ArticlePDF Available

Laser-induced endothelial cell activation supports fibrin formation

Authors:
  • Intelligent Imaging Innovations

Abstract and Figures

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 fluorochrome, into the mouse circulation resulted in dye uptake in the endothelium and circulating hematopoietic cells. Laser injury in mice treated with eptifibatide to inhibit platelet accumulation resulted in rapid calcium mobilization within the endothelium. Calcium mobilization correlated with the secretion of lysosomal-associated membrane protein 1, a marker of endothelium activation. In the absence of eptifibatide, endothelium activation preceded platelet accumulation. 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 (10 μM) 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 formation that was inhibited by an inhibitory monoclonal anti-tissue factor antibody. Thus laser injury leads to rapid endothelial 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.
Content may be subject to copyright.
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 VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
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 Ca2to 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 60oil 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
60water-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 Ca2levels 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
(Kd345nM). 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 VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
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 Ca2levels 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 Ca2elevation 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 Ca2flux 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 Ca2mobiliza-
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 Ca2levels were observed
by excitation at 488 nm. (A) Representative composite
fluorescence and brightfield images after vessel injury
show Ca2elevation 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 Ca2elevation 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.
ENDOTHELIAL CELLACTIVATION IN THROMBUS FORMATION 4677BLOOD, 25 NOVEMBER 2010 VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
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 Ca2elevation on the targeted side of the vessel
was approximately 3-fold higher than on the opposite side of the
vessel. Maximal Ca2elevation 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 Ca2elevation (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.
4678 ATKINSON et al BLOOD, 25 NOVEMBER 2010 VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
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 Ca2elevation, 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 Ca2after laser injury. At 0.5 seconds after the laser
pulse Ca2elevation 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 Ca2level 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 Ca2elevation 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 Ca2elevation
within seconds of their addition to the cells (Figure 4C). Thrombin
induced the highest peak Ca2elevation followed by ADP and
histamine. A comparison of these modes of activation revealed
similar kinetics for both agonist and laser stimulation. Decay of the
Ca2signal was most rapid in thrombin-activated cells and slowest
in laser-activated cells. The peak Ca2responses 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.
ENDOTHELIAL CELLACTIVATION IN THROMBUS FORMATION 4679BLOOD, 25 NOVEMBER 2010 VOLUME 116, NUMBER 22
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
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 Ca2elevation 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 Ca2elevation and activation as the distance
from the laser-injured cell increased. The spread of Ca2mobiliza-
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
n85 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 Ca2concen-
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 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 Ca2flux 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 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 Ca2mobiliza-
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 Ca2flux to neighboring cells. Rather these authors observed
that addition of culture medium from injured cells could elevate
Ca2levels 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 Ca2mobilization 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 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.
References
1. Ignarro LJ, Buga GM, Wood KS, Byrns RE,
Chaudhuri G. Endothelium-derived relaxing factor
produced and released from artery and vein is
nitric oxide. Proc Natl Acad Sci U S A. 1987;
84(24):9265-9269.
2. Radomski MW, Palmer RM, Moncada S. The
anti-aggregating properties of vascular endothe-
lium: interactions between prostacyclin and nitric
oxide. Br J Pharmacol. 1987;92(3):639-646.
3. Marcus AJ, Broekman MJ, Pinsky DJ. COX
inhibitors and thromboregulation. N Eng J Med.
2002;347(13):1025-1026.
4. Marcus AJ, Broekman MJ, Drosopoulos JH, et al.
Role of CD39 (NTPDase-1) in thromboregulation,
cerebroprotection, and cardioprotection. Semin
Thromb Hemost. 2005;31(2):234-246.
5. Griffin JH, Ferna´ndez JA, Gale AJ, Mosnier LO.
Activated Protein C. J Thromb Haemost. 2007;
5(S1):73-80.
6. Rosen ED, Raymond S, Zollman A, et al.
Laser-induced noninvasive vascular injury
models in mice generate platelet- and
coagulation-dependent thrombi. Am J Pathol.
2001;158(5):1613-1622.
7. Dubois C, Panicot-Dubois L, Merrill-Skoloff G,
Furie B, Furie BC. Glycoprotein VI-dependent
and -independent pathways of thrombus forma-
tion in vivo. Blood. 2006;107(10):3902-3906.
8. Furie B, Furie BC. In vivo thrombus formation.
J Thromb Haemost. 2007;5(S1):12-17.
9. Vandendries ER, Hamilton JR, Coughlin SR,
Furie B, Furie BC. Par4 is required for platelet
thrombus propagation but not fibrin generation in
a mouse model of thrombosis. Proc Natl Acad Sci
USA.2007;104(1):288-292.
