; ADP stimulation, n ⫽82 cells from
7 experiments, histamine n ⫽45 cells from 3 experiments; thrombin
n⫽85 cells from 6 experiments; vehicle n ⫽10 cells from 2 experiments.
Figure 5. Rapid propagation of endothelial cell acti-
vation within a confluent cell population follows
laser-induced activation in vitro. HUVECs were loaded
with Fluo-4 AM. Cells were observed for 1 minute prior to
activation to confirm a stable baseline then subjected to
a single pulse from a nitrogen laser and continuous
imaging. (A) Images from different time points of a
representative cell population. The first frame shows the
cell monolayer in its basal state just prior to activation
and the X marks the point upon which the laser is
focused. The subsequent time points show activation of
the target cell (within 1 second of laser firing), closely
followed by activation of surrounding cells and then
steady return toward a basal cytoplasmic Ca2⫹concen-
tration. (B) The mean pixel intensity of the 4 cells is
plotted versus time to yield the corresponding 4 kinetic
traces shown on the right. Cell 1, solid line; Cell 2, -.-.;
Cell 3, - - -; Cell 4, ...
.
4680 ATKINSON et al BLOOD, 25 NOVEMBER 2010 䡠VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
both rapid activation of the target cell and neighboring cells. These
data confirm that the Ca2⫹flux observed in vivo after laser-induced
endothelial cell injury is a consequence of the injury.
Laser-activated endothelial cells can induce
thrombin generation
To determine whether laser-induced activation of endothelial cells
alters the coagulant potential of the cells we activated cultured
endothelial cells in the presence of human plasma. Plasma was
anticoagulated with sodium citrate, and subjected to centrifugation
twice to remove platelets. The supernatant plasma was subjected to
ultracentrifugation to remove any subcellular elements. Corn
trypsin inhibitor was added at a final concentration of 0.1 mg/mL to
inhibit factor XIIa-mediated initiation of blood coagulation, and
specific antibodies or control antibodies were added to the plasma.
No fibrin clot formed in this plasma for more than 45 minutes after
recalcification. Plasma was overlayed upon washed, confluent,
Fluo-4–loaded HUVECs growing on gelatin-coated photo-etched
coverslips and calcium was added. The cells were subjected to laser
injury or sham. Cells were fixed in situ 15 minutes after addition of
calcium and the presence of fibrin detected by immunofluorescence
using an Alexa 647–labeled fibrin specific antibody. Background
signal was calculated using a similarly labeled isotype matched
control antibody (Figure 6D). With unstimulated cells no fibrin was
generated (Figure 6A). In contrast, laser stimulation of cells in the
presence of plasma led to rapid formation of fibrin strands visible
over the stimulated cells and neighboring cells (Figure 6B). Fibrin
formation was blocked when an inhibitory monoclonal anti–tissue
factor antibody, cH36, was included in the plasma but not when
an isotype matched control antibody was included (Figure 6C and
E, respectively).
Discussion
Intravital models are an essential tool in the process of investigat-
ing hemostasis and thrombosis. They provide an in vivo system of
verifying in vitro data and a more holistic means of investigation of
a process that has many interdependent components. Most intravi-
tal thrombosis models lead to disruption or denudation of the
endothelium, and studies in these models have concentrated on
responses to the subendothelial matrix.16 The subendothelial matrix
undoubtedly plays a critical role in hemostasis. However, we were
interested in exploring cases where the endothelium may be fully
intact but diseased or activated in some other way. In this study, we
have extended our ability to image platelet activation, fibrin
generation and thrombus formation to include monitoring of
activation of the endothelium in vivo.
