|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 507-515
Platelet-Endothelial Cell Interactions During
Ischemia/Reperfusion: The Role of P-Selectin
By
Steffen Massberg,
Georg Enders,
Rosmarie Leiderer,
Simone Eisenmenger,
Dietmar Vestweber,
Fritz Krombach, and
Konrad Messmer
From the Ludwig-Maximilians-University, Institute for Surgical
Research, Klinikum Grosshadern, Munich, Germany; and the University of
Münster, Department for Molecular Biology of Inflammation,
Münster, Germany.
 |
ABSTRACT |
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.
| |
INTRODUCTION |
RESTORATION OF BLOOD flow after prolonged
ischemia initiates a series of events that may accumulate in
microvascular and parenchymal cell injury. Leukocyte adhesion to the
endothelium and subsequent leukocyte emigration to the interstitial
compartments are hallmarks of ischemia/reperfusion (I/R)
injury.1-4 Once adherent or emigrated, leukocytes release
toxic oxygen products and proteases, thus contributing to the
manifestation of I/R-induced vascular and tissue damage.5 Whereas many of the mechanisms underlying the I/R-induced inflammatory response still remain unknown, growing evidence suggests a role for
platelets in the pathogenesis of postischemic reperfusion injury.
Ischemia leads to the accumulation and activation of platelets within
vascular beds early after reperfusion.6 Correspondingly, several studies have demonstrated beneficial effects of platelet depletion during I/R.7,8 Although platelets are anuclear, they possess a cellular machinery comparable to leukocytes in many
aspects. Platelets have a cytoskeleton that allows cell motion. Upon
activation, they generate oxygen radicals9-11 and release proinflammatory mediators such as thromboxane A2,
leukotrienes, serotonine, platelet factor 4, and platelet-derived
growth factor (PDGF).12-17 In addition, platelets have the
potential to modulate leukocyte functional responses.18,19
Hence, the adhesion and activation of platelets to postischemic
endothelial cells might significantly aggravate endothelial cell damage
and contribute to leukocyte activation and recruitment at the site of
injury. However, the exact role of platelets in the manifestation of
I/R injury, in particular the behavior of platelets during I/R in vivo,
and the molecular mechanisms whereby platelets accumulate in
postischemic tissues have not been identified so far.
Platelets carry several adhesion molecules required for cell-cell
interaction, such as P-selectin, a putative P-selectin ligand (CD 162 ?), PECAM-1, and several integrins (LFA-1, glycoprotein IIb/IIIa),
which play a key role in mediating platelet adhesion to subendothelial
matrix proteins.20,21 P-selectin, which is stored in
 -granules of platelets and Weibel-Palade bodies of endothelial
cells, is rapidly expressed on the cell surface upon activation, eg, by
hypoxia/reoxygenation.22 P-selectin is a key mediator of
leukocyte rolling and subsequent extravasation during
I/R.4,23 Concomitantly, there is growing evidence that platelets also interact with stimulated endothelial cells via this
particular selectin.24 This finding, together with a rapid expression upon stimulation, makes P-selectin a likely candidate involved in mediating platelet-endothelial cell interactions during initial postischemic reperfusion in which the endothelial barrier is
not necessarily destroyed. However, the role of endothelial/platelet P-selectin for postischemic thrombocyte adhesion has not been investigated thus far.
The aim of the present study, therefore, was to characterize the
nature of platelet-endothelial cell interactions in vivo under
physiologic conditions and to assess whether platelet interactions with
the microvascular endothelium are altered during intestinal I/R. The
role of P-selectin as a molecular determinant of I/R-induced platelet-endothelial cell interactions was assessed by in vivo immunoinhibition with anti-P-selectin monoclonal antibodies (MoAbs). Because both endothelial cells and platelets express P-selectin, the
contribution of P-selectin from either platelets or endothelial cells
was established by the use of P-selectin-deficient mice.
| |
MATERIALS AND METHODS |
Animals.
Female Balb/c mice (Charles River, Sulzfeld, Germany) and
C57BL/6J/129Sv mice (wild-type and P-selectin-deficient25),
aged 5 to 7 weeks, were used (50 experimental animals and 36 platelet donors). P-selectin-deficient mice were generated and kindly provided by Prof A.L. Beaudet's group (Houston, TX).25 All
experimental procedures performed on mice were approved by the German
legislation on protection of animals.
Surgical procedure.
The animals were anesthetized by inhalation of
isoflurane-N2O (FiO2 0.35, 0.015 L/L
isoflurane; Forene; Abbott GmbH, Wiesbaden, Germany) and placed in
supine position on a heating pad for maintenance of body temperature
between 36°C and 37°C. Polyethylene catheters (PE 50, ID 0.28 mm; Portex, Hythe, UK) were inserted into the left carotid artery and
jugular vein for recording of mean arterial and central venous blood
pressure, for blood sampling, for injection of fluorescent dye, and for
infusion of fluorescent platelets, respectively. After a transverse
laparotomy, a segment of the jejunum was gently exteriorized. Segmental
intestinal ischemia was induced by isolating and clamping the arterial
vessels supplying the jejunum for 1 hour. During the entire
experimental procedure, the tissue was constantly superfused with
37°C Ringer's lactate to avoid temperature changes and exposure to
ambient air.
Blood sampling and platelet preparation.
Blood from P-selectin-deficient or wild-type mice was harvested by
cardiac puncture and collected in 1-mL polypropylene tubes containing
0.1 mL volume of 38 mmol/L citric acid/75 mmol/L trisodium citrate/100
mmol/L dextrose. Rhodamine-6G (0.05%; 50 µL/mL whole blood, MW 479;
Sigma, St Louis, MO) was added to label platelets in vitro, and the
blood was centrifuged at 250g for 10 minutes. Platelet-rich
plasma was gently transferred to a fresh tube, and the centrifugation
was repeated at 2,000g for 10 minutes. The platelet pellet was
resuspended in 0.5 mL phosphate-buffered saline (PBS; Seromed, Berlin,
Germany). Fluorescent platelets (50 × 106) were
infused intravenously (IV) over 10 minutes. The purity of the platelet
suspension was confirmed before infusion using a Coulter
ACT Counter (Coulter Corp, Miami, FL).
Intravital fluorescence microscopy.
As described previously (Massberg et al),25a
the exposed segment was scanned from the oral to the aboral section
starting 5 minutes after the onset of reperfusion. Five nonoverlapping regions of interest were selected randomly. Within these regions, 1 to
2 second order (A2) arterioles and postcapillary venules were
visualized in the intestinal submucosa using a modified Leitz-Orthoplan microscope with a 100W HBO mercury lamp, attached to a Ploemo-Pak illuminator (Leitz, Wetzlar, Germany) for
epi-illumination.26 With a 25× water immersion
objective (W 25x/0.6; Leitz), the magnification on the video screen
(PVM-1442 QM, diagonal 33 cm; Sony, Munich, Germany) was 450×.
The microscopic images were recorded by a CCD video camera (FK 6990, Cohu; Prospective Measurements, San Diego, CA) and transferred to a
video system for off-line evaluation. Leukocytes were stained in vivo
by intravenous injection of 0.05% acridine orange (0.05 mL IV, MW 302;
Merck, Darmstadt, Germany). Platelets (50 × 106)
labeled with rhodamine-6G ex vivo were infused via the jugular vein
with a syringe pump (S250i Pump; WPI, Sarasota, FL) for 10 minutes starting 5 minutes before reperfusion. Both platelet- and
leukocyte-endothelial cell interactions were visualized in the same
animal by the use of two different excitation and emission filter sets
(Leitz).26
Video analysis.
Quantitative assessment of microcirculatory parameters was performed
off-line by frame-to-frame analysis of the videotaped images. Vessel
diameters were determined by a computer-assisted image analysis system
(CAP IMAGE; Dr H. Zeintl, Heidelberg,
Germany).27 Platelet-endothelial cell and
leukocyte-endothelial cell interactions were analyzed within
A2-arterioles (mean diameter, 40 µm) and postcapillary venules (mean
diameter, 60 µm) per animal, respectively. Platelets and leukocytes
were classified according to their interaction with the endothelial
cell lining as free flowing, rolling, and adherent cells. Rolling
platelets or leukocytes were defined as cells crossing an imaginary
perpendicular through the vessel at a velocity significantly lower than
the centerline velocity in the microvessel; their numbers are given as
cells per second per vessel diameter. Adherent cells were defined in
each vessel segment as cells that did not move or detach from the
endothelial lining within an observation period of 30 seconds.