10. Zwaal RFA, Confurius P, Bevers EM.
Lipid–protein interactions in blood coagulation.
Biochim Biophys Acta. 1998;1376:433-453.
11. Falati S, Liu Q, Gross P, et al. Accumulation of
tissue factor into developing thrombi in vivo is de-
pendent upon microparticle P-selectin glycopro-
tein ligand 1 and platelet P-selectin. J Exp Med.
2003;197:1585-1598.
12. Falati S, Gross P, Merrill-Skoloff G, Furie BC,
Furie B. Real-time in vivo imaging of platelets,
tissue factor and fibrin during arterial thrombus
formation in the mouse. Nat Med. 2002;8:
1175-1181.
13. National Institutes of Health National Cancer
Institute Protein Reviews on the Web. CD107a.
http://prow.nci.nih.gov/guide/1115712236_g.htm.
Accessed October 14, 1999.
14. Luttman W, Bratke K, Kupper M, Myrtek D. Immu-
nology. Burlington, MA: Academic; 2006.
15. Febbraio M, Silverstein RL. Identification
and characterization of LAMP-1 as an
activation-dependent platelet surface glycopro-
tein. J Biol Chem. 1990;265:18531-18537.
16. Rumbaut RE, Slaff DW, Burns AR. Microvascular
thrombosis models in venules and arterioles in
vivo. Microcirculation. 2005;12(3):259-274.
17. Sammak PJ, Hinman LE, Tran PO, Sjaastad MD,
Machen TE. How do injured cells communicate
with the surviving cell monolayer? J Cell Sci.
1997;110(4):465-475.
18. Dubois C, Panicot-Dubois L, Gainor JF, Furie BC,
Furie B. Thrombin-initiated platelet activation in
vivo is vWF independent during thrombus forma-
tion in a laser injury model. J Clin Invest. 2007;
117(4):953-960.
19. Bombeli T, Schwartz BR, Harlan JM. Endothelial
cells undergoing apoptosis become proadhesive
for nonactivated platelets. Blood. 1999;93(11):
3831-3838.
20. Frenette PS, Moyna C, Hartwell DW, Lowe JB,
Hynes RO, Wagner DD. Platelet-endothelial inter-
actions in inflamed mesenteric venules. Blood.
1998;91(4):1318-1324.
21. Massberg S, Enders G, Leiderer R, et al. Platelet-
endothelial cell interactions during ischemia/
reperfusion: the role of P-selectin. Blood. 1998;
92(2):507-515.
22. Cho J, Furie BC, Coughlin SR, Furie B. A critical
role for extracellular protein disulfide isomerase
during thrombus formation in mice. J Clin Invest.
2008;118:1123-1131.
23. Jasuja R, Furie B, Furie BC. Endothelium-derived
but not platelet-derived protein disulfide isomer-
ase is required for thrombus formation in vivo.
Blood. 2010;116(22):4665-4674.
24. Rodgers GM, Shuman MA. Prothrombin is acti-
vated on vascular endothelial cells by factor Xa
and calcium. Proc Natl Acad Sci U S A. 1983;
80(22):7001-7005.
25. van Heerde WL, Poort S, van’t Veer C, Reuteling-
sperger CP, de Groot PG. Binding of recombinant
Annexin V to endothelial cells: affect of annexin V
binding on endothelial cell-mediated thrombin
formation. Biochem J. 1994;302(1):305-312.
ENDOTHELIAL CELLACTIVATION IN THROMBUS FORMATION 4683BLOOD, 25 NOVEMBER 2010 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
http://www.bloodjournal.org/content/116/22/4675.full.html
Updated information and services can be found at:
(479 articles)Vascular Biology (902 articles)Thrombosis and Hemostasis
Articles on similar topics can be found in the following Blood collections
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Information about subscriptions and ASH membership may be found online at:
Copyright 2011 by The American Society of Hematology; all rights reserved.
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
For personal use only.on October 22, 2015. by guest www.bloodjournal.orgFrom
... vessel or cell type injury method key observations references cremaster arteriole laser activation laser activated endothelial cells trigger thrombus formation; neutrophil slow rolling on thrombus mediated by P-selectin-PSGL-1 [49] cremaster arteriole and HUVEC laser activation endothelial activation precedes platelet accumulation; normal fibrin formation observed in Par4 -/mice [50] cremaster arteriole laser activation prothrombinase found on activated endothelial cells [51] cremaster arteriole laser activation neutrophils contain and express tissue factor at the site of laser injury; neutrophils accumulated before platelets [52] venule TNF-α TNF-α activated endothelial cells recruit neutrophils; platelets bind adherent neutrophils rather than endothelium [49] artery and HUVEC TNF-α and IFN-γ fractalkine causes degranulation, activation, and expression of platelet P-selectin on adherent platelets, mediating neutrophil recruitment [53] HUVEC TNF-α endothelial TF drives fibrin deposition and coagulation; upregulated ICAM can be targeted for delivering recombinant thrombomodulin to inflamed cells [47] cremaster arteriole CCL2, TNF-α, IL-1β, or IFN-γ platelets guide neutrophils to extravasation points via P-selectin-PSGL-1 and CD40/ CD40 L [54] artery and HUVEC ApoE -/mice increased endothelial stiffness causes enhanced leucocyte transendothelial migration [55] artery ApoE -/mice reduced glycocalyx thickness and increased platelet adhesion occur at bifurcation point [56] artery ApoE -/mice endothelial dysfunction and glycocalyx impairment coincide with endothelial-dependent vasodilation, permeability, and increases in atherosclerotic biomarkers [4] royalsocietypublishing.org/journal/rsob Open Biol. 10: 200161 cells to inflammation, thrombosis and fibrosis [48] (table 1). ...
... Similarly, studies using alternative stimuli have evidenced endothelial cells as sources of tissue factor (TF) [47,50,63], a protein that is primarily secreted by activated monocytes to initiate platelet deposition and thrombin formation [64] and thereby actuate thrombogenesis and tissue fibrosis [65,66]. Endothelial-derived TF has been reported in response to TNF-α [47,63] and laser activation [50,51], a technique which can elicit endothelial activation without vessel denudation that has been used as a model of vascular thrombosis and atherosclerosis [67]. ...
... Similarly, studies using alternative stimuli have evidenced endothelial cells as sources of tissue factor (TF) [47,50,63], a protein that is primarily secreted by activated monocytes to initiate platelet deposition and thrombin formation [64] and thereby actuate thrombogenesis and tissue fibrosis [65,66]. Endothelial-derived TF has been reported in response to TNF-α [47,63] and laser activation [50,51], a technique which can elicit endothelial activation without vessel denudation that has been used as a model of vascular thrombosis and atherosclerosis [67]. Endothelial TF has been shown to cause fibrin deposition, upregulation of ICAM-1 and vascular cell adhesion molecule-1, and increased platelet binding. ...
Article
Full-text available
Severe fibrotic and thrombotic events permeate the healthcare system, causing suffering for millions of patients with inflammatory disorders. As late-state consequences of chronic inflammation, fibrosis and thrombosis are the culmination of pathological interactions of activated endothelium, neutrophils and platelets after vessel injury. Coupling of these three cell types ensures a pro-coagulant, cytokine-rich environment that promotes the capture, activation and proliferation of circulating immune cells and recruitment of key pro-fibrotic cell types such as myofibroblasts. As the first responders to sterile inflammatory injury, it is important to understand how endothelial cells, neutrophils and platelets help create this environment. There has been a growing interest in this intersection over the past decade that has helped shape the development of therapeutics to target these processes. Here, we review recent insights into how neutrophils, platelets and endothelial cells guide the development of pathological vessel repair that can also result in underlying tissue fibrosis. We further discuss recent efforts that have been made to translate this knowledge into therapeutics and provide perspective as to how a compound or combination therapeutics may be most efficacious when tackling fibrosis and thrombosis that is brought upon by chronic inflammation.
... Calcium mobilization in endothelial cell layer was quantified as previously described. [17] Briefly, platelets were depleted using the R300 antibody and mice were pretreated with Fluo-4 AM. The endothelial cell layer in the arterioles of the cremaster muscle were damaged by laser injury and calcium mobilization was quantified for up to 180 seconds. ...
... The endothelial cell layer in the arterioles of the cremaster muscle were damaged by laser injury and calcium mobilization was quantified for up to 180 seconds. [17] Tail blood loss measurement Male or female mice (6 to 8 weeks old) were anesthetized by an intraperitoneal (i.p.) injection with 100 mg/kg ketamine, 12.5 mg/kg xylazine and 0.25 mg/kg atropine. Tails were transected 3 mm from the tip with a scalpel blade and immediately immersed into a saline solution at 37 °C. ...
... A. WT C57BL/6J Mouse B. P2Y12 KO Mouse Mice were treated with R300 to deplete platelets and pretreated with Fluo-4 AM(17), and the endothelial cell layer in arterioles of the cremaster muscle was damaged by laser injury. Calcium mobilization was quantified for up to 180 seconds. ...
Article
Full-text available
Introduction Selatogrel is a reversible antagonist of the P2Y12 receptor. In rat thrombosis/haemostasis models, selatogrel was associated with lower blood loss than clopidogrel or ticagrelor at equivalent anti-thrombotic effect. Material and Methods We sought to elucidate the mechanism underlying the observed differences in blood loss, using real-time intravital microscopy in mouse. Results Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to hemostatic seals, were present. The phenotype of these hemostatic seals was dependent on the type of P2Y12 receptor antagonist. In the presence of clopidogrel and ticagrelor, detachment of platelets from the hemostatic seals was increased, indicative of reduced stability. In contrast, in the presence of selatogrel, platelet detachment was not increased. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel reduced laser-induced calcium mobilization in the endothelium, restricted neutrophil adhesion and subsequent fibrin formation and thus reduced fibrin-mediated stabilization of the hemostatic seals. The effects of ticagrelor were also observed in P2Y12 receptor-deficient mice, indicating that the effects are off-target and independent of the P2Y12 receptor. In contrast, selatogrel did not interfere with these elements of haemostasis in wild-type or in P2Y12 receptor-deficient mice. Conclusion In the presence of selatogrel the stability of hemostatic seals was unperturbed, translating to an improved blood loss profile. Our data suggest that the mechanism underlying the differences in blood loss profiles of P2Y12 receptor antagonists is by off-target interference with endothelial activation, neutrophil function and thus, fibrin-mediated stabilization of haemostatic seals.
... The advantages of this thrombosis model are numerous: (1) the ability to monitor thrombus formation in time and space, (2) the ability to generate multiple thrombi in the same animal, (3) the ability to follow the kinetics of thrombus formation in vivo, and (4) the ability to quantify several thrombosis parameters simultaneously [72]. The use of a calcium mobilization reporter compound, Fluo-4-AM, demonstrates that laser injury induces rapid activation of targeted endothelial cells [73]. This activation also generates degranulation of the endothelium expressing the molecules Lysosomal-Associated membrane protein-1 (LAMP-1) [73], PDI [74], and ERp5 [75] on its surface. ...
... The use of a calcium mobilization reporter compound, Fluo-4-AM, demonstrates that laser injury induces rapid activation of targeted endothelial cells [73]. This activation also generates degranulation of the endothelium expressing the molecules Lysosomal-Associated membrane protein-1 (LAMP-1) [73], PDI [74], and ERp5 [75] on its surface. The endothelium is a key player in this model of thrombosis, with the exception of the subendothelial matrix and its pro-thrombotic components. ...
Article
Full-text available
Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus formation is a major challenge for scientists today. For this purpose, new imaging technologies (such as confocal intravital microscopy, electron microscopy, holotomography, etc.) coupled with animal models of thrombosis (mouse, rat, rabbit, etc.) allow a better overview of this complex physiopathological process. Each of the cellular components is known to participate, including the subendothelial matrix, the endothelium, platelets, circulating cells, and, notably, neutrophils. Initially known as immune cells, neutrophils have been considered to be part of the landscape of thrombosis for more than a decade. They participate in this biological process through their expression of tissue factor (TF) and protein disulfide isomerase (PDI). Moreover, highly activated neutrophils are described as being able to release their DNA and thus form chromatin networks known as “neutrophil extracellular traps” (NETs). Initially, described as “dead sacrifices for a good cause” that prevent the dissemination of bacteria in the body, NETs have also been studied in several human pathologies, such as cardiovascular and respiratory diseases. Many articles suggest that they are involved in platelet thrombus formation and the activation of the coagulation cascade. This review presents the models of thrombosis in which neutrophils and NETs are involved and describes their mechanisms of action. We have even highlighted the medical diagnostic advances related to this research.
... [10][11][12][13][14] Platelet accumulation, fibrin formation, tissue factor and phosphatidylserine externalization, intraplatelet calcium flux, coagulation factor binding, and thrombo-embolization have all been measured using this technique. 9,[15][16][17][18][19] The model has also been used to evaluate the architecture of the platelet mass and to assess the relationship between signalling pathways and clot structure. 20,21 A major limitation of the laser-injury model of thrombus formation, however, is the significant degree of variability observed among injuries. ...
... Vascular injury in the cremaster arteriole laser injury model is thought to occur through laser injury-mediated activation of the endothelium. 16 The absence of detectable levels of subendothelial collagen exposure indicates that this model does not result in endothelial denudation. 24 The percentage power of the laser pulse applied was adjusted to cause an injury that resulted in distortion of the vessel wall (as shown in Figure S2) and formation of a nonocclusive platelet thrombus. ...
Article
Background The cremaster arteriole laser‐induced injury model is a powerful technique with which to investigate the molecular mechanisms that drive thrombus formation. This model is capable of direct visualization and quantification of accumulation of thrombus constituents, including both platelets and fibrin. However, a large degree of variability in platelet accumulation and fibrin formation is observed between thrombi. Strategies to understand this variability will enhance performance and standardization of the model. We determined whether ablation injury size contributes to variation in platelet accumulation and fibrin formation and, if so, whether incorporating ablation injury size into measurements reduces variation. Methods Thrombus formation was initiated by laser‐induced injury of cremaster arterioles of mice (n = 59 injuries). Ablation injuries within the vessel wall were consistently identified and quantified by measuring the length of vessel wall injury observed immediately following laser‐induced disruption. Platelet accumulation and fibrin formation as detected by fluorescently labeled antibodies were captured by digital intravital microscopy. Results Laser‐induced disruption of the vessel wall resulted in ablation injuries of variable length (18‐95 µm) enabling interrogation of the relationship between injury severity and thrombus dynamics. Strong positive correlations were observed between vessel injury length and both platelet accumulation and fibrin formation when the data are transformed as area under the curve (Spearman r = .80 and .76, respectively). Normalization of area under the curve measurements by injury length reduced intraclass coefficients of variation among thrombi and improved hypothesis testing when comparing different data sets. Conclusions Measurement of vessel wall injury length provides a reliable and robust marker of injury severity. Injury length can effectively normalize measurements of platelet accumulation and fibrin formation improving data interpretation and standardization.
... Under physiological conditions, endothelial cells form a non-adhesive surface that prevents platelet activation and coagulation cascades [17]. Once the vascular endothelium is damaged and exfoliated, the exposed subendothelial tissue (collagen) and the platelets in contact with it will be activated, resulting in thrombosis [18]. The damage of endothelial cells leads to the activation of coagulation factors, which dominates the coagulation mechanism in the body and leads to thrombosis [19]. ...
Article
Sargassum fucoidan is a kind of sulfated heteropolysaccharide with a variety of biological activities. The aim of this study was to investigate the extraction, purification, physicochemical characterization and in vitro antithrombotic activity of fucoidan from Sargassum henslowianum C.Agardh. Hot-water-assisted ultrasound was used to extract fucoidan (F). Fucoidan was purified by DEAE cellulose 52 (F1), Vc-H2O2 (FD1) and Superdex 75 gel (FDS1). The physical and chemical properties of fucoidans were analyzed by chemical composition, monosaccharide composition, average molecular weight (Mw) and FTIR. The sulfate contents of F, F1, FD1 and FDS1 were 11.45%, 16.35% and 17.52%, 9.66%, respectively; the Mw was 5.677 × 105, 4.393 × 105, 2.176 × 104 and 6.166 × 103, respectively. The results of monosaccharide composition showed that the four fucoidans contained l-fucose, d-galactose, l-mannose, d-xylose, l-rhamnose and d-glucose, but the mass fraction ratio was different. The results of FTIR showed that fucoidan contained characteristic peaks of sugar and sulfate. In vitro, F1, FD1 and FDS1 could alleviate HUVEC damage induced by adrenaline (Adr). F1, FD1 and FDS1 decreased vWF and TF and increased the ratio of t-PA/PAI-1 in Adr-induced HUVEC.
... Next, we studied the relationship between cancer progression and thrombus formation. We used an orthotopic model of pancreatic cancer to evaluate the kinetics of platelet accumulation and fibrin generation after a laser-induced injury in a vessel wall using real-time intravital microscopy (Furie Model) (31,37). Remarkably, there was an evident prothrombotic phenotype at day 10 (Panc02 mice D10) after tumor implantation in comparison to the control mice (Figure 2A). ...
Article
Full-text available
Platelet function can be modified by cancer cells to support tumor growth, causing alterations in the delicate hemostatic equilibrium. Cancer-cell and platelet interactions are one of the main pillars of Trousseau’s syndrome: a paraneoplastic syndrome with recurring and migrating episodes of thrombophlebitis. Altogether, this leads to a four-fold risk of thrombotic events in cancer patients, which in turn, portend a poor prognosis. We previously demonstrated that anti-P2RY12 drugs inhibit cancer-associated-thrombosis and formation of tumor metastasis in pancreatic cancer models. Here, we aimed to (1) compare the effects of aspirin and clopidogrel on pancreatic cancer prevention, (2) characterize the effects of clopidogrel (platelet P2RY12 inhibitor) on cancer-associated thrombosis and cancer growth in vivo , (3) determine the effect of P2RY12 across different digestive-tract cancers in vitro , and (4) analyze the expression pattern of P2RY12 in two different cancer types affecting the digestive system. Clopidogrel treatment resulted in better survival rates with smaller primary tumors and less metastasis than aspirin treatment. Clopidogrel was also more effective than aspirin at dissolving spontaneous endogenous thrombi in our orthotopic advanced cancer mouse model. P2RY12 expression gives pancreatic adenocarcinomas proliferative advantages. In conclusion, we propose the hypothesis that clopidogrel should be further studied to target and prevent Trousseau’s syndrome; as well as diminish cancer growth and spread. However, more studies are required to determine the implicated pathways and effects of these drugs on cancer development.
... This injury may compound the loss ACE2's vasoprotective effects (174). This is particularly concerning given that even limited injury to the endothelium of cerebral vessels can initiate in situ thrombosis and lead to strokes (175,176). ...
Article
Full-text available
As the life expectancy of people living with HIV (PLWH) on combination antiretroviral therapy (cART) increases, so does morbidity from cerebrovascular disease and neurocognitive disorders. Brain arterial remodeling stands out as a novel investigational target to understand the role of HIV in cerebrovascular and neurocognitive outcomes. We therefore conducted a review of publications in PubMed, EMBASE, Web of Science and Wiley Online Library, from inception to April 2021. We included search terms such as HIV, cART, brain, neuroimmunity, arterial remodeling, cerebrovascular disease, and neurocognitive disorders. The literature shows that, in the post-cART era, PLWH continue to experience an increased risk of stroke and neurocognitive disorders (albeit milder forms) compared to uninfected populations. PLWH who are immunosuppressed have a higher proportion of hemorrhagic strokes and strokes caused by opportunistic infection and HIV vasculopathy, while PLWH on long-term cART have higher rates of ischemic strokes, compared to HIV-seronegative controls. Brain large artery atherosclerosis in PLWH is associated with lower CD4 nadir and higher CD4 count during the stroke event. HIV vasculopathy, a form of non-atherosclerotic outward remodeling, on the other hand, is associated with protracted immunosuppression. HIV vasculopathy was also linked to a thinner media layer and increased adventitial macrophages, suggestive of non-atherosclerotic degeneration of the brain arterial wall in the setting of chronic central nervous system inflammation. Cerebrovascular architecture seems to be differentially affected by HIV infection in successfully treated versus immunosuppressed PLWH. Brain large artery atherosclerosis is prevalent even with long-term immune reconstitution post-cART. HIV-associated changes in brain arterial walls may also relate to higher rates of HIV-associated neurocognitive disorders, although milder forms are more prevalent in the post-cART era. The underlying mechanisms of HIV-associated pathological arterial remodeling remain poorly understood, but a role has been proposed for chronic HIV-associated inflammation with increased burden on the vasculature. Neuroimaging may come to play a role in assessing brain arterial remodeling and stratifying cerebrovascular risk, but the data remains inconclusive. An improved understanding of the different phenotypes of brain arterial remodeling associated with HIV may reveal opportunities to reduce rates of cerebrovascular disease in the aging population of PLWH on cART.
Article
Full-text available
The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)–activated neutrophils play a key role in initiating the tissue factor–dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.
Article
Full-text available
Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations. Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay. Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells. Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.
Article
Classical antithrombotics and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. The platelet-independent fibrin generation by activated endothelium highlights the importance of vascular protection in addition to platelet inhibition in thrombosis prevention. Dihydromyricetin (DHM), the most abundant flavonoid in Ampelopsis grossedentata, has unique vasoprotective effects. This study aims to characterize the antithrombotic potential of DHM. The effects of DHM on the activation of platelets and endothelial cells were evaluated in vitro. Calcium mobilization and activation of mitogen-activated protein kinases (MAPKs) were examined as the potential targets of DHM based on molecular docking analysis. The in vivo effects of DHM were determined in FeCl3-injured carotid arteries and laser-injured cremasteric arterioles. The results showed that DHM suppressed a range of platelet responses including aggregation, secretion, adhesion, spreading and integrin activation, and inhibited exocytosis, phosphatidylserine exposure and tissue factor expression in activated endothelial cells. Mechanistically, DHM attenuated thrombin-induced calcium mobilization and phosphorylation of ERK1/2 and p38 both in platelets and endothelial cells. Intravenous treatment with DHM delayed FeCl3-induced carotid arterial thrombosis. Furthermore, DHM treatment inhibited both platelet accumulation and fibrin generation in the presence or absence of eptifibatide in the laser injury-induced thrombosis model, without prolonging ex vivo plasma coagulation or tail bleeding time. DHM represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation as a result of endothelial protection.
Article
Full-text available
Platelets normally circulate in a quiescent state. When activated, they undergo biochemical and morphological changes which greatly alter their function and contribute to their role in thrombosis and hemostasis. We have identified, cloned, and sequenced a cDNA from a human unbilical vein endothelial cell library that encodes a 110-kDa integral membrane protein. This protein is present on the surface of activated but not resting platelets and has previously been identified as lysosomal-associated membrane protein 1 (LAMP-1). Half-maximal surface expression of platelet LAMP-1 was induced by concentrations of thrombin that resulted in lysosome enzyme release, not alpha-, or dense granule release. Also consistent with lysosome enzyme studies, there was little surface expression of LAMP-1 in response to the weak agonists ADP and epinephrine. In addition, sucrose density gradient fractionation of platelet granules showed colocalization of LAMP-1 with the lysosomal enzyme, beta-galactosidase, and not with markers of alpha- or dense granules. While we found virtually no LAMP-1 on the resting platelet surface (0-90 molecules/cell), we estimated a mean of 1175 LAMP-1 molecules on the thrombin-activated platelet surface. The translocation of this heavily glycosylated protein to the platelet surface upon stimulation may play a role in the adhesive, prothrombic nature of these cells.
Article
Full-text available
Mechanically scratching cell monolayers relieves contact inhibition and induces surviving cells near the wound edge to move and proliferate. The present work was designed to test whether surviving cells passively respond to newly available space, or whether cells are actively stimulated by signals from injured cells nearby. We monitored intracellular free Ca2+ ([Ca2+]i) while scratching confluent monolayers of bovine pulmonary endothelial cells and mouse mammary epithelial cells. Within seconds after wounding, a transient elevation of [Ca2+]i was observed in surviving cells. In endothelial cells, the [Ca2+]i elevation propagated into the monolayer for a distance of 10 to 12 cell rows at a speed of 20 to 28 microm/second. The amplitude of the wave of [Ca2+]i was reduced as it propagated into the monolayer, but the velocity of the wave was nearly constant. Cells that experienced the [Ca2+]i elevation had intact plasma membranes, and survived for over 24 hours post wounding. Removing extracellular Ca2+ decreased the amplitude by two-thirds and reduced the propagation rate by half, suggesting that Ca2+ influx contributed to the increased [Ca2+]i. To determine how [Ca2+]i waves were stimulated, we blocked extracellular communication by fluid perfusion or intercellular communication by breaks in the monolayer. In bovine pulmonary artery endothelial cultures, the [Ca2+]i wave passed over breaks in the monolayer, and was prevented from traveling upstream in a perfusion chamber. Conditioned media from injured cells also elevated [Ca2+]i in unwounded reporter cultures. In mouse mammary epithelial monolayers with established cell-cell contacts, the [Ca2+]i wave passed over breaks in the monolayer, but was only partially prevented from traveling upstream during perfusion. These experiments showed that mechanical wounds lead to long distance, [Ca2+]i-dependent communication between the injured cells and the surviving cell monolayer through at least two mechanisms: first, extracellular release of a chemical stimulus from wounded cells that diffused to neighboring cells (present in both monolayers); second, transmission of an intercellular signal through cell-cell junctions (present in the mammary epithelial monolayers). Thus, mechanical injury provided a direct, chemical stimulus to nearby cells which have not themselves been damaged.
Article
Under normal conditions, platelets do not adhere to endothelium. However, when platelets or endothelial cells are stimulated by thrombin or cytokines, respectively, platelets bind avidly to endothelium. Because there is accumulating evidence that endothelial cells may become apoptotic under certain proinflammatory or prothrombotic conditions, we investigated whether endothelial cells undergoing apoptosis may become proadhesive for nonactivated platelets. Human umbilical vein endothelial cells (HUVEC) were induced to undergo apoptosis by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum-deprivation. After treatment of HUVEC or platelets with different receptor antagonists, nonactivated, washed human platelets were allowed to adhere to HUVEC for 20 minutes. To exclude matrix involvement, platelet binding was measured in suspension by using flow cytometry. Independent of the method of apoptosis induction, there was a marked increase in platelet binding to apoptotic HUVEC. Although HUVEC exhibited maximal adhesiveness for platelets after 2 to 4 hours, complete DNA fragmentation of HUVEC occurred only several hours later. Adhesion assays after blockade of different platelet receptors showed only involvement of β1-integrins. Platelet binding to apoptotic HUVEC was inhibited by more than 70% when platelets were treated with blocking anti-β1 antibodies. Treatment of apoptotic HUVEC with blocking antibodies to different potential platelet receptors, including known ligands for β1-integrins, did not affect platelet binding. As assessed by determination of β-thromboglobulin and platelet factor 4 in the supernatants, platelets bound to apoptotic HUVEC became slightly activated. However, significant expression of platelet P-selectin (CD62P) was not found. These data provide further evidence that endothelial cells undergoing apoptosis may contribute to thrombotic events.
Article
Growing evidence supports a pathophysiological role for platelets during the manifestation of postischemic reperfusion injury; in the current study, we investigated the nature and the molecular determinants of platelet-endothelial cell interactions induced by ischemia/reperfusion (I/R). Platelet-endothelium and leukocyte-endothelium interactions after 1 hour of ischemia were monitored in vivo within mouse small intestine. By intravital fluorescence microscopy, we observed that platelets, like leukocytes, roll along or firmly adhere to postischemic microvascular endothelial cells. In contrast, few leukocyte-endothelial cell interactions were detected in sham-operated controls. Monoclonal antibodies against P-selectin significantly attenuated platelet rolling and adherence in response to I/R. To identify whether platelet or endothelial P-selectin plays the major role in mediating postischemic platelet-endothelial cell interactions, P-selectin-deficient or wild-type platelets were transfused into wild-type or P-selectin-deficient mice, respectively. Whereas platelets lacking P-selectin rolled along or adhered to postischemic wild-type endothelium, interactions between wild-type platelets with mutant endothelium were nearly absent, indicating that I/R-induced platelet-endothelium interactions are dependent on the expression of P-selectin by endothelial cells. Concomitantly, P-selectin expression in the intestinal microvasculature was enhanced in response to I/R, whereas no upregulation of P-selectin was observed on circulating platelets. In summary, we provide first in vivo evidence that platelets accumulate in the postischemic microvasculature early after reperfusion via P-selectin-ligand interactions. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of I/R injury.
Article
The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.
Article
Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is detected after vessel wall injury, and fibrin deposition is normal. Treatment of mice with a function blocking anti-PDI antibody completely inhibits fibrin generation in eptifibatide-treated mice. These results indicate that, although both platelets and endothelial cells secrete PDI after laser-induced injury, PDI from endothelial cells is required for fibrin generation in vivo.
Article
The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.
Article
1. The interactions between endothelium-derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined. 2. Porcine aortic endothelial cells treated with indomethacin and stimulated with bradykinin (10-100 nM) released NO in quantities sufficient to account for the inhibition of platelet aggregation attributed to endothelium-derived relaxing factor (EDRF). 3. In the absence of indomethacin, stimulation of the cells with bradykinin (1-3 nM) released small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation. 4. EDRF and authentic NO also caused disaggregation of platelets aggregated either with collagen or with U46619. 5. A reciprocal potentiation of both the anti- and the dis-aggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells. 6. It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet-vessel wall interactions.
Article
Vascular endothelial cells derived from adult bovine aorta (ABAE) treated with factor Xa and calcium were found to activate prothrombin. In contrast, nonvascular cells (human foreskin fibroblasts, bovine corneal endothelial cells, or human fetal lung cells) had either no or very little effect on prothrombin activation. In the presence of 6 X 10(5) ABAE cells, 20 ng of factor Xa converted 90 micrograms of prothrombin into 80 units of thrombin after 45 min at 37 degrees C. Exogenous factor V was not required for prothrombin activation, but thrombin generation was enhanced 2- to 4-fold by the addition of factor V (500-2,500 ng/ml). Treatment of ABAE cells with anti-bovine factor V IgG markedly inhibited prothrombin activation by factor Xa and calcium. In cells grown in serum-free medium for 3 months, the amount of factor V activity was equivalent to that found in cells grown with serum, which suggests that these cells probably synthesize factor V. Sparse ABAE cells increased prothrombin activation by factor Xa 6-fold compared to activation in confluent cells. Although previous thrombin treatment of ABAE cells did not enhance prothrombin activation, addition of dansyl arginine-4-ethyl piperidine amide markedly inhibited activation of 125I-labeled prothrombin by factor Xa, indicating that thrombin formation is necessary for optimal prothrombin activation. These data indicate that aortic endothelium may provide a physiologically important surface for activation of prothrombin as well as a mechanism for optimal formation of clots at sites of vascular injury.
Article
Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation.