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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3829-3838
Fibrinogen Deposition at the Postischemic Vessel Wall Promotes Platelet
Adhesion During Ischemia-Reperfusion In Vivo
By
Steffen Massberg,
Georg Enders,
Francine Cláudia de Melo Matos,
Luciana Inês Domschke Tomic,
Rosmarie Leiderer,
Simone Eisenmenger,
Konrad Messmer, and
Fritz Krombach
From the Ludwig-Maximilians-University, Institute for Surgical
Research, Klinikum Grosshadern, Munich, Germany.
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ABSTRACT |
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  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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
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  IIb/3
integrins on adjacent platelets is believed to represent the molecular
substrate for platelet aggregation during primary hemostasis and to
promote the adhesion of platelets to immobilized fibrinogen and to
HUVEC monolayers in vitro.20,21 Fibrinogen accumulation
might, therefore, directly contribute to platelet recruitment during
postischemic reperfusion. However, the causative role of fibrinogen in
mediating platelet adhesion during I/R has not been clearly
established. The aim of the present in vivo study, therefore, was (1)
to investigate the time course of fibrinogen accumulation during I/R,
(2) to study the impact of fibrinogen deposition on platelet adhesion to the postischemic endothelium, and (3) to evaluate the role of ICAM-1
in fibrinogen accumulation during I/R.
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MATERIALS AND METHODS |
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
Before infusion, the platelet count and the purity of each platelet
suspension (murine or human) were assessed by a Coulter AC
T Counter (Coulter Corp, Miami, FL) and by flow cytometry. Platelet separation by differential centrifugation yielded a platelet suspension with negligible amounts of other cellular components. More than 99% of
all platelets were labeled with rhodamine-6G. As reported earlier,
platelet preparation did not increase platelet P-selectin expression,
indicating the absence of platelet activation due to the separation
procedure.23 A total of 100 × 106
platelets stained with rhodamine-6G were transfused to achieve a
labeled fraction in the recipient mouse of approximately 10% of all
circulating 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.
Quantitative assessment of platelet-endothelial cell interactions
within these vessels was performed off-line by frame-to-frame analysis
of the videotaped images. Within both arterioles and postcapillary
venules, platelet-endothelial cell interactions were classified as
rolling (intermittent platelet adhesion) or firm adhesion of platelets.
Rolling platelets were defined as platelets crossing an imaginary
perpendicular line 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. Firm platelet adhesion
was defined in each vessel segment as number of cells that did not move
or detach from the endothelial lining within an observation period of
30 seconds. Platelet adhesion is quantified as number of cells per square millimeter endothelial surface, calculated from the diameter and
length of the vessel segment observed.
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 IIb/3 integrin
(CD41/CD61), as a mediator of postischemic platelet-endothelial cell
interactions was determined using the Arg-Gly-Asp (RGD) peptide
Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP, molecular weight, 587.7; BIOMOL,
Hamburg, Germany), which is known to inhibit the interaction of
fibrinogen with the platelet IIb/3
integrin (n = 6). Purified GRGDSP peptide (10 mg/kg) was injected
intravenously before the infusion of fluorescent platelets. As an
independent approach to examine the role of the
IIb/3 integrin in mediating postischemic
platelet-endothelial cell interactions, human platelets derived from a
patient with homozygous Glanzmann's disease were infused into
wild-type mice (n = 6). Platelets derived from healthy human volunteers
served as controls (n = 6).
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.
For electron microscopy, tissue was excised from sham-operated control
and I/R experiments (groups A and B). The samples were fixed and
processed as described earlier.6 Ultrathin sections were
cut and stained with uranyl acetate and lead citrate and examined under
a Zeiss EM 900 transmission electron microscope (Zeiss,
Oberkochen, Germany) operating at 80 kV.
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.
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RESULTS |
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).
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| Fig 1.
Platelet adhesion in response to I/R of the small
intestine in vivo (ischemia time, 90 minutes). Platelet-endothelial
cell interactions were investigated in arterioles (A) and venules (B)
using IVM. Platelet adhesion was assessed following I/R, after
pretreatment with an antibody directed to fibrinogen (-Fbg), or with
control antibody (Ctrl-Ab), as well as in ICAM-1-deficient mice
(CD54 /), or after infusion of GRGDSP peptide (RGD), respectively.
Sham-operated animals (Sham) served as controls. The number of adherent
platelets is given per mm2 vessel surface. Mean ± SEM, n
= 6 experimental animals per group. *P < .01 v
sham, Dunn's method.
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| Fig 2.
Sequence of photographs documenting I/R-induced
platelet-endothelial cell interactions in vivo. Using IVM,
rhodamine-6G-labeled platelets are visualized within 3 postcapillary
venules before ischemia (A), as well as following 90 minutes of
ischemia and 30 minutes of reperfusion (B). Few platelets adhere to the
venular endothelium before I/R (A); the majority passes the vessel
segment without interacting with the endothelial surface. In contrast,
a large number of platelets are seen interacting with the endothelium
30 minutes after reperfusion (B). Monitor magnification 450×; bars
represent 50 µm.
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| Fig 3.
Platelets in postischemic microvasculature visualized by
electron microscopy. There are no defects in the endothelial cell layer
(arrowheads) Platelets (arrows) attach directly to endothelial cells
(arrowheads). Bars represent 1 µm. Original
magnifications: 7,000× (A) and 12,000× (B). NCL, endothelial cell
nucleus.
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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).
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| Fig 4.
Accumulation of fibrinogen and platelets during I/R in
vivo. Alexa 488-conjugated human fibrinogen (17 mg/kg) was administered
intravenously 30 minutes before the induction of ischemia (left
column). Rhodamin-labeled platelets were visualized in identical
arterioles and venules using a different filter set (right column; see
Materials and Methods). In wild-type animals (A,B), fibrinogen is bound
unevenly to the vascular wall of arterioles and venules in the
postischemic microvasculature. Areas with large amounts of fibrinogen
(A, large arrow) can be seen besides regions without detectable
fibrinogen deposition (A, small arrow). The accumulation of large
amounts of fibrinogen colocalizes with platelet adhesion (B,
arrowhead). In mice lacking ICAM-1 (C,D), the I/R-induced accumulation
of fibrinogen and platelets is attenuated. Monitor magnification,
450×.
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To evaluate the causative role of endothelial fibrinogen deposition for
postischemic platelet adhesion in vivo, an affinity-purified polyclonal
antibody directed against mouse fibrinogen was administered immediately
before the onset of reperfusion. Whereas the antifibrinogen antibody
had no effects on platelet rolling in arterioles and venules, the
number of adherent platelets was significantly reduced. A mean of 135 ± 79 and 61 ± 22 platelets/mm2 was seen firmly
attached to the endothelial cell surface of arterioles and venules,
respectively, indicating that fibrinogen promotes platelet adhesion
during I/R (Fig 1). In contrast, the goat anti-human IgG polyclonal
control antibody had no effects on postischemic platelet accumulation
in both arterioles and venules, respectively (Fig 1).
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.
To assess whether alterations in ICAM-1 expression might account for
the changes in fibrinogen sequestration observed during I/R,
immunohistochemistry was performed on cryostat sections of wild-type
mice using anti-ICAM-1 monoclonal antibody. In sham-operated animals,
ICAM-1 was constitutively expressed by the vascular endothelium of both
venules and arterioles. However, no alteration in the expression of
ICAM-1 was observed in response to I/R (not shown).
Involvement of the platelet
IIb/3
integrin in postischemic platelet-endothelial cell interactions.
To study the role of the platelet fibrinogen receptor
(IIb/3 integrin) for postischemic
platelet adhesion, GRGDSP-peptide, an antagonist to the
IIb/3 integrin, was administered
intravenously. While GRGDSP had no significant effects on the number of
rolling platelets in postischemic arterioles and venules (18 ± 6 and 24 ± 5 platelets/s/mm), platelet adhesion in response to I/R
was significantly attenuated (23 ± 10 and 24 ± 12 platelets/mm2 in arterioles and venules, Fig 1).
To examine the contribution of the IIb/3
integrin to postischemic platelet adhesion, human platelets derived
from a patient with homozygous Glanzmann's disease were infused into
wild-type mice during I/R. Whereas CD31, CD36, CD42, and CD63 were
present on the surface of these platelets, they completely lacked the IIb/3 integrin complex (CD41/CD61), as
confirmed by flow cytometry (data not shown). As a control, platelets
derived from healthy human volunteers were transfused into mice before
and after I/R. Under baseline conditions without I/R, no adhesion of
normal human platelets to murine endothelium was observed (Fig
5). However, I/R significantly increased
the number of adherent platelets in both arterioles and venules (296 ± 68 and 298 ± 96 platelets/mm2, respectively). In
contrast, when platelets lacking the
IIb/3 integrin complex were transfused,
only 24 ± 11 and 59 ± 16 adherent platelets/mm2
were seen in postischemic arterioles and venules, respectively. Taken
together, these results suggest that platelet adhesion to postischemic
endothelial cells in vivo is IIb/3
integrin-dependent and involves fibrinogen and endothelial ICAM-1.
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| Fig 5.
Adhesion of human platelets in response to 90 minutes of
ischemia and subsequent reperfusion. Human platelets derived from
healthy human subjects or from a patient suffering from Glanzmann's
disease ( IIb/3/) were labeled with
rhodamine-6G and transfused into wild-type mice. Platelet-endothelial
cell interactions were investigated in arterioles (A) and venules (B)
using IVM. Sham-operated animals served as controls. The number of
adherent platelets is given per mm2 vessel surface. Mean ± SEM, n = 6 experimental animals per group. *P < .01 v sham, Dunn's method.
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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.
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| Fig 6.
Effect of platelet adhesion on tyrosine phosphorylation
in HUVECs. HUVECs were exposed to hypoxia (4 hours, 1 vol%
O2) and reoxygenation (1 hour; 21 vol% O2, 5 vol% CO2). Upon onset of reoxygenation, 2 mL PBS (group
1), PBS containing isolated platelets (150 × 106
platelets/mL), or platelets plus fibrinogen (500 mg/dL) were added to
the HUVECs and incubated for 60 minutes (for details see Materials and
Methods). HUVECs without hypoxia/reoxygenation served as controls.
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DISCUSSION |
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 accumulation of fibrin(ogen) has been demonstrated following
myocardial, hepatic, renal, and cerebral ischemia.17,27,28 While in many instances, microvascular fibrinogen accumulation is
associated with the recruitment of platelets,29 the
causative role of fibrinogen in promoting platelet adhesion during
postischemic reperfusion has not been established thus far. The present
study provides several lines of evidence indicating that the deposition of fibrinogen onto the postischemic vessel wall significantly contributes to platelet adhesion in both arterioles and venules: (1)
the microvascular accumulation of fibrinogen was a prominent phenomenon
following I/R; (2) fibrinogen deposition colocalized with platelet
adherence in vivo; and (3) treatment with an antifibrinogen antibody
attenuated platelet adhesion in both arterioles and venules. Immunoinhibition of fibrinogen-receptor interactions did not affect platelet rolling or transient attachment. Therefore, in agreement with
previous findings,20 fibrinogen appears to support firm and
irreversible platelet attachment.
It appears noteworthy that the absolute number of adherent platelets
was higher in arterioles as compared with venules. In vitro platelet
adhesion onto fibrinogen is less efficient at the high wall shear
rates, which occur in arterioles, but also in small postcapillary
venules.20 Therefore, an initial capturing process is
required to allow irreversible binding of platelets to fibrinogen.
Platelet capturing or intermittent platelet adhesion can be mediated
via P-selectin6,30 and by binding of the leucine-rich glycoprotein Ib (GPIb) to von Willebrand factor.20 The
latter interaction, unlike the fibrinogen-GPIIb/IIIa interaction, is particularly efficient at high shear rates.20 Once
captured, activation of platelets can occur, leading to an increase in
the fibrinogen-binding affinity of the GPIIb/IIIa receptor complex and
initiating firm and irreversible platelet adhesion. Similar to platelet
capturing per se, the activation of platelets under flow is shear
rate-dependent in that the shear rate must be above a threshold
limit.31-33 Both, shear-dependent platelet capturing and
activation may explain why platelet recruitment is more efficient in
arterioles than in venules, despite a low resistance of the fibrinogen-GPIIb/IIIa interaction to tensile stress. Interestingly, after transfusion of human platelets, there was no difference in the
number of adherent platelets in arterioles as compared with venules
(Fig 5). This suggests that the molecular mechanisms that underlie
shear-dependent platelet capturing function only within one species,
but might be less effective in the xenogeneic setting.
Endothelial denudation was not observed in the present study, as
demonstrated by electron microscopy. Instead, the deposition of
fibrinogen occurred directly on the endothelial cell surface, indicating that the attachment of fibrinogen to endothelial cells might
be mediated by a fibrinogen receptor present on the endothelium. Recently, the functional role of ICAM-1 as a novel fibrinogen receptor
has been emphasized. ICAM-1 has been shown to contain a fibrinogen
recognition site that is distinct from previously recognized ICAM-1
ligand binding regions.34 Through its ICAM-1 recognition,
fibrinogen enhances the adhesion of leukocytes to endothelial cells in
vitro and in vivo,18,19 and supports transendothelial monocyte migration.35 In addition, fibrinogen-ICAM-1
bridging has recently been demonstrated to mediate platelet adhesion to HUVECs in vitro.21 In the present study, the lack of
endothelial ICAM-1 expression attenuated fibrinogen deposition to the
postischemic vessel wall. In addition, in the absence of ICAM-1
platelet adhesion was significantly reduced, indicating that through
its fibrinogen recognition, ICAM-1 promotes platelet adhesion to the
postischemic endothelium in vivo. This mechanism might be of particular
importance in mediating platelet adhesion to atherosclerotic lesions,
which are characterized by the large deposition of fibrin(ogen) and the
increased expression of ICAM-1.36-38 The established role
of both fibrinogen and soluble ICAM-1 as a major risk factor for myocardial infarction further highlights the potential pathophysiologic relevance of platelet adhesion to endothelial cells via fibrinogen bridging to ICAM-1.39-41
Although both fibrinogen and ICAM-1 are constitutively expressed,
fibrinogen deposition was absent under physiologic conditions and
during ischemia. In contrast, within minutes after the onset of
reperfusion, fibrinogen accumulation was drastically enhanced, which
suggests that reoxygenation/reperfusion is required to promote endothelial recognition of the clotting factor. The mechanisms that
initiate the interaction between soluble fibrinogen and endothelial cells remain unclear. An upregulation of the endothelial fibrinogen receptor might explain the increase in fibrinogen binding following ischemia. However, since an enhancement of ICAM-1 or fibrinogen expression requires de novo mRNA and protein synthesis, this mechanism is unlikely to contribute to fibrinogen deposition observed after 1.5 hours of ischemia. Accordingly, we were not able to detect any
differences in the ICAM-1 expression before and after I/R. This
suggests that changes in the affinity of fibrinogen to its receptor (or
vice versa), rather than alterations in ICAM-1 surface expression, are
involved in the regulation of fibrinogen-endothelial cell interaction.
An increase in the adhesive properties of ICAM-1 might explain
fibrinogen binding to the endothelium in response to I/R. However, in
contrast to integrins that are known to undergo conformational changes
upon activation,42 similar mechanisms leading to an
increased adhesiveness of ICAM-1 have not been documented so far.
Hence, fibrinogen is most likely to be modified in a way that increases
its binding affinity to the endothelial surface. Endothelial cell
activation, eg, by hypoxia/reoxygenation or by oxygen free radicals, is
known to induce rapid upregulation of tissue factor
expression13,14,43 and to suppress thrombomodulin activity
in endothelial cells.15,16 Both tissue factor activation and suppression of thrombomodulin activity lead to thrombin
release/activation. Thrombin generated by the postischemic endothelium
and released by recruited platelets initiates proteolytic fibrinogen
degradation, which results in fibrin attachment to the
endothelium.13,14,44 Since Alexa-fibrinogen retains its
fluorescence after incubation with thrombin, we cannot exclude that
fibrin instead of fibrinogen deposition was visualized by in vivo
fluorescence microscopy. In addition, the polyclonal antifibrinogen
antibody used in the present study recognizes both fibrinogen and its
degradation product fibrin. However, we were not able to identify the
exact molecular characteristics of fibrinogen present after
reperfusion, since an antibody that recognizes exclusively murine
fibrin but not fibrinogen is currently not available. Nevertheless, in
a baboon model of cerebral I/R, the presence of microvascular fibrin
has been clearly demonstrated by Okada et al using the murine
anti-human fibrin monoclonal antibody MH-1.17 Therefore,
I/R-induced fibrinogen degradation, leading to fibrin deposition to the
vessel wall, might in fact be the mechanism that initiates platelet
adhesion during postischemic reperfusion. It is unclear thus far how
proteolytic processing enhances fibrin(ogen) deposition to the
endothelium. Gardiner et al45 proposed that proteolysis of
soluble fibrinogen might increase the binding affinity of the clotting
factor to ICAM-1 by complete exposure of the  (117-133) sequence,
which mediates the association between fibrin(ogen) and endothelial ICAM-1.46 Alternatively, small amounts of fibrinogen might
be constitutively bound to ICAM-1. The degradation of soluble
fibrinogen during reperfusion could lead to a polymerization of the
resultant fibrin to the fibrinogen bound to ICAM-1. This polymerization would then enhance the deposition of fibrin- (ogen) to the
endothelial cell layer, promoting platelet adhesion during I/R.
The IIb/3 integrin appears to be the
platelet receptor that binds to fibrinogen and initiates platelet
adhesion to the postischemic vessel wall. Both treatment with the
RGD-containing peptide GRGDSP, which inhibits ligand binding to
IIb/3 integrin, as well as the absence of
functional IIb/3 integrin on the surface
of platelets derived from patients with Glanzmann's disease,
attenuated platelet adhesion during I/R. This strongly suggests that
platelets bind to postischemic endothelial cells in a process that
involves IIb/3 integrin bridging to
endothelium-bound fibrinogen. The relevance of this mechanism is
stressed by the protective effects of
IIb/3 antagonists in patients with
unstable angina pectoris or myocardial infarction.47
Rupture of an atherosclerotic plaque initiates platelet adhesion and
subsequent thrombus formation and ischemia of the supplied organ. As
reported here, reperfusion after an ischemic episode also induces
platelet adhesion, a process that may lead to recurrence of luminal
narrowing and eventually reocclusion. It is now clear that the
resultant reperfusion injury is one form of acute
inflammation1-4 in which platelets might play an important
role. As demonstrated earlier,6 platelets are among the
first cells recruited to the postischemic microvasculature. Activated
platelets release oxygen radicals and generate a variety of
proinflammatory mediators, such as platelet-activating factor (PAF),
interleukin-1 (IL-1), epithelial-derived neutrophil-activating
factor-78 (ENA-78), neutrophil-activating peptide-2 (NAP-2), and
RANTES.9-12,48-50 In the present study, we have demonstrated that platelet adhesion enhances tyrosine
phosphorylation in endothelial cells. Supporting a role of platelets in
inflammatory reactions, Kaplanski et al have reported that activated
platelets induce IL-8 secretion from human umbilical vein endothelial
cells.51 Although this indicates that platelet-endothelial
cell interactions may modulate endothelial cell function and initiate
endothelial cell activation, the mechanisms that underlie
platelet-dependent signaling in endothelial cells have not been
established thus far. Consistent with a role of ICAM-1 in vascular cell
signal transduction, binding of fibrinogen to ICAM-1 expressed on the endothelial surface of the saphenous vein modulates vascular tone in an
NO-independent mechanism.52 In addition, platelet adhesion to the endothelial cell layer allows close cell-to-cell contact. This
might facilitate juxtacrine activation of the endothelial cells by
soluble platelet mediators or by direct ligand-receptor interactions.53
In conclusion, we have demonstrated in vivo that fibrin(ogen)
accumulates in the postischemic microvasculature early after the onset
of reperfusion. This deposition of fibrin(ogen) onto the endothelial
cell surface promotes platelet adhesion, involving fibrin(ogen)-IIb/3 integrin interactions.
Since adherent platelets induce tyrosine phosphorylation, platelet
recruitment is likely to significantly contribute to the manifestation
of microvascular I/R injury.
| |
ACKNOWLEDGMENT |
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.
| |
FOOTNOTES |
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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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]
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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]
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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]
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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]
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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]
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P. Lagadec, O. Dejoux, M. Ticchioni, F. Cottrez, M. Johansen, E. J. Brown, and A. Bernard
Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow
Blood,
June 15, 2003;
101(12):
4836 - 4843.
[Abstract]
[Full Text]
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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]
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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]
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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]
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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]
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A. E. May, T. Kalsch, S. Massberg, Y. Herouy, R. Schmidt, and M. Gawaz
Engagement of Glycoprotein IIb/IIIa ({alpha}IIb{beta}3) on Platelets Upregulates CD40L and Triggers CD40L-Dependent Matrix Degradation by Endothelial Cells
Circulation,
October 15, 2002;
106(16):
2111 - 2117.
[Abstract]
[Full Text]
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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]
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D. Jarrar, J. F. Kuebler, L. W. Rue III, S. Matalon, P. Wang, K. I. Bland, and I. H. Chaudry
Alveolar macrophage activation after trauma-hemorrhage and sepsis is dependent on NF-kappa B and MAPK/ERK mechanisms
Am J Physiol Lung Cell Mol Physiol,
October 1, 2002;
283(4):
L799 - L805.
[Abstract]
[Full Text]
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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]
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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]
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P Andrassy, M Zielinska, R Busch, A Schomig, and C Firschke
Myocardial blood volume and the amount of viable myocardium early after mechanical reperfusion of acute myocardial infarction: prospective study using venous contrast echocardiography
Heart,
April 1, 2002;
87(4):
350 - 355.
[Abstract]
[Full Text]
[PDF]
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J.-C. Murciano, D. Harshaw, D. G. Neschis, L. Koniaris, K. Bdeir, S. Medinilla, A. B. Fisher, M. A. Golden, D. B. Cines, M. T. Nakada, et al.
Platelets inhibit the lysis of pulmonary microemboli
Am J Physiol Lung Cell Mol Physiol,
March 1, 2002;
282(3):
L529 - L539.
[Abstract]
[Full Text]
[PDF]
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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]
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H.-A. LEHR, J. BRUNNER, R. RANGOONWALA, and C. JAMES KIRKPATRICK
Particulate Matter Contamination of Intravenous Antibiotics Aggravates Loss of Functional Capillary Density in Postischemic Striated Muscle
Am. J. Respir. Crit. Care Med.,
February 15, 2002;
165(4):
514 - 520.
[Abstract]
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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]
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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]
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V. K. Lishko, V. P. Yakubenko, K. M. Hertzberg, G. Grieninger, and T. P. Ugarova
The alternatively spliced {alpha}EC domain of human fibrinogen-420 is a novel ligand for leukocyte integrins {alpha}M{beta}2 and {alpha}X{beta}2
Blood,
October 15, 2001;
98(8):
2448 - 2455.
[Abstract]
[Full Text]
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J. Lou, R. Lucas, and G. E. Grau
Pathogenesis of Cerebral Malaria: Recent Experimental Data and Possible Applications for Humans
Clin. Microbiol. Rev.,
October 1, 2001;
14(4):
810 - 820.
[Abstract]
[Full Text]
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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]
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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]
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TOP
ABSTRACT
INTRODUCTION