Adherence is quantified as number of cells per square millimeter of
endothelial surface, calculated from the diameter and length of the
vessel segment observed, assuming cylindrical geometry. In every
experimental animal, platelet/leukocyte rolling and
adhesion were analyzed within 5 to 7 arterioles and venules. Video
sequences of 30 seconds were recorded of each vessel using the two
different excitation and emission filter sets. Twenty-five minutes were
required to complete intravital microscopy.
Experimental protocol.
To investigate the effects of I/R on platelet-endothelial cell
interaction, the mouse small intestine was subjected to 1 hour of
ischemia (group B, n = 6). Intravital microscopy was performed 5 to 30 minutes after the onset of reperfusion. Sham-operated animals without
intestinal I/R served as controls (group A, n = 6). In group C,
antimouse-P-selectin antibody (2 mg/kg body weight IV, n = 6) was
administered before reperfusion to study the role of P-selectin for
I/R-induced platelet-endothelial cell interactions. A separate group of
animals (group D, n = 6) received the equivalent dose of
isotype-matched control antibody before the onset of reperfusion
(ChromPure rat IgG; Dianova, Hamburg, Germany). To assess the role of
P-selectin from either platelets or endothelium in mediating
I/R-induced platelet-endothelial cell interactions, interaction of
wild-type platelets with P-selectin-deficient endothelium (group E, n = 6) and interaction of P-selectin-deficient platelets with wild-type
endothelium (group F, n = 6) were investigated by intravital
fluorescence microscopy. For each experiment, 1 separate platelet donor
was required.
Histology and immunostaining for P-selectin and von Willebrand
factor.
Tissue samples from the intestinal segment studied were taken 60 minutes after the onset of reperfusion. The biopsies were frozen in
liquid nitrogen and stored at  80°C. Acetone-fixed cryostat sections (6 µm) were stained for P-selectin and von Willebrand factor
(vWF), respectively, by applying the peroxidase technique in a three-stage procedure. Rabbit antimouse vWF and P-selectin MoAbs
and commercially available immunohistochemistry kits (Vectastain; Camon, Wiesbaden, Germany) were used. Control sections were incubated with an isotype-matched primary antibody. Endogenous peroxidase activity was blocked with methanol-H2O2 for 10 minutes at room temperature. An easily detectable reddish-brown-colored
end product was obtained by development in
H2O2/3-amino-9-ethylcarbazol. The sections were
counter-stained with Mayer's hemalaun.
Electron microscopy.
Tissue was excised for electron microscopy from sham-operated control
and I/R-experiments (groups A and B). After sampling for light
microscopy, a perfusion with 10 mL Ringer's lactate followed by
Karnovsky's solution (0.04 mL/g body weight) was performed via the
carotid artery for 5 minutes. After this perfusion, small, approximately 1 × 2 mm pieces were taken from the jejunal segment and further fixed in Karnovsky's solution for 2 to 3 days. The samples
were postfixed with 2% osmium tetroxide in Sörensen buffer (pH
7.4) at room temperature and then dehydrated and embedded in Araldite
monomer solution for 24 hours at 60°C. Ultrathin sections were cut
and stained with uranyl acetate and lead citrate and examined under a
Zeiss EM 900 transmission electron microscope operating at 80 kV.
Flow cytometry.
The expression of CD11b and L-selectin on circulating granulocytes and
P-selectin on circulating platelets during I/R was determined ex vivo
in a separate set of experiments. The left carotid artery was
cannulated, a transverse laparotomy was performed, and a segmental
intestinal ischemia (1 hour) was induced as described above (n = 7).
Sham-operated animals without I/R served as control (n = 7).
Blood samples were drawn from the carotid artery before ischemia as
well as 30 and 60 minutes after the onset of reperfusion. Expression of
CD11b, L-selectin (CD62L) on leukocytes, and P-selectin (CD62P) on
platelets was assessed by direct immunofluorescence using rat antimouse
phycoerythrin- or fluorescein isothiocyanate (FITC)-coupled monoclonal
IgG (Pharmingen, Hamburg, Germany). Fluorochrome-coupled
IgG1 and IgG2 isotype-matched control
antibodies were used to exclude unspecific binding. The cells were
incubated with saturating amounts of MoAb for 30 minutes at 4°C.
After incubation, cells were washed twice with PBS. A commercially
available lysing solution (FACS Lysing solution; Becton Dickinson,
Heidelberg, Germany) was added for the removal of erythrocytes and
fixation of cells. For determination of platelet P-selectin expression, lysis and fixation were omitted. Analysis of 10,000 events was performed on a FACSort flow cytometer (Becton Dickinson). Granulocytes and platelets were selectively analyzed for their fluorescence properties using a Lysis II data handling program (Becton Dickinson). The relative fluorescence intensity of a given sample was calculated by
subtracting the signal obtained when cells were incubated with the
corresponding isotype control from the signal generated by cells
incubated with the test antibody.
MoAbs.
Unlabeled rat antimouse-P-selectin antibodies for in vivo
immunoinhibition studies were generated as described by Weller et al.28 Isotype-matched control IgG was obtained from
Dianova. For flow cytometry, commercially available FITC- and
phycoerythrin (PE)-labeled rat antimouse CD11b, L-selectin, P-selectin
MoAb, and isotype- matched rat IgG1 and IgG2
were used (Pharmingen). Immunohistology was performed using monoclonal
rat antimouse P-selectin and vWF antibodies, respectively, purchased
from Pharmingen.
Statistics.
Data analysis was performed with a statistical software package
(SigmaStat for Windows; Jandel Scientific, Erkrath,
Germany). The Kruskal-Wallis test followed by Dunn's method was used
for the estimation of stochastic probability in intergroup comparisons. Mean values ± standard error of the mean (SEM) are given. P
values less than .05 were considered significant.
| |
RESULTS |
Platelet preparation for intravital fluorescence microscopy.
The purity and fluorescent labeling of the platelet sample was
ascertained before infusion by flow cytometry. Platelet separation by
differential centrifugation yielded a platelet suspension with negligible amounts of other cellular components. Less than 5 leukocytes/106 platelets were found in the platelet
preparations used for the present study. P-selectin expression (mean
fluorescence intensity) after blood sampling was not increased when
additional differential centrifugation was performed (P < .788), indicating that significant platelet activation during the
preparation procedure did not occur. However, in response to thrombin
(1 U/mL), a 10-fold increase of P-selectin expression on identically
treated platelets was observed (P < .05). A concomitant
increase in the forward scatter light signal showed that the cells
sustained the ability to form aggregates (data not shown). No adverse
effects on heart rate or blood pressure were observed upon IV infusion
of 50 × 106 labeled platelets.
Leukocyte-endothelial cell interactions in vivo.
Leukocyte behavior in the intestinal microcirculation was investigated
in control animals and after segmental I/R. Rolling and firm adhesion
of leukocytes were never observed in arterioles and only rarely in
postcapillary venules of the intestinal submucosa of sham-operated
wild-type mice (Table 1). In contrast, I/R
markedly enhanced leukocyte-endothelial cell interactions in
postcapillary venules. Both leukocyte rolling and firm adherence were
significantly increased 15 minutes after the onset of reperfusion.
Immunoinhibition by anti-P-selectin MoAb almost completely prevented
rolling and attenuated firm adhesion of leukocytes. Concomitantly,
microvascular leukocyte accumulation in response to I/R was nearly
absent in mice deficient in P-selectin (data not shown), indicating a
key role of P-selectin for early postischemic leukocyte recruitment at
the site of injury.
Platelet-endothelial cell interactions in vivo.
