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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3829-3838
By
From the Ludwig-Maximilians-University, Institute for Surgical
Research, Klinikum Grosshadern, Munich, Germany.
Following ischemia-reperfusion (I/R), platelet adhesion is thought
to represent the initial event leading to remodeling and reocclusion of
the vasculature. The mechanisms underlying platelet adhesion to the
endothelium have not been completely established. Endothelial cells
rendered ischemic acquire a procoagulant phenotype, characterized by
fibrinogen accumulation. Therefore, we evaluated whether fibrinogen
deposition during I/R mediates platelet adhesion. Using fluorescence
microscopy, fibrinogen deposition and the accumulation of platelets
were assessed in vivo in a model of intestinal I/R (1.5 hours/60
minutes). Fibrinogen accumulated in arterioles and venules early after
the onset of reperfusion. The deposition of fibrinogen colocalized with
large numbers of adherent platelets (520 ± 65 and 347 ± 81 platelets/mm2 in arterioles and venules). Pretreatment with
an antifibrinogen antibody attenuated platelet adhesion. Intracellular
adhesion molecule (ICAM)-1 served as a major receptor for fibrinogen,
since fibrinogen deposition and platelet adhesion to the endothelial cell surface were markedly decreased in ICAM-1-deficient mice. The
platelet
IN INDUSTRIALIZED COUNTRIES,
ischemia-reperfusion (I/R)-induced organ injury underlies many of the
most important diseases, including angina pectoris, myocardial
infarction, and stroke. Although many of the ischemic episodes can be
reversed by medical interventions, the reintroduction of oxygenated
blood initiates an inflammatory reaction (reperfusion injury),
associated with leukocyte adhesion and emigration.1-4 In
addition to leukocytes, platelets are recruited to the postischemic
microvasculature very early after the onset of
reperfusion.5,6 Upon activation, platelets generate oxygen
radicals and release a variety of chemokines and growth
factors.7-12 Therefore, platelet adhesion to the
endothelium and intravascular platelet aggregation are thought to be
the initial events leading to remodeling and reocclusion of the
postischemic vessel segment. However, the mechanisms that underlie the
interactions between platelets and endothelial cells during
ischemia-reperfusion (I/R) have not been completely elucidated thus far.
Although originally envisioned as passive and inert, the endothelium
has been shown to actively respond to stimuli. Following hypoxia/reoxygenation or I/R, vascular endothelial cells, which act as
a nonthrombogenic surface under physiologic conditions, acquire a
procoagulant phenotype. This procoagulant endothelial activity arises
from changes in the synthesis and surface expression of endothelial
proteins. Endothelial cell activation, eg, by hypoxia/reoxygenation, is
known to induce tissue factor expression and to suppress thrombomodulin activity, leading to thrombin activation and promoting fibrin(ogen) deposition.13-16
While the accumulation of fibrin(ogen) driven by tissue factor has been
reported during reperfusion of ischemic organs,17 the role
of fibrin(ogen) in the pathogenesis of I/R injury has not been clearly
defined thus far. Leukocytes, endothelial cells, and platelets are
known to associate with fibrinogen. Fibrinogen binding to intercellular
adhesion molecule (ICAM)-1 expressed on the endothelial cell surface
has been demonstrated to mediate the adhesion of leukocytes to human
umbilical vein endothelial cells (HUVECs) in
vitro18 and to induce the attachment of monocytic HL-60
cells to the vascular wall of mesenteric venules in vivo.19 Moreover, fibrinogen binding to Animals.
Female Balb/c mice (Charles River, Sulzfeld, Germany) and C57BL/6J mice
(wild-type, or ICAM-1-deficient), aged between 5 and 7 weeks (54 experimental animals and 54 platelet donors) were used. The ICAM-1
mutant strain was generated in the laboratory of Professor A.L. Beaudet
(Houston, TX)22 and purchased from The Jackson Laboratory
(Bar Harbor, ME). All experimental procedures were approved by the
German legislation on protection of animals.
Surgical procedure.
The surgical procedure has been described in detail
elsewhere.23 In brief, the mice were anesthetized by
inhalation of isoflurane-N2O (FiO2 0.35, 0.015 l/l isoflurane; Forene, Abbott GmbH, Wiesbaden, Germany), and
polyethylene catheters (PE 50, inner diameter [ID] 0.28 mm; Portex,
Hythe, UK) were inserted into the left carotid artery and jugular vein.
A segment of the jejunum was exteriorized and subjected to 1.5 hours of
normothermic ischemia. Platelet-endothelial cell interactions in the
postischemic microvasculature were investigated by intravital
microscopy (IVM) 10 to 30 minutes after the onset of reperfusion.
Blood sampling and platelet preparation.
For IVM, murine platelets were isolated from whole blood and labeled
with rhodamine-6G (50 µL 0.05% per mL whole blood; molecular weight, 479; Sigma-Aldrich, Deisenhofen,
Germany), as previously described.23 For experiments with
human platelets, whole blood was obtained from healthy, nonsmoking
volunteers or from a patient with Glanzmann's disease. The donors had
not received acetylsalicylic acid or any other platelet inhibitor for
at least 14 days before the experiments. Human platelets were isolated
by centrifugation and labeled with rhodamine-6G as described for murine
platelets.23
Intravital fluorescence microscopy.
Using IVM, platelet-endothelial cell interactions were analyzed within
submucosal arterioles and postcapillary venules of the exposed segment.
The fluorescent platelets were infused via the jugular vein for 5 minutes starting 5 minutes after the onset of reperfusion. In each
animal, the jejunal segment was scanned from the oral to the aboral
section 10 to 30 minutes after the onset of reperfusion. Five
nonoverlapping regions of interest were selected randomly, using a
microscopic setup previously described.23 In each animal, 5 to 7 arterioles (mean diameter, 40 µm) and 5 to 7 postcapillary
venules (mean diameter, 60 µm) were recorded on videotape.
Experimental groups.
Platelet-endothelial cell interactions were investigated under sham
conditions without ischemia (n = 6) or in response to 1.5 hours
ischemia (n = 6), respectively. IVM was performed 10 to 30 minutes
after the onset of reperfusion. The role of fibrinogen for postischemic
platelet-endothelial cell interaction was determined using a goat
polyclonal antibody directed against mouse fibrinogen (4 mg/kg; Nordic
Immunology, Tilburg, Netherlands; n = 6). The antibody was
affinity-purified with fibrinogen-Sepharose
(Sigma-Aldrich) before use and infused intravenously immediately before
injection of fluorescent platelets. Control animals were subjected to
I/R as described earlier. Goat anti-human IgG polyclonal control
antibody (n = 3; 4 mg/kg; DAKO, Glostrup, Denmark) was infused
intravenously after the onset of reperfusion. To assess the involvement
of endothelial ICAM-1 in mediating postischemic platelet accumulation,
wild-type platelets were transfused into ICAM-1-deficient animals in a
separate group (n = 6). The possible role of the platelet
fibrinogen receptor, the Fibrinogen binding to postischemic endothelial cells.
In a separate set of experiments, the binding of fibrinogen to
endothelial cells (wild-type or ICAM-1-deficient; n = 3 per group) was
investigated in vivo by video fluorescence microscopy according to
Witte.24 Alexa 488-conjugated fibrinogen (17 mg/kg; Molecular Probes, Eugene, OR) was administered intravenously 30 minutes
before the induction of ischemia. To assess the microvascular distribution of fibrinogen under physiologic conditions and in response
to I/R, 5 arterioles and venules were recorded before ischemia, at the
end of the ischemic period, as well as 5 and 30 minutes following
reperfusion. To evaluate whether fibrinogen deposition is associated
with platelet adhesion during reperfusion, rhodamine-6G-labeled
platelets were prepared and infused, as described earlier. Using two
different filter sets, both fibrinogen binding to the microvascular
wall and platelet-endothelial cell interactions were visualized in
identical vessel segments during initial reperfusion. Sham-operated
animals without I/R followed the same protocol and served as controls
(n = 2).
Immunohistology and electron microscopy.
Samples from the intestinal segment were taken 60 minutes after the
onset of reperfusion and immediately placed in O.C.T compound (Tissue-Tek; Miles Inc, Elkhart, IN) and frozen in liquid nitrogen or
fixed in paraformaldehyde, respectively. Endogenous peroxidase activity
was blocked with methanol-H2O2 for 10 minutes
at room temperature. For immunostaining of ICAM-1, acetone-fixed
cryostat sections (6 µm) were incubated with biotin-conjugated
hamster-anti-mouse ICAM-1 (3E2; Pharmingen, Hamburg,
Germany) monoclonal antibodies. For staining of fibrinogen, rabbit
anti-human fibrinogen polyclonal antibody (lot no. 097; DAKO) was added
on paraffin sections (6 µm). This antibody cross-reacts with the
corresponding mouse antigen. Tissue sections, prepared with
isotype-matched primary antibodies, served as controls. Following
incubation with the primary antibody, the sections were stained with
commercially available peroxidase immunohistochemistry kits
(Vectastain; Camon, Wiesbaden, Germany). An easily detectable
reddish-brown-colored end product was obtained by development in
H2O2/3-amino-9-ethylcarbazol. Counterstaining of the sections was performed using Mayer's hemalaun.
Endothelial cell culture and exposure of cells to
hypoxia/reoxygenation.
To investigate the effects of platelet adhesion on
hypoxia/reoxygenation-induced endothelial cell activation, HUVECs were isolated from fresh umbilical cords and cultured in Endothelial Cell
Growth Medium (Promo Cell, Heidelberg, Germany) with 2% fetal calf
serum. For experimental use, HUVECs were plated (4.0 × 106 cells/flask) and grown to confluence (3 to 5 days) in
Falcon Primaria culture flasks with 25 cm2 growth area
(Becton Dickinson, Heidelberg, Germany). The cells were
identified by their typical morphology. The endothelial cells were
exposed to hypoxia (4 hours, 1 vol% O2) and reoxygenation (1 hour, respectively; 21 vol% O2, 5 vol%
CO2) using an anaerobic jar (BBL Gas Pak System; Pierce,
Rockford, IL).
Assessment of tyrosine phosphorylation.
Upon the onset of reoxygenation, 2 mL phosphate-buffered saline
(PBS) (group 1), PBS containing isolated platelets (150 × 106 platelets/mL), or platelets plus fibrinogen (500 mg/dL) were added to the HUVECs. After 60 minutes of
incubation, the plates were washed twice with PBS. The cells were lysed
in 150 µL lysis buffer (pH 7.4, 10 µmol/l TRIS buffer, 10% sodium
dodecyl sulfate [SDS], 1 mmol/L sodium vanadate, 10 mmol/L EDTA, and
Complete protease inhibitor [Boehringer, Ingelheim,
Germany] as recommended). It appears noteworthy that
after washing of the plates (see above), only very few platelets
remained attached to the endothelial cell culture. This implicates that
the amount of platelet protein contained within the cell lysates is
negligible. The whole-cell protein lysates (20 µg protein per lane)
were separated on a 10% SDS-polyacrylamide gel electropheresis (PAGE)
and transferred to a PVDF membrane (Boehringer). The
membranes were blocked with PBS containing 0.3% gelatin and incubated
for 1 hour with a mouse-anti-human phosphotyrosine antibody (1 µg/mL
PBS, PY20; Transduction Laboratories, Lexington, UK). The membranes
were washed 4 times and exposed to peroxidase-conjugated rabbit-anti-mouse IgG secondary antibody (diluted 1:2,000 in PBS; DAKO). Finally, the membranes were washed, incubated with Supersignal (Pierce), and exposed to x-ray film.
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 ± SEM are given. P values less than .05 were
considered significant.
Platelet-endothelial cell interactions in response to I/R.
In the physiologic state without I/R, circulating platelets rarely
interacted with the microvascular endothelium (Figs
1 and 2). Few
platelets were observed rolling along the endothelial cell lining of
arterioles and postcapillary venules (0 ± 0 and 3 ± 1 platelets/s/mm, respectively). At the same time, only 26 ± 14 and
28 ± 11 platelets were found firmly attached per mm2
endothelial cell surface of arterioles and venules, respectively. In
contrast, 1.5 hours of ischemia dramatically enhanced
platelet-endothelial cell interactions immediately after postischemic
reperfusion (Figs 1 and 2). As reported earlier, postischemic platelet
accumulation involved arterioles, as well as venules. More than 15 platelets/s/mm vessel diameter were seen rolling along the arteriolar
and venular vessel wall, respectively. At the same time, the number of
firmly adherent platelets had increased 20- and 12-fold in arterioles and venules, compared with sham-operated animals (Fig 1). Platelet aggregation was a prominent phenomenon. Electron microscopy
demonstrated that single or aggregated platelets adhered directly to
endothelial cells; obvious defects in the endothelial cell layer were
not detected (Fig 3).
Role of fibrinogen for platelet-endothelial cell interactions during
I/R.
To assess fibrinogen deposition during I/R, the vascular distribution
of fibrinogen was determined using immunohistology. In sham-operated
animals small amounts of fibrinogen were detectable within the vessel
lumen. Fibrinogen accumulation on the endothelial surface was not
observed. In contrast, fibrinogen sequestration on the luminal surface
of arterioles and venules was a prominent phenomenon after I/R. To
investigate the time course of fibrinogen binding to the endothelial
cell surface during I/R in vivo, fluorescent fibrinogen was
administered intravenously before the induction of 1.5 hours of
ischemia. In the physiologic state (baseline conditions), Alexa
488-conjugated fibrinogen was found homogeneously distributed in the
plasma. No fibrinogen deposition was detectable in arterioles or
venules (Fig 4). Similarly, during the
ischemic period, no significant accumulation of fibrinogen on the
endothelial surface was observed. In contrast, reperfusion dramatically
enhanced fibrinogen binding to the endothelium in the postischemic
microvasculature. Within 1 to 5 minutes after the onset of reperfusion,
streaks of fluorescent fibrinogen were observed along the endothelial lining. After 10 minutes of reperfusion, the gaps between the streaks
were partially filled with fibrinogen, resulting in a heterogenous
fibrinogen coat on the endothelial cell surface of the majority of all
vessels studied. In most instances, significant fibrinogen deposition
at the arteriolar and venular endothelial surface coincided with the
adhesion of platelets in these areas (Fig 4).
Role of ICAM-1 (CD54) for postischemic platelet-endothelial cell
interactions.
To determine the role of ICAM-1 as an endothelial fibrinogen receptor,
the accumulation of Alexa 488-conjugated fibrinogen during I/R was
investigated in mice deficient in ICAM-1. No fibrinogen binding was
seen under control conditions or during ischemia. During reperfusion, a
moderate increase in endothelial fibrinogen binding occurred in few
arterioles and venules (Fig 4), while the majority of the vessels
studied showed no significant fibrinogen accumulation, indicating that
ICAM-1 is in fact involved in mediating the deposition of fibrinogen at
the postischemic endothelium. To evaluate whether ICAM-1-dependent
fibrinogen sequestration on the endothelial surface might mediate
platelet-endothelial cell interactions during postischemic reperfusion,
fluorescent wild-type platelets were infused into ICAM-1-deficient
animals (Fig 1). Whereas the number of rolling platelets did not differ from wild-type mice (23 ± 5 and 36 ± 4 platelets/s/mm in
arterioles and venules, respectively), platelet adhesion was
significantly reduced in the absence of endothelial ICAM-1: 125 ± 42 and 71 ± 27 platelets were seen firmly attached per
mm2 endothelial cell surface of arterioles and venules,
respectively, indicating that ICAM-1 is in fact involved in platelet
recruitment during postischemic reperfusion.
Involvement of the platelet
Platelet adhesion enhances tyrosine phosphorylation in reoxygenated
HUVECs.
To determine whether platelet adhesion affects signaling pathways in
endothelial cells, tyrosine phosphorylation in HUVECs was assessed in
the absence or presence of platelets and exogenous fibrinogen. Under
normoxic conditions in the absence of platelets, small amounts of
phosphotyrosine were detectable (Fig 6). In
the presence of platelets, phosphorylation of tyrosine residues was markedly enhanced. The antiphosphotyrosine antibody recognized several
proteins, including a 65-kD, a 95-kD, and a 97-kD protein. Tyrosine
phosphorylation of the 65-kD protein was enhanced further when
exogenous fibrinogen was added to the HUVECs together with the
platelets. Hypoxia/reoxygenation increased tyrosine phosphorylation in
HUVECs. Despite the absence of platelets, a 65-kD protein, as well as a
95- and a 97-kD protein, was detectable. However, tyrosine
phosphorylation of the 65-kD protein was markedly enhanced, in
particular in the presence of both platelets and exogenous fibrinogen.
Atherosclerosis, a major cause of morbidity in industrialized
countries, causes luminal narrowing of the affected blood vessel and
can lead to a compromised perfusion of the supplied organ. Following
ischemia, restoration of nutritive blood flow induces the adhesion of
platelets to the injured endothelium.6 Platelet recruitment
to the postischemic vasculature might represent the initial event,
contributing to recurrence of luminal narrowing or even complete
reocclusion.25,26 However, the molecular mechanisms underlying platelet-endothelial cell interactions in response to I/R
have not been completely elucidated thus far.
The authors thank Elke Schütze, Katrin Baltzer-Quoast, and Sylvia
Münzing for their excellent and skillful technical assistance. We
are grateful to Dr Karin Auberger (Dr von Haunersches Kinderspital der
Universität München, Munich, Germany) for providing the CD41/CD61-deficient platelets.
Submitted December 9, 1998; accepted July 27, 1999.
S.M. and G.E. contributed equally to this work.
Supported by research grant Biomed 2 Contract No. BMH4-CT95-0875
(DG12-SSMA).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Steffen Massberg, MD, Deutsches Herzzentrum
München, Technical University of Munich, Lazarettstr 36, 80636 Munich, Germany; e-mail: massberg{at}icf.med.uni-muenchen.de.
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TOP
ABSTRACT
INTRODUCTION
IIb/
3 integrin plays a key role
in fibrinogen-dependent platelet accumulation, because (1) platelet
adhesion involved RGD-recognition sequences, and (2) platelets isolated
from a patient with Glanzmann's disease showed decreased interaction
with the postischemic endothelium. Since platelets are demonstrated
here to induce tyrosine phosphorylation in endothelial cells, platelet recruitment might contribute to the development of an inflammatory reaction during I/R.
IIb/
/
(117-133) sequence,
which mediates the association between fibrin(ogen) and endothelial ICAM-1.