Figure 6. Laser activated endothelial cells can induce thrombin generation. HUVECs plated on photo-etched coverslips were loaded with Fluo-4 AM and incubated with
plasma in presence of calcium and corn trypsin inhibitor for 15 minutes. Cells were either stimulated with laser or left unstimulated in the presence or absence of various
antibodies. After incubation with plasma, cells were fixed and immunostained for fibrin (red), phalloidin (green) and DAPI (blue). (A) Representative images of cells not
stimulated by laser and incubated with recalcified plasma show minimal fibrin specific Alexa 647 signal (red). (B) Detection of fibrin-specific signal (red) after laser induced injury
of a single cell in a field in presence of recalcified plasma. (C) Representative image showing lack of fibrin formation after laser injury when the cells were incubated with
recalcified plasma containing 100 g/mL function blocking tissue factor monoclonal antibody cH36. (D) No signal was detected when laser stimulated cells were
immunostained with an isotype matched control IgG instead of fibrin antibody in the presence of plasma. (E) Fibrin meshwork was detected on cells activated with laser and
incubated with recalcified plasma in the presence of an isotype matched control human IgG instead of the monoclonal antibody cH36. (F) Mean integrated fluorescence signal
intensity of fibrin (n ⫽29). Data are from 2 independently performed experiments. The mean ⫾SEM is plotted for each group. A background mask was created for all images
from panel D. Mean of the maximum signal intensities from 29 images in panel D was used as a constant background number to create a threshold segment mask for all other
conditions and integrated fluorescence intensity was calculated. The means of the integrated fluorescence intensity show a significant decrease in fibrin generation when laser
stimulated endothelial cells are incubated with recalcified plasma in presence of function blocking tissue factor antibody.
ENDOTHELIAL CELLACTIVATION IN THROMBUS FORMATION 4681BLOOD, 25 NOVEMBER 2010 䡠VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
Activation of the endothelium and its subsequent interactions
with platelets may be important in cases of venous thrombosis, as
well as in failure of arterially transplanted vein grafts, stents or
artificial valves. We thus employed a laser-induced injury model of
thrombosis to examine thrombus formation in the presence of
intact but activated endothelium.16 We have previously demon-
strated in our model that collagen-exposure and GPVI do not play a
role in platelet aggregation and thrombus development.7Therefore,
the initiating events of thrombus formation in this model appear to
be dominated by thrombin generation.
In this study we have investigated the possible role of endothe-
lial cell activation in thrombus formation and have demonstrated
rapid activation of the vascular endothelium prior to thrombus
formation in vivo in response to laser injury. Both Ca2⫹mobiliza-
tion and granule secretion leading to LAMP-1 antigen surface
expression were detected after laser injury but before platelet
accumulation in our intravital model. The activation propagated
locally but was limited to the region immediately adjacent to the
injury site. These findings were supported by the results of
laser-induced endothelial cell activation in vitro that led to rapid
calcium mobilization in the injured cell and surrounding cells.
Sammak and colleagues have previously shown that mechanical
disruption of a cultured endothelial monolayer leads to Ca2⫹
mobilization within seconds of injury and subsequent propagation
of activation to surrounding cells.17 As in our studies, these authors
showed that cell–cell contact was not required for propagation of
the Ca2⫹flux to neighboring cells. Rather these authors observed
that addition of culture medium from injured cells could elevate
Ca2⫹levels in unwounded reporter cultures, suggesting a soluble
mediator secreted from activated endothelial cells. Dispersion of a
soluble mediator from the injured cell would be consistent with our
in vitro results that show a decrease in level of calcium mobiliza-
tion in cells circumferentially from the point of the injured cell in a
nonflow system. If a similar mechanism occurs in vivo, then blood
flow would be expected to rapidly disperse the signaling molecule
consistent with our observation that endothelial cell activation is
limited to a small region of the injured vessel around the injury site.
Our results suggest that the endothelium plays a role in
thrombus initiation or the early stages of thrombus formation. We
have previously demonstrated that in our laser-induced thrombosis
model resting platelets are recruited to the site of vessel injury
where they become activated.18 In the current study we show that
both Ca2⫹mobilization and LAMP-1 expression precede platelet
accumulation at the site of injury in the cremaster arteriolar
endothelium. While we observe both calcium elevation and LAMP-1
expression on both sides of the vessel around the injury site,
thrombus formation only occurs on the side of the injury. One
explanation for focal thrombus growth at the injury site may be the
magnitude of the activation of the cells. In our analysis of calcium
mobilization along the vessel wall the endothelium shows greater
activation closer to the site of injury indicating that there may be a
threshold level of activation needed to initiate platelet adhesion.
A number of mechanisms are known to support recruitment of
resting platelets to the endothelial cell surface. For example
apoptotic endothelial cells that may be present under some
inflammatory or prothrombotic conditions have been shown to be
proadhesive for resting platelets.19 However, induction of apoptosis
is a slow process not consistent with the kinetics of the events
observed after laser vessel wall injury. Endothelial cell P-selectin
has been implicated in recruitment of platelets to endothelium
stimulated with inflammatory mediators such as tumor necrosis
factor-␣20 and in response to ischemia-reperfusion injury21 but
platelets accumulate after laser induced injury in P-selectin⫺/⫺
mice making it unlikely that this protein is playing an important
role in recruiting platelets to the activated endothelium in this
model.11 Like P-selectin, von Willebrand factor is stored in the
Weibel-Palade bodies of endothelial cells and is released when
these cells are activated. Although von Willebrand factor is known
to play a role in platelet adhesion to the endothelium it is not
required for recruitment of platelets after laser injury.18 Thus, the
mechanism of recruitment of resting platelets to laser-activated
endothelium remains to be elucidated.
LAMP-1, a lysosomal membrane protein, is not present on the
cell surface under basal conditions; secretion is required to
translocate the protein to the plasma membrane. Therefore we
suggest that activation and subsequent secretion of endothelial cell
granule contents upon laser injury leads to the release of mediators
and surface presentation of proteins that locally transform the
endothelium into a pro-thrombotic surface. One such mediator
could be the thiol isomerase protein disulfide isomerase, which has
recently been shown to play a critical role in thrombus formation
and fibrin generation in vivo.22 Protein disulfide isomerase is
present in endothelial cell granules and is secreted upon laser-
induced vessel wall injury in our mouse model of thrombosis.23
The activated platelet surface is generally considered the
primary site for assembly of the tenase and prothrombinase
complexes required for thrombin production. Thus, it was perplex-
ing to find that fibrin generation was normal in Par4⫺/⫺mice—
lacking the platelet thrombin receptor—in our laser-induced throm-
bosis model where only a minimal platelet aggregate of unactivated
platelets forms at the site of injury.9Our in vitro results indicate that
laser-induced activation of endothelial cells can convert these cells
from a quiescent noncoagulant state to an activated procoagulant
state that supports thrombin generation indicated by fibrin deposi-
tion. The components of this thrombin generation system include
washed cultured HUVECs and platelet depleted plasma that has
been treated with corn trypsin inhibitor to inhibit the intrinsic
pathway to thrombin generation. Alternative sources of membranes
from plasma were removed by ultracentrifugation. In the absence
of another source of membrane, our results demonstrate that
activated endothelial cells support the formation of the tenase and
prothrombinase complexes. Endothelial cells activated with throm-
bin, phorbol 12-myristate 13-acetate, or tumor necrosis factor-␣
have been shown to support assembly of these complexes.24,25
The fibrin deposition on HUVECs was demonstrated to be
tissue factor dependent by use of an inhibitory anti–tissue factor
antibody. The absence of clot formation after recalcification of the
citrated ultracentrifuged plasma used in these experiments indi-
cates that any tissue factor activity retained in the plasma is below
the level required to form fibrin. Thus, our in vitro results may also
implicate activated endothelial cells as a source of the tissue factor
required to form the initiating tissue factor-factor VIIa complex
necessary for thrombin generation. In vivo thrombin formed from
the enzymatic reactions supported by the activated endothelial cell
membrane may initiate activation of the initial platelets recruited to
the activated endothelial surface. Taken together these results
suggest an important role for the activated endothelium in throm-
bus formation through provision of active tissue factor and a
membrane surface for tenase and prothrombinase formation under
conditions where extravascular cells and vascular matrix compo-
nents are not exposed.
Acknowledgments
We thank Glenn Merrill-Skoloff for expert technical assistance.
This work was supported by grants from the National Institutes
of Health to B.F. and B.C.F. R.J. is a recipient of a fellowship
from the American Heart Association and V.M.C. is a recipient
4682 ATKINSON et al BLOOD, 25 NOVEMBER 2010 䡠VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
of a grant from the National Health and Medical Research
Council of Australia.
Authorship
Contribution: B.T.A., R.J, and V.M.C. designed and performed the
experiments, analyzed the results, and wrote the manuscript; P.N.
performed the experiments and analyzed the results; and B.F. and
B.C.F. designed the experiments and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Barbara C. Furie, Beth Israel Deaconess
Medical Center, 330 Brookline Ave, E/CLS 905, Boston, MA
02215; e-mail: bfurie1@bidmc.harvard.edu.
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ENDOTHELIAL CELLACTIVATION IN THROMBUS FORMATION 4683BLOOD, 25 NOVEMBER 2010 䡠VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
online July 30, 2010 originally publisheddoi:10.1182/blood-2010-05-283986
2010 116: 4675-4683
Furie
Ben T. Atkinson, Reema Jasuja, Vivien M. Chen, Prathima Nandivada, Bruce Furie and Barbara C.
Laser-induced endothelial cell activation supports fibrin formation
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