Platelet interactions with the microvascular endothelium showed
striking parallels to leukocyte-endothelial cell interactions; both
intermittent adhesion (rolling) and firm adhesion of platelets were
seen. However, platelet-endothelial cell interactions were not confined
to postcapillary venules, but were also prominent in smaller arterioles
(mean diameter, 40 µm) within the intestinal submucosa
(Figs 1 and
2). Similar to leukocytes,
platelets rarely interacted with the microvascular endothelium in
sham-operated wild-type mice. Only a few platelets were encountered
rolling along the endothelial cell lining of arterioles and
postcapillary venules, respectively. Firm adherence was 60.0 ± 25.7 and 23.8 ± 8.8 mm-2 in arterioles and venules,
respectively, indicating that platelet-endothelial cell interactions
play a minor role under physiologic conditions. In contrast, I/R
induced platelet interactions with the endothelial cell surface already
5 minutes after the onset of reoxygenation without further increase
throughout the remaining observation period. There were
26.9 ± 3.2 and 42.0 ± 5.8 platelets/sec/mm vessel diameter
rolling along the arteriolar and venular vessel wall, respectively. In
parallel, the number of firmly adherent platelets increased 10-fold,
compared with sham-operated animals. Frequently aggregated leukocytes
coated with rhodamine-labeled platelets were observed. Anti-P-selectin MoAb almost completely prevented platelet rolling and firm adhesion in
arterioles and venules in response to I/R, indicating that P-selectin
is the molecular determinant of postischemic platelet-endothelial cell
interactions during initial postischemic reperfusion. Moreover, anti-P-selectin MoAb inhibited the interaction of platelets with leukocytes. These effects were specific inasmuch as an isotype-matched control MoAb did not attenuate I/R-induced platelet interactions with
either endothelium or leukocytes.
View larger version (11K):
[in this window]
[in a new window]
View larger version (12K):
[in this window]
[in a new window]
| Fig 1.
Platelet-endothelial cell interactions during I/R of the
small intestine in vivo. Platelet-endothelial cell interactions were investigated in arterioles (A) and venules (B) using intravital fluorescence microscopy. Sham-operated animals served as controls. According to their interaction with the endothelial cell lining, platelets were classified into rolling or firmly adherent cells. Rolling platelets (upper panels) are presented as the number of cells
per second and millimeters of vessel diameter; adherent platelets
(lower panels) are given per square millimeter of vessel surface. Mean ± SEM, n = 6 experimental animals per group. *P < .01 versus sham, #P < .01 versus anti-CD62P MoAb,
Dunn's method.
|
|
View larger version (57K):
[in this window]
[in a new window]
| Fig 2.
I/R-induced platelet-endothelial cell
interactions in vivo. Rhodamine-6G-labeled platelets are visualized
within postcapillary venules using intravital fluorescence microscopy.
Whereas few platelets adhere to the venular endothelium in
sham-operated animals (group B, left panel), platelet attachment to the
endothelium is significantly enhanced in response to I/R already 5 minutes after the onset of reoxygenation (group A, right panel, arrow). Original monitor magnification × 450.
|
|
Because both platelets and endothelial cells express P-selectin, it was
important to clarify whether endothelial or platelet P-selectin was
required for I/R-dependent platelet-endothelial cell interactions. To
study this, wild-type or P-selectin-deficient platelets were separated
and transfused into P-selectin-deficient or wild-type mice,
respectively. During postischemic reperfusion, P-selectin-deficient
platelets interacted similarly with wild-type endothelium as described
above for wild-type platelets. In contrast, I/R-induced platelet
rolling and firm adhesion was almost absent when wild-type platelets
were transfused into P-selectin-deficient mice (Fig 1). These results
provide evidence that endothelial P-selectin interacting with a
putative P-selectin ligand on the platelet is the key mediator for
postischemic platelet-endothelial cell interaction. Because aggregation
of mutant platelets with wild-type leukocytes was not observed, our
results support the notion that platelet P-selectin interacting with a
ligand on myeloid cells, presumably PSGL-1, is responsible for
platelet-leukocyte interaction in response to I/R.
Electron microscopy.
Electron microscopy confirmed the presence of platelets adherent to
endothelial cells in response to I/R. Platelets in various states of
activation were frequently observed attached to the postischemic
microvascular endothelium (Fig 3).
Adherent, single, or aggregated platelets appeared degranulated
extending pseudopodia to the endothelial cell surfaces. In most
instances, platelets adhered directly to endothelial cells, and obvious
endothelial denudation was not detected. Occasionally, neutrophils
carrying activated platelets adherent on the surface could be
demonstrated (Fig 3). In animals not exposed to I/R, few platelets with
direct contact with the endothelium were seen. Platelets in these
animals generally retained a discoid shape with prominent -granules.
View larger version (200K):
[in this window]
[in a new window]
View larger version (168K):
[in this window]
[in a new window]
| Fig 3.
Platelets in postischemic microvasculature visualized by
electron microscopy. Electron microscopy demonstrated platelets
attached to both endothelial cells (arrowhead) and neutrophils (arrows) in response to I/R (upper panel, original magnification × 3,000). Platelets bind directly to endothelial cells (lower panel,
original magnification × 20,000).
|
|
Immunohistochemistry.
To investigate the expression of P-selectin within the postischemic
microvasculature, immunohistochemistry was performed on cryostat
sections using MoAb against P-selectin. In addition, vWF
immunoreactivity was investigated to identify vascular structures within the serial sections. In sham-operated wild-type mice, P-selectin was only weakly expressed by vascular endothelium of venules and to a
lesser extent of arterioles (Fig 4). I/R markedly
enhanced immunoreactivity for P-selectin in both arterioles and venules (Fig 4). Because of the large increase in platelet adhesion to the
postischemic endothelium, we were not able to clearly distinguish by
light microscopy whether the enhanced P-selectin expression was indeed
present on the endothelial cells or whether it was due to platelets
attached to the vessel wall. Formation of platelet aggregates,
identified by an intense staining for P-selectin, was a prominent
phenomenon after I/R. In contrast, platelet aggregates, which in some
instances contained polymorphonuclear leukocytes, were never seen in
sham-operated control animals or in animals treated with
anti-P-selectin MoAb. Similarly, in P-selectin-deficient mice,
immunoreactivity for P-selectin was never detected, neither after
sham-operation nor after I/R.
View larger version (164K):
[in this window]
[in a new window]
| Fig 4.
P-selectin
expression in arterioles and venules of mouse small intestine. Cryostat
sections of intestinal specimens were incubated with a MoAb against
P-selectin and stained using the peroxidase-antiperoxidase technique as
described in the Materials and Methods. Counterstaining was performed
using Mayer's hemalaun. In control animals (A, C, and E), P-selectin
was weakly expressed by vascular endothelium of arterioles (C) and
venules (E). In contrast, I/R (B, D, and F) markedly enhanced
immunoreactivity for P-selectin in both arterioles (D) and venules (F).
Formation of platelet aggregates, identified by an intense staining for
P-selectin, was a prominent phenomenon after I/R (arrowheads). The
magnification of the objective was 40× in (A) and (B) and 100× in
(C) through (F), respectively.
|
|
Flow cytometry.
Based on our in vivo results, endothelial cell activation with
upregulation of P-selectin by exocytosis of Weibel-Palade bodies appears to be the primary mechanism for postischemic
platelet-endothelial cell interaction. Nonetheless, it was important to
determine whether I/R would lead to an activation of platelets. To
study this, P-selectin expression on circulating platelets in response
to intestinal I/R was evaluated by flow cytometry. No change in
platelet P-selectin immunoreactivity was observed during
postischemic reperfusion; 25.3% ± 4.2%, 22.2% ± 6.8%, and 24.6% ± 5.8% of all platelets stained positive for
P-selectin before ischemia as well as 30 and 60 minutes after the onset
of reperfusion, respectively. Furthermore, there was no shift in mean
fluorescence intensity when compared with baseline conditions or to
sham-operated control animals. In contrast, I/R was associated with
activation of circulating leukocytes, as indicated by a 1.5-fold
enhanced CD11b expression 30 and 60 minutes after the onset of
reperfusion (data not shown). Shedding of L-selectin was not observed
(data not shown).
| |
DISCUSSION |
Platelets might play an important role during the manifestation of I/R
injury. Platelets, like leukocytes, accumulate in
postischemic/reperfused tissues and contribute to endothelial and
parenchymal cell injury.6-8 Whereas several distinct
adhesive interactions involved in the process of I/R-induced leukocyte
accumulation have been described,4 the mechanisms that
initiate platelet accumulation in the postischemic microvasculature
have not been identified to date. The present study, therefore, has
investigated the in vivo interactions of platelets with the
microvascular endothelium in response to intestinal I/R. We found that
platelets similar to leukocytes roll along and firmly adhere to the
endothelial surface of microvessels during postischemic reperfusion.
Local accumulation of platelets was observed reaching a maximum within
minutes after the onset of reperfusion, indicating that platelets are
among the first cells recruited to the site of injury.
Because P-selectin, which is stored in intracellular granules of
endothelial cells and platelets, can be rapidly mobilized to the cell
surface in response to various stimuli independent of de novo mRNA or
protein synthesis,29,30 we evaluated the role of this
lectin-like adhesion molecule in mediating initial postischemic
platelet-endothelial cell interactions. Our results provide
strong evidence that P-selectin expressed by the endothelium is the
molecular determinant of I/R-induced platelet-endothelial cell
interactions: An anti-P-selectin MoAb attenuated rolling and subsequent
firm adherence of platelets. Moreover, rolling and adherence of
wild-type platelets were nearly absent in mice with
P-selectin-deficient endothelium, whereas P-selectin-deficient and
wild-type platelets interacted similarly with postischemic wild-type
endothelium. No increase in the P-selectin expression was found on
circulating wild-type platelets during postischemic reperfusion. In
contrast, I/R clearly enhanced P-selectin immunoreactivity in the
postischemic microvasculature. This is likely due either to endothelial
platelet adhesion or the result of a redistribution of the P-selectin
to the endothelial cell surface. Although we were not able to
demonstrate P-selectin mobilization to the endothelial cell surface by
immunohistochemistry studies, both in vitro and ex vivo experiments
have previously documented that hypoxia/reoxygenation22 or
thrombin31,32 that is generated during I/R33
induces rapid mobilization of P-selectin onto the endothelial cell
surface. At the same time, it has been demonstrated that the expression of P-selectin on the endothelium upon endothelial activation induces platelet-endothelial cell interactions in
vivo.24 Hence, in line with our findings, this indicates
that the P-selectin present on the endothelium mediates platelet
adhesion to stimulated/injured endothelial cells in response to I/R.
This is in contrast to platelet adhesion to the exposed subendothelial
matrix via integrins such as the GPIIb/IIIa complex (CD41/CD61) and via
the leucin-rich glycoprotein complex GPIb-X (CD42a/CD42b), which occurs
when the endothelium is denuded.21
The interactions between platelets and endothelium in contrast to
leukocyte-endothelial cell interactions were not restricted to venules,
but were also prominent in arterioles. Several reasons may explain the
presence of platelet-endothelial cell interactions in arterioles. It
has been shown in vivo that near the vessel wall the amount of
platelets per unit volume is significantly higher in arterioles
compared with venules.34 Based on estimates of wall shear
rates in vivo,35 platelets near the vessel wall are exposed
to much lower shear forces as compared with the leukocytes due to their
smaller diameter (2 µm). This means that even weak receptor-ligand
interactions may be sufficient to allow platelet-endothelial cell
interactions in arterioles. Differences in the properties of the
P-selectin ligand expressed on platelets and leukocytes could also
contribute to the different behavior of these cell types. However,
there have been no studies that have addressed this question, and the
exact nature of the ligand on platelets that binds to endothelial
P-selectin remains unclear. PSGL-1, a glycoprotein that avidly binds to
P-selectin, is expressed predominantly on myeloid cells and mediates
leukocyte-endothelium interactions and leukocyte-platelet interactions
in vitro and in vivo.12,36-39 Although PSGL-1 might also be
involved in platelet-endothelial cell interactions, the presence of
this mucin-like glycoprotein on the platelet membrane has not yet been
documented.40 Hence, platelets might also carry a
different, undefined P-selectin ligand. This ligand would be most
likely a sialylated carbohydrate determinant, such as sialyl
Lewisx.41
Although platelet P-selectin appears to play a minor role for
I/R-induced platelet-endothelial cell interactions, the complex adhesive apparatus expressed on the platelet cell surface allows them
to cooperate and synergize with leukocytes during I/R. Firm adherence
of circulating leukocytes to inflamed vascular endothelium is an
essential component of a cascade that results in the adhesion and
eventual emigration of leukocytes through the vessel wall. Platelets
and leukocytes colocalize in areas of infarction,42 indicating that platelets might significantly contribute to I/R-induced inflammatory responses. By adhesion to endothelial cells or
subendothelial structures, platelets might occupy a position analogous
to endothelium with respect to leukocyte accumulation and emigration.
P-selectin expressed by surface adherent, activated platelets mediates
rolling of leukocytes under flow in vitro and is involved in leukocyte attachment to artificial vessel surfaces in vivo.43-47
Rolling leukocytes may sequentially develop firm adhesion and
transmigrate across surface-adherent platelets via the
 2-integrin CD11b/CD18,46 indicating that
platelets are actually involved in the multistep process of I/R-induced
leukocyte accumulation and extravasation. However, leukocytes do not
attach solely to surface adherent platelets. As our results
demonstrate, circulating leukocyte-platelet aggregates are formed in
vivo after exposure to I/R, and neutralization of P-selectin diminishes
these interactions. Similarly, aggregates are absent in
P-selectin-deficient mice, providing further evidence that
platelet-leukocyte aggregation involves platelet P-selectin interacting
with a functional ligand on the leukocyte, presumably PSGL-1.11
P-selectin-mediated platelet interactions with leukocytes or
endothelial cells may modify and directly promote functional responses.
Adhesion of activated platelets to leukocytes induces nuclear
translocation of NF- B, and enhances expression of CD11b/CD18 and
generation of monocyte chemotactic protein (MCP)-1 and superoxide anions.12,44,48 In all instances, binding of platelet
P-selectin to its ligand on the leukocyte is required. Although similar
studies have not yet been performed with regard to platelet-endothelial cell-interactions, platelet attachment to postischemic endothelial cells might modulate endothelial cell function. Platelets release proinflammatory and chemotactic mediators such as PAF,
epithelial-derived neutrophil activating factor-78 (ENA-78),
neutrophil-activating peptide-2 (NAP-2), RANTES, platelet factor 4, serotonin, interleukin-8, and monocyte chemotactic protein-3
(MCP-3),12-17,49 whereby they may activate both endothelium
and leukocytes. Hence, it is conceivable that platelet-endothelium
interaction via P-selectin facilitates juxtacrine activation of the
cells by mediating close cell-to-cell contact.50 Whether
P-selectin-ligand interactions may also directly initiate intracellular
signaling pathways that effect further endothelial cell functional
responses has not been elucidated so far.
In conclusion, we have demonstrated that platelets, similarly to
leukocytes, roll along and firmly adhere to microvascular endothelium
during postischemic reperfusion. I/R-induced platelet-endothelial cell
interactions are mediated via endothelial P-selectin, whereas platelet
P-selectin promotes platelet interactions with leukocytes. Because
platelets release potent proinflammatory chemokines and modulate
leukocyte function, platelet accumulation in the postischemic microvasculature might significantly contribute to the manifestation of
I/R injury.
| |
FOOTNOTES |
Submitted December 27, 1997;
accepted March 17, 1998.
Supported by Research Grant Biomed 2 Contract No. BMH4-CT95-0875
(DG12-SSMA).
Address reprint requests to Steffen Massberg, MD, Institute for
Surgical Research, Ludwig-Maximilians-University, Klinikum Grosshadern, Marchioninistrasse 15, D-81366 Munich, Germany;
e-mail: massberg{at}icf.med.uni-muenchen.de.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Sylvia Münzing, Elke Schütze, Katrin
Baltzer, and Bärbel Lorenz for their excellent and skillful
technical assistance.
| |
REFERENCES |
1.
Harris AG,
Leiderer R,
Peer F,
Messmer K:
Skeletal muscle microvascular and tissue injury after varying durations of ischemia.
Am J Physiol
271:H2388,
1996[Abstract/Free Full Text]
2.
Nolte D,
Hecht R,
Schmid P,
Botzlar A,
Menger MD,
Neumueller C,
Sinowatz F,
Vestweber D,
Messmer K:
Role of Mac-1 and ICAM-1 in ischemia-reperfusion injury in a microcirculation model of BALB/C mice.
Am J Physiol
267:H1320,
1994[Abstract/Free Full Text]
3.
Lehr HA,
Menger MD,
Messmer K:
Impact of leukocyte adhesion on myocardial ischemia/reperfusion injury: Conceivable mechanisms and proven facts.
J Lab Clin Med
121:539,
1993[Medline]
[Order article via Infotrieve]
4.
Ley K:
Molecular mechanisms of leukocyte recruitment in the inflammatory process.
Cardiovasc Res
32:733,
1996[Medline]
[Order article via Infotrieve]
5.
Eppihimer MJ,
Granger DN:
Ischemia/reperfusion-induced leukocyte-endothelial interactions in postcapillary venules.
Shock
8:16,
1997[Medline]
[Order article via Infotrieve]
6.
Chintala MS,
Bernardino V,
Chiu PJ:
Cyclic GMP but not cyclic AMP prevents renal platelet accumulation after ischemia-reperfusion in anesthetized rats.
J Pharmacol Exp Ther
271:1203,
1994[Abstract/Free Full Text]
7.
Kuroda T,
Shiohara E:
Leukocyte and platelet depletion protects the liver from damage induced by cholestasis and ischemia-reperfusion in the dog.
Scand J Gastroenterol
31:182,
1996[Medline]
[Order article via Infotrieve]
8.
Kuroda T,
Shiohara E,
Homma T,
Furukawa Y,
Chiba S:
Effects of leukocyte and platelet depletion on ischemia Reperfusion injury to dog pancreas.
Gastroenterology
107:1125,
1994[Medline]
[Order article via Infotrieve]
9.
Marcus AJ:
Pathways of oxygen utilization by stimulated platelets and leukocytes.
Semin Hematol
16:188,
1979[Medline]
[Order article via Infotrieve]
10.
Leo R,
Pratico D,
Iuliano L,
Pulcinelli FM,
Ghiselli A,
Pignatelli P,
Colavita AR,
FitzGerald GA,
Violi F:
Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated.
Circulation
95:885,
1997[Abstract/Free Full Text]
11.
Forde RC,
Fitzgerald DJ:
Reactive oxygen species and platelet activation in reperfusion injury.
Circulation
95:787,
1997[Free Full Text]
12.
Weyrich AS,
Elstad MR,
McEver RP,
McIntyre TM,
Moore KL,
Morrissey JH,
Prescott SM,
Zimmerman GA:
Activated platelets signal chemokine synthesis by human monocytes.
J Clin Invest
97:1525,
1996[Medline]
[Order article via Infotrieve]
13.
Bubel S,
Wilhelm D,
Entelmann M,
Kirchner H,
Kluter H:
Chemokines in stored platelet concentrates.
Transfusion
36:445,
1996[Medline]
[Order article via Infotrieve]
14.
Barry OP,
Pratico D,
Lawson JA,
FitzGerald GA:
Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles.
J Clin Invest
99:2118,
1997[Medline]
[Order article via Infotrieve]
15.
Piccardoni P,
Evangelista V,
Piccoli A,
Degaetano G,
Walz A,
Cerletti C:
Thrombin activated human platelets release two NAP 2 variants that stimulate polymorphonuclear leukocytes.
Thromb Haemost
76:780,
1996[Medline]
[Order article via Infotrieve]
16.
Deuel TF,
Senior RM,
Chang D,
Griffin GL,
Heinrikson RL,
Kaiser ET:
Platelet factor 4 is chemotactic for neutrophils and monocytes.
Proc Natl Acad Sci USA
78:4584,
1981[Abstract/Free Full Text]
17.
Hervig TA,
Farstad M:
Human blood platelet serotonin studied in vitro: Endogenous serotonin may stimulate thrombin induced serotonin release in stored platelets.
Platelets
7:53,
1996
18.
Ruf A,
Patscheke H:
Platelet-induced neutrophil activation: Platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18.
Br J Haematol
90:791,
1995[Medline]
[Order article via Infotrieve]
19.
Faint RW:
Platelet-neutrophil interactions: Their significance.
Blood Rev
6:83,
1992[Medline]
[Order article via Infotrieve]
20.
Williams MJ,
Du X,
Loftus JC,
Ginsberg MH:
Platelet adhesion receptors.
Semin Cell Biol
6:305,
1995[Medline]
[Order article via Infotrieve]
21. (suppl 1)
Body SC:
Platelet activation and interactions with the microvasculature.
J Cardiovasc Pharmacol
27:S13,
1996
22.
Pinsky DJ,
Naka Y,
Liao H,
Oz MC,
Wagner DD,
Mayadas TN,
Johnson RC,
Hynes RO,
Heath M,
Lawson CA,
Stern DM:
Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.
J Clin Invest
97:493,
1996[Medline]
[Order article via Infotrieve]
23.
Ley K,
Bullard DC,
Arbones ML,
Bosse R,
Vestweber D,
Tedder TF,
Beaudet AL:
Sequential contribution of L- and P-selectin to leukocyte rolling in vivo.
J Exp Med
181:669,
1995[Abstract/Free Full Text]
24.
Frenette PS,
Johnson RC,
Hynes RO,
Wagner DD:
Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin.
Proc Natl Acad Sci USA
92:7450,
1995[Abstract/Free Full Text]
25.
Bullard DC,
Qin L,
Lorenzo I,
Quinlin WM,
Doyle NA,
Bosse R,
Vestweber D,
Doerschuk CM,
Beaudet AL:
P-selectin/ICAM-1 double mutant mice: Acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli.
J Clin Invest
95:1782,
1995
25a. Massberg S, Eisenmenger S, Enders G, Krombach F, Messmer
K: Quantitative analysis of small intestinal
microcirculation in the mouse. Res Exp Med (in press)
26.
Massberg S,
Leiderer R,
Gonzalez AP,
Menger MD,
Messmer K:
Carolina Rinse attenuates postischemic microvascular injury in rat small bowel isografts.
Surgery
123:181,
1998[Medline]
[Order article via Infotrieve]
27.
Klyscz T,
Jünger M,
Jung F,
Zeintl H:
CAP IMAGE: A newly developed computer aided videoframe analysis system for dynamic capillaroscopy.
Biomedizinische Technik
42:168,
1997 [Medline]
[Order article via Infotrieve]
28.
Weller A,
Isenmann S,
Vestweber D:
Cloning of the mouse endothelial selectins. Expression of both E- and P-selectin is inducible by tumor necrosis factor alpha.
J Biol Chem
267:15176,
1992[Abstract/Free Full Text]
29.
Stenberg PE,
McEver RP,
Shuman MA,
Jacques YV,
Bainton DF:
A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation.
J Cell Biol
101:880,
1985[Abstract/Free Full Text]
30.
Hattori R,
Hamilton KK,
Fugate RD,
McEver RP,
Sims PJ:
Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140.
J Biol Chem
264:7768,
1989[Abstract/Free Full Text]
31.
Czervionke RL,
Hoak JC,
Fry GL:
Effect of aspirin on thrombin-induced adherence of platelets to cultured cells from the blood vessel wall.
J Clin Invest
62:847,
1978
32.
Venturini CM,
Fenton JW,
Minnear FL,
Kaplan JE:
Rat platelets adhere to human thrombin-treated rat lungs under flow conditions.
Thromb Haemost
62:1006,
1989[Medline]
[Order article via Infotrieve]
33.
Hoffmeister HM,
Jur M,
Wendel HP,
Heller W,
Seipel L:
Alterations of coagulation and fibrinolytic and kallikrein-kinin systems in the acute and postacute phases in patients with unstable angina pectoris.
Circulation
91:2520,
1995[Abstract/Free Full Text]
34.
Woldhuis B,
Tangelder GJ,
Slaaf DW,
Reneman RS:
Concentration profile of blood platelets differs in arterioles and venules.
Am J Physiol
262:H1217,
1992[Abstract/Free Full Text]
35.
Tangelder GJ,
Slaaf DW,
Arts T,
Reneman RS:
Wall shear rate in arterioles in vivo: Least estimates from platelet velocity profiles.
Am J Physiol
254:H1059,
1988[Abstract/Free Full Text]
36.
Borges E,
Tietz W,
Steegmaier M,
Moll T,
Hallmann R,
Hamann A,
Vestweber D:
P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.
J Exp Med
185:573,
1997[Abstract/Free Full Text]
37.
Moore KL,
Patel KD,
Bruehl RE,
Fugang L,
Johnson DA,
Lichtenstein HS,
Cummings RD,
Bainton DF,
McEver RP:
P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.
J Cell Biol
128:661,
1995[Abstract/Free Full Text]
38.
Norman KE,
Moore KL,
McEver RP,
Ley K:
Leukocyte rolling in vivo is mediated by P-selectin glycoprotein ligand-1.
Blood
86:4417,
1995[Abstract/Free Full Text]
39.
Furie B,
Furie BC:
The molecular basis of platelet and endothelial cell interaction with neutrophils and monocytes: Role of P-selectin and the P-selectin ligand, PSGL-1.
Thromb Haemost
74:224,
1995[Medline]
[Order article via Infotrieve]
40.
Laszik Z,
Jansen PJ,
Cummings RD,
Tedder TF,
McEver RP,
Moore KL:
P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells.
Blood
88:3010,
1996[Abstract/Free Full Text]
41.
Springer TA,
Lasky LA:
Sticky sugars for selectins.
Nature
349:196,
1991[Medline]
[Order article via Infotrieve]
42.
Bednar M,
Smith B,
Pinto A,
Mullane KM:
Neutrophil depletion suppresses 111In-labeled platelet accumulation in infarcted myocardium.
J Cardiovasc Pharmacol
7:906,
1985[Medline]
[Order article via Infotrieve]
43.
Buttrum SM,
Hatton R,
Nash GB:
Selectin-mediated rolling of neutrophils on immobilized platelets.
Blood
82:1165,
1993[Abstract/Free Full Text]
44.
Yeo EL,
Sheppard JA,
Feuerstein IA:
Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model).
Blood
83:2498,
1994[Abstract/Free Full Text]
45.
Lalor P,
Nash GB:
Adhesion of flowing leucocytes to immobilized platelets.
Br J Haematol
89:725,
1995[Medline]
[Order article via Infotrieve]
46.
Diacovo TG,
Roth SJ,
Buccola JM,
Bainton DF,
Springer TA:
Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18.
Blood
88:146,
1996[Abstract/Free Full Text]
47.
Palabrica T,
Lobb R,
Furie BC,
Aronovitz M,
Benjamin C,
Hsu YM,
Sajer SA,
Furie B:
Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets.
Nature
359:848,
1992[Medline]
[Order article via Infotrieve]
48.
Neumann FJ,
Marx N,
Gawaz M,
Brand K,
Ott I,
Rokitta C,
Sticherling C,
Meinl C,
May A,
Schömig A:
Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets.
Circulation
95:2387,
1997[Abstract/Free Full Text]
49.
Power CA,
Clemetson JM,
Clemetson KJ,
Wells TN:
Chemokine and chemokine receptor mRNA expression in human platelets.
Cytokine
7:479,
1995[Medline]
[Order article via Infotrieve]
50.
Lorant DE,
Topham MK,
Whatley RE,
McEver RP,
McIntyre TM,
Prescott SM,
Zimmerman GA:
Inflammatory roles of P-selectin.
J Clin Invest
92:559,
1993
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. Osman, J. Russell, and D. N. Granger
Lymphocyte-derived interferon-{gamma} mediates ischemia-reperfusion-induced leukocyte and platelet adhesion in intestinal microcirculation
Am J Physiol Gastrointest Liver Physiol,
March 1, 2009;
296(3):
G659 - G663.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. van Gils, J. J. Zwaginga, and P. L. Hordijk
Molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases
J. Leukoc. Biol.,
February 1, 2009;
85(2):
195 - 204.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Wagner and P. S. Frenette
The vessel wall and its interactions
Blood,
June 1, 2008;
111(11):
5271 - 5281.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Siegel-Axel, K. Daub, P. Seizer, S. Lindemann, and M. Gawaz
Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis
Cardiovasc Res,
April 1, 2008;
78(1):
8 - 17.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Stellos, H. Langer, K. Daub, T. Schoenberger, A. Gauss, T. Geisler, B. Bigalke, I. Mueller, M. Schumm, I. Schaefer, et al.
Platelet-Derived Stromal Cell-Derived Factor-1 Regulates Adhesion and Promotes Differentiation of Human CD34+ Cells to Endothelial Progenitor Cells
Circulation,
January 15, 2008;
117(2):
206 - 215.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Danese, E. Dejana, and C. Fiocchi
Immune Regulation by Microvascular Endothelial Cells: Directing Innate and Adaptive Immunity, Coagulation, and Inflammation
J. Immunol.,
May 15, 2007;
178(10):
6017 - 6022.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. von Hundelshausen and C. Weber
Platelets as Immune Cells: Bridging Inflammation and Cardiovascular Disease
Circ. Res.,
January 5, 2007;
100(1):
27 - 40.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Kuligowski, A. R. Kitching, and M. J. Hickey
Leukocyte Recruitment to the Inflamed Glomerulus: A Critical Role for Platelet-Derived P-Selectin in the Absence of Rolling.
J. Immunol.,
June 1, 2006;
176(11):
6991 - 6999.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Xu, Y. Huo, M.-C. Toufektsian, S. I. Ramos, Y. Ma, A. D. Tejani, B. A. French, and Z. Yang
Activated platelets contribute importantly to myocardial reperfusion injury
Am J Physiol Heart Circ Physiol,
February 1, 2006;
290(2):
H692 - H699.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Fung, R. R. Fiscus, A. P. C. Yim, G. D. Angelini, and A. A. Arifi
The Potential Use of Type-5 Phosphodiesterase Inhibitors in Coronary Artery Bypass Graft Surgery
Chest,
October 1, 2005;
128(4):
3065 - 3073.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Ferenc and F.-J. Neumann
Efficacy of primary PCI: the microvessel perspective
Eur. Heart J. Suppl.,
October 1, 2005;
7(suppl_I):
I4 - I9.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Massberg, K. Schurzinger, M. Lorenz, I. Konrad, C. Schulz, N. Plesnila, E. Kennerknecht, M. Rudelius, S. Sauer, S. Braun, et al.
Platelet Adhesion Via Glycoprotein IIb Integrin Is Critical for Atheroprogression and Focal Cerebral Ischemia: An In Vivo Study in Mice Lacking Glycoprotein IIb
Circulation,
August 23, 2005;
112(8):
1180 - 1188.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Khandoga, J. S. Kessler, H. Meissner, M. Hanschen, M. Corada, T. Motoike, G. Enders, E. Dejana, and F. Krombach
Junctional adhesion molecule-A deficiency increases hepatic ischemia-reperfusion injury despite reduction of neutrophil transendothelial migration
Blood,
July 15, 2005;
106(2):
725 - 733.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mori, J. W. Salter, T. Vowinkel, C. F. Krieglstein, K. Y. Stokes, and D. N. Granger
Molecular determinants of the prothrombogenic phenotype assumed by inflamed colonic venules
Am J Physiol Gastrointest Liver Physiol,
May 1, 2005;
288(5):
G920 - G926.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. C. van der Heyde, I. Gramaglia, G. Sun, and C. Woods
Platelet depletion by anti-CD41 ({alpha}IIb) mAb injection early but not late in the course of disease protects against Plasmodium berghei pathogenesis by altering the levels of pathogenic cytokines
Blood,
March 1, 2005;
105(5):
1956 - 1963.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Barrabes, D. Garcia-Dorado, M. Mirabet, J. Inserte, L. Agullo, B. Soriano, A. Massaguer, F. Padilla, R.-M. Lidon, and J. Soler-Soler
Antagonism of selectin function attenuates microvascular platelet deposition and platelet-mediated myocardial injury after transient ischemia
J. Am. Coll. Cardiol.,
January 18, 2005;
45(2):
293 - 299.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. C. Wood, R. P. Hebbel, and D. N. Granger
Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice
Am J Physiol Heart Circ Physiol,
May 1, 2004;
286(5):
H1608 - H1614.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Cooper, J. Russell, K. D. Chitman, M. C. Williams, R. E. Wolf, and D. N. Granger
Leukocyte dependence of platelet adhesion in postcapillary venules
Am J Physiol Heart Circ Physiol,
May 1, 2004;
286(5):
H1895 - H1900.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. N. Granger, T. Vowinkel, and T. Petnehazy
Modulation of the Inflammatory Response in Cardiovascular Disease
Hypertension,
May 1, 2004;
43(5):
924 - 931.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Khandoga, A. Stampfl, S. Takenaka, H. Schulz, R. Radykewicz, W. Kreyling, and F. Krombach
Ultrafine Particles Exert Prothrombotic but Not Inflammatory Effects on the Hepatic Microcirculation in Healthy Mice In Vivo
Circulation,
March 16, 2004;
109(10):
1320 - 1325.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nishijima, J. Kiryu, A. Tsujikawa, K. Miyamoto, M. Honjo, H. Tanihara, A. Nonaka, K. Yamashiro, H. Katsuta, S. Miyahara, et al.
Platelets Adhering to the Vascular Wall Mediate Postischemic Leukocyte-Endothelial Cell Interactions in Retinal Microcirculation
Invest. Ophthalmol. Vis. Sci.,
March 1, 2004;
45(3):
977 - 984.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Gawaz
Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium
Cardiovasc Res,
February 15, 2004;
61(3):
498 - 511.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Massberg, S. Gruner, I. Konrad, M. I. Garcia Arguinzonis, M. Eigenthaler, K. Hemler, J. Kersting, C. Schulz, I. Muller, F. Besta, et al.
Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)-deficient mice
Blood,
January 1, 2004;
103(1):
136 - 142.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Wagner and P. C. Burger
Platelets in Inflammation and Thrombosis
Arterioscler. Thromb. Vasc. Biol.,
December 1, 2003;
23(12):
2131 - 2137.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Sun, W.-L. Chang, J. Li, S. M. Berney, D. Kimpel, and H. C. van der Heyde
Inhibition of Platelet Adherence to Brain Microvasculature Protects against Severe Plasmodium berghei Malaria
Infect. Immun.,
November 1, 2003;
71(11):
6553 - 6561.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Nishiwaki, T. Ueda, S. Ugawa, S. Shimada, and Y. Ogura
Upregulation of P-selectin and Intercellular Adhesion Molecule-1 after Retinal Ischemia-Reperfusion Injury
Invest. Ophthalmol. Vis. Sci.,
November 1, 2003;
44(11):
4931 - 4935.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Kojima, H. Kanada, S. Shimizu, E. Kasama, K. Shibuya, H. Nakauchi, T. Nagasawa, and A. Shibuya
CD226 Mediates Platelet and Megakaryocytic Cell Adhesion to Vascular Endothelial Cells
J. Biol. Chem.,
September 19, 2003;
278(38):
36748 - 36753.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Kalvegren, M. Majeed, and T. Bengtsson
Chlamydia pneumoniae Binds to Platelets and Triggers P-Selectin Expression and Aggregation: A Causal Role in Cardiovascular Disease?
Arterioscler. Thromb. Vasc. Biol.,
September 1, 2003;
23(9):
1677 - 1683.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Miyahara, J. Kiryu, A. Tsujikawa, H. Katsuta, K. Nishijima, K. Miyamoto, K. Yamashiro, A. Nonaka, and Y. Honda
Argatroban Attenuates Leukocyte- and Platelet-Endothelial Cell Interactions After Transient Retinal Ischemia
Stroke,
August 1, 2003;
34(8):
2043 - 2049.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Ishikawa, D. Cooper, J. Russell, J. W. Salter, J. H. Zhang, A. Nanda, and D. N. Granger
Molecular Determinants of the Prothrombogenic and Inflammatory Phenotype Assumed by the Postischemic Cerebral Microcirculation
Stroke,
July 1, 2003;
34(7):
1777 - 1782.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Cooper, K. D. Chitman, M. C. Williams, and D. N. Granger
Time-dependent platelet-vessel wall interactions induced by intestinal ischemia-reperfusion
Am J Physiol Gastrointest Liver Physiol,
June 1, 2003;
284(6):
G1027 - G1033.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-L. Chang, J. Li, G. Sun, H.-L. Chen, R. D. Specian, S. M. Berney, D. N. Granger, and H. C. van der Heyde
P-Selectin Contributes to Severe Experimental Malaria but Is Not Required for Leukocyte Adhesion to Brain Microvasculature
Infect. Immun.,
April 1, 2003;
71(4):
1911 - 1918.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
A. Tailor and D. N. Granger
Hypercholesterolemia Promotes P-Selectin-Dependent Platelet-Endothelial Cell Adhesion in Postcapillary Venules
Arterioscler. Thromb. Vasc. Biol.,
April 1, 2003;
23(4):
675 - 680.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. C. Manegold, J. Hutter, S. A. Pahernik, K. Messmer, and M. Dellian
Platelet-endothelial interaction in tumor angiogenesis and microcirculation
Blood,
March 1, 2003;
101(5):
1970 - 1976.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. H. Cerwinka, D. Cooper, C. F. Krieglstein, C. R. Ross, J. M. McCord, and D. N. Granger
Superoxide mediates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules
Am J Physiol Heart Circ Physiol,
February 1, 2003;
284(2):
H535 - H541.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Massberg, M. Gawaz, S. Gruner, V. Schulte, I. Konrad, D. Zohlnhofer, U. Heinzmann, and B. Nieswandt
A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo
J. Exp. Med.,
January 6, 2003;
197(1):
41 - 49.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nishijima, J. Kiryu, A. Tsujikawa, M. Honjo, A. Nonaka, K. Yamashiro, H. Kamizuru, Y. Ieki, H. Tanihara, Y. Honda, et al.
Inhibitory Effects of Antithrombin III on Interactions between Blood Cells and Endothelial Cells during Retinal Ischemia-Reperfusion Injury
Invest. Ophthalmol. Vis. Sci.,
January 1, 2003;
44(1):
332 - 341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Russell, D. Cooper, A. Tailor, K. Y. Stokes, and D. N. Granger
Low venular shear rates promote leukocyte-dependent recruitment of adherent platelets
Am J Physiol Gastrointest Liver Physiol,
January 1, 2003;
284(1):
G123 - G129.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. J. M. Molenaar, C. C. M. Appeldoorn, S. A. M. de Haas, I. N. Michon, A. Bonnefoy, M. F. Hoylaerts, H. Pannekoek, T. J. C. van Berkel, J. Kuiper, and E. A. L. Biessen
Specific inhibition of P-selectin-mediated cell adhesion by phage display-derived peptide antagonists
Blood,
November 15, 2002;
100(10):
3570 - 3577.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Massberg, K. Brand, S. Gruner, S. Page, E. Muller, I. Muller, W. Bergmeier, T. Richter, M. Lorenz, I. Konrad, et al.
A Critical Role of Platelet Adhesion in the Initiation of Atherosclerotic Lesion Formation
J. Exp. Med.,
October 7, 2002;
196(7):
887 - 896.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Schober, D. Manka, P. von Hundelshausen, Y. Huo, P. Hanrath, I. J. Sarembock, K. Ley, and C. Weber
Deposition of Platelet RANTES Triggering Monocyte Recruitment Requires P-Selectin and Is Involved in Neointima Formation After Arterial Injury
Circulation,
September 17, 2002;
106(12):
1523 - 1529.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Kupatt, R. Wichels, J. Horstkotte, F. Krombach, H. Habazettl, and P. Boekstegers
Molecular mechanisms of platelet-mediated leukocyte recruitment during myocardial reperfusion
J. Leukoc. Biol.,
September 1, 2002;
72(3):
455 - 461.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Khandoga, G. Enders, P. Biberthaler, and F. Krombach
Poly(ADP-ribose) polymerase triggers the microvascular mechanisms of hepatic ischemia-reperfusion injury
Am J Physiol Gastrointest Liver Physiol,
September 1, 2002;
283(3):
G553 - G560.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. H. Cerwinka, D. Cooper, C. F. Krieglstein, M. Feelisch, and D. N. Granger
Nitric oxide modulates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules
Am J Physiol Heart Circ Physiol,
March 1, 2002;
282(3):
H1111 - H1117.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Barrabes, D. Garcia-Dorado, M. Mirabet, R.-M. Lidon, B. Soriano, M. Ruiz-Meana, P. Pizcueta, J. Blanco, Y. Puigfel, and J. Soler-Soler
Lack of effect of glycoprotein IIb/IIIa blockade on myocardial platelet or polymorphonuclear leukocyte accumulation and on infarct size after transient coronary occlusion in pigs
J. Am. Coll. Cardiol.,
January 2, 2002;
39(1):
157 - 165.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. W. Salter, C. F. Krieglstein, A. C. Issekutz, and D. N. Granger
Platelets modulate ischemia/reperfusion-induced leukocyte recruitment in the mesenteric circulation
Am J Physiol Gastrointest Liver Physiol,
December 1, 2001;
281(6):
G1432 - G1439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. SINGBARTL, S. B. FORLOW, and K. LEY
Platelet, but not endothelial, P-selectin is critical for neutrophil-mediated acute postischemic renal failure
FASEB J,
November 1, 2001;
15(13):
2337 - 2344.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Morigi, M. Galbusera, E. Binda, B. Imberti, S. Gastoldi, A. Remuzzi, C. Zoja, and G. Remuzzi
Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress
Blood,
September 15, 2001;
98(6):
1828 - 1835.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nishijima, J. Kiryu, A. Tsujikawa, M. Honjo, A. Nonaka, K. Yamashiro, H. Tanihara, S. J. Tojo, Y. Ogura, and Y. Honda
In Vivo Evaluation of Platelet-Endothelial Interactions after Transient Retinal Ischemia
Invest. Ophthalmol. Vis. Sci.,
August 1, 2001;
42(9):
2102 - 2109.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Nagai, M. De Mol, B. Van Hoef, M. Verstreken, and D. Collen
Depletion of circulating {alpha}2-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization
Blood,
May 15, 2001;
97(10):
3086 - 3092.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Dickfeld, E. Lengyel, A. E May, S. Massberg, K. Brand, S. Page, C. Thielen, K. Langenbrink, and M. Gawaz
Transient interaction of activated platelets with endothelial cells induces expression of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway: Implications for atherogenesis
Cardiovasc Res,
January 1, 2001;
49(1):
189 - 199.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Lefer
Platelets : Unindicted Coconspirators in Inflammatory Tissue Injury
Circ. Res.,
December 8, 2000;
87(12):
1077 - 1078.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Carvalho-Tavares, M. J. Hickey, J. Hutchison, J. Michaud, I. T. Sutcliffe, and P. Kubes
A Role for Platelets and Endothelial Selectins in Tumor Necrosis Factor-{alpha}-Induced Leukocyte Recruitment in the Brain Microvasculature
Circ. Res.,
December 8, 2000;
87(12):
1141 - 1148.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Andre, C. V. Denis, J. Ware, S. Saffaripour, R. O. Hynes, Z. M. Ruggeri, and D. D. Wagner
Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins
Blood,
November 15, 2000;
96(10):
3322 - 3328.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Kupatt, H. Habazettl, P. Hanusch, R. Wichels, D. Hahnel, B. F. Becker, and P. Boekstegers
c7E3Fab Reduces Postischemic Leukocyte-Thrombocyte Interaction Mediated by Fibrinogen : Implications for Myocardial Reperfusion Injury
Arterioscler. Thromb. Vasc. Biol.,
October 1, 2000;
20(10):
2226 - 2232.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Tsujikawa, J. Kiryu, K. Yamashiro, A. Nonaka, K. Nishijima, Y. Honda, and Y. Ogura
Interactions Between Blood Cells and Retinal Endothelium in Endotoxic Sepsis
Hypertension,
August 1, 2000;
36(2):
250 - 258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Katayama, Y. Ikeda, M. Handa, T. Tamatani, S. Sakamoto, M. Ito, Y. Ishimura, and M. Suematsu
Immunoneutralization of Glycoprotein Ib{alpha} Attenuates Endotoxin-Induced Interactions of Platelets and Leukocytes With Rat Venular Endothelium In Vivo
Circ. Res.,
May 26, 2000;
86(10):
1031 - 1037.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. S. Frenette, C. V. Denis, L. Weiss, K. Jurk, S. Subbarao, B. Kehrel, J. H. Hartwig, D. Vestweber, and D. D. Wagner
P-Selectin Glycoprotein Ligand 1 (PSGL-1) Is Expressed on Platelets and Can Mediate Platelet-Endothelial Interactions In Vivo
J. Exp. Med.,
April 18, 2000;
191(8):
1413 - 1422.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kakutani, T. Masaki, and T. Sawamura
A platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1
PNAS,
January 4, 2000;
97(1):
360 - 364.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Massberg, G. Enders, F. C. d. M. Matos, L. I. D. Tomic, R. Leiderer, S. Eisenmenger, K. Messmer, and F. Krombach
Fibrinogen Deposition at the Postischemic Vessel Wall Promotes Platelet Adhesion During Ischemia-Reperfusion In Vivo
Blood,
December 1, 1999;
94(11):
3829 - 3838.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F.-J. Neumann, D. Zohlnhofer, L. Fakhoury, I. Ott, M. Gawaz, and A. Schomig
Effect of glycoprotein IIb/IIIa receptor blockade on platelet-leukocyte interaction and surface expression of the leukocyte integrin Mac-1 in acute myocardial infarction
J. Am. Coll. Cardiol.,
November 1, 1999;
34(5):
1420 - 1426.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Tsujikawa, J. Kiryu, A. Nonaka, K. Yamashiro, H. Nishiwaki, S. J. Tojo, Y. Ogura, and Y. Honda
In Vivo Evaluation of Platelet-Endothelial Interactions in Retinal Microcirculation of Rats
Invest. Ophthalmol. Vis. Sci.,
November 1, 1999;
40(12):
2918 - 2924.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. M. Romo, J.-F. Dong, A. J. Schade, E. E. Gardiner, G. S. Kansas, C. Q. Li, L. V. McIntire, M. C. Berndt, and J. A. Lopez
The Glycoprotein Ib-IX-V Complex Is a Platelet Counterreceptor for P-Selectin
J. Exp. Med.,
September 20, 1999;
190(6):
803 - 814.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. von Dobschuetz, T. Hoffmann, and K. Messmer
Inhibition of Neutrophil Proteinases by Recombinant Serpin Lex032 Reduces Capillary No-Reflow in Ischemia/Reperfusion-Induced Acute Pancreatitis
J. Pharmacol. Exp. Ther.,
August 1, 1999;
290(2):
782 - 788.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. Bombeli, B. R. Schwartz, and J. M. Harlan
Endothelial Cells Undergoing Apoptosis Become Proadhesive for Nonactivated Platelets
Blood,
June 1, 1999;
93(11):
3831 - 3838.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Massberg, M. Sausbier, P. Klatt, M. Bauer, A. Pfeifer, W. Siess, R. Fassler, P. Ruth, F. Krombach, and F. Hofmann
Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3',5'-Monophosphate Kinase I
J. Exp. Med.,
April 19, 1999;
189(8):
1255 - 1264.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract