© 1998 American Heart Association, Inc.
Original Contributions |
Priming of Platelet 
IIbß3 by Oxidants Is Associated With Tyrosine Phosphorylation of ß3
From the Division of Cardiology, Department of Medicine (K.I., N.K., C.C.W.) and the Division of Hematology, Departments of Medicine and Pathology (L.D.C., P.F.B.), Johns Hopkins University School of Medicine, Baltimore, Md; and the Heart and Lung Institute and Cardiology Division, Department of Medicine (Y.P., C.R., G.E.C., A.M., P.J.G.-C.), Ohio State University, Columbus.
Correspondence to Pascal J. Goldschmidt-Clermont, Heart and Lung Institute, Medical Research Facility, Suite 514, Ohio State University, 420 W 12th St, Columbus, OH 43210. E-mail Goldschmidt-1{at}medctr.osu.edu
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Abstract |
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Abstract—Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced by H2O2 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin
IIbß3 (glycoprotein IIb-IIIa).
A specific inhibitor of IIbß3
blocked platelet aggregation induced by
H2O2 and NaVO4, thus confirming
that aggregation requires this receptor. In the presence of
H2O2 and NaVO4, multiple
platelet substrates were phosphorylated on
tyrosine. Such tyrosine kinase response was necessary but not
sufficient to activate IIbß3, as
detected by binding of soluble fibrinogen to platelets. Stirring of
the platelets exposed to H2O2 and
NaVO4 was also needed to allow for binding of fibrinogen to
IIbß3. The tyrosine kinase
inhibitor genistein was able to block platelet
aggregation induced by H2O2 and
NaVO4, thus confirming that tyrosine kinase activity was
needed to trigger IIbß3 activation on
stirring. N-Acetyl-L-cysteine, a
cell-permeant antioxidant, blocked the tyrosine
phosphorylation of platelet substrates and also the
platelet aggregation induced by H2O2 and
NaVO4. We found that ß3 was
phosphorylated on tyrosine in platelets exposed to
H2O2 and NaVO4, even in the absence
of aggregation. Hence, tyrosine phosphorylation of
ß3 might contribute to the "priming" of
IIbß3 induced by
H2O2 and NaVO4, whereby the
receptor can become activated on stirring of the
platelets.
Key Words: reactive oxygen species • platelets • tyrosine kinases • glycoprotein IIb-IIIa • shear
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Introduction |
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Platelet adhesion and aggregation represent key events of coronary thrombotic processes.1 The presence of leukocytes within unstable coronary plaques has been linked to an increased risk of thrombosis.2 Leukocytes and platelets have been found adjacent during acute coronary syndromes and even circulate as clusters in the blood of patients with unstable angina.3 4 5 Leukocytes and, in particular, neutrophils produce large amounts of reactive oxygen species (ROS), which could influence platelet function in a paracrine fashion.6 Moreover, superoxide and hydroxyl radicals have been shown to be produced by anoxic and subsequently reoxygenated platelets.7 Previous studies on the effects of ROS and, in particular, hydrogen peroxide (H2O2) on platelets indicated that ROS might exert a dual effect on platelets. In the presence of nitric oxide (NO), H2O2 seems to potentiate the inhibitory effect of NO on agonist-mediated platelet activation.8 In contrast, in the presence of arachidonate, H2O2 enhances aggregation induced by phospholipid-derived arachidonate.9 10 To further characterize the effect of H2O2 on platelet reactivity, we sought to characterize the effect of ROS and, in particular, of H2O2 on platelet signaling pathways.
Integrin IIbß3, the
classic platelet fibrinogen receptor, can exist in resting or
activated states, and the latter can occur with or without
ligand engagement. Activation corresponds to a structural change,
whereby the affinity of
IIbß3 for fibrinogen
and other adhesive molecules in solution is markedly
increased.11 Moreover, integrin
IIbß3 can be found in
2 different compartments relative to extracellular ligands: exposed (to
extracellular ligands) or cryptic (not accessible to extracellular
ligands).12 Receptor molecules can be recruited
from cryptic storage to the platelet surface or removed from the
surface through a process of endocytosis.12 13 14
The molecular reactions that lead to receptor activation have been
studied: ß3 seems to play the role of a
regulatory subunit of the receptor, whereas
IIb appears to mediate receptor
specificity.15 16 There is good evidence that the
activation process utilizes ATP15 and involves
interaction of the short cytoplasmic domain of
ß3 with a regulatory
factor.15 17 18
H2O2 can induce a strong
tyrosine kinase response in smooth muscle cells, especially in cells
whose tyrosine phosphatases are concurrently inhibited with
orthovanadate.19
H2O2 was also shown to be a
necessary mediator for receptor tyrosine kinases in their
mitogenic effects.19 We and others
have shown that the activation of
IIbß3 can be
antagonized by inhibitors of tyrosine kinases, such as
genistein.20 In this study, we show that
H2O2, in the presence of
orthovanadate, induces a strong tyrosine
phosphorylation reaction and triggers a change in the
reactivity of the fibrinogen receptor, whereby it becomes capable of
mediating aggregation when platelets are exposed to shear in the
form of stirring. This state of
IIbß3 is different
from the activated state; we call it "primed," and it is
associated with the tyrosine phosphorylation of
ß3.
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Materials
Pepstatin, antipain, chymostatin, leupeptin, apyrase, N-acetyl-L-cysteine (NAC), prostaglandin E1, genistin, and genistein were from Sigma Chemical Co. ADP and other reagents used for the aggregation assay, including the aggregometer, were from Chrono-Log Corp. The 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) protease inhibitor was from Calbiochem-Novabiochem Corp. Lyophilized human fibrinogen was from AB Kabi. FITC for labeling fibrinogen was from Pierce, and FITC calibration beads (Quantum Series) were from Flow Cytometry Standards Corp. The thrombin receptor–tethered (activating) peptide (TRAP) Ser-Phe-Leu-Leu-Arg-Asn was from Bachem Bioscience Inc. Monoclonal antibodies to phosphotyrosine and anti-phosphotyrosine–agarose beads were from Upstate Biotechnology. The anti–P-selectin (S12) monoclonal antibody was the generous gift of Roger McEver (University of Oklahoma, Oklahoma City). The anti-ß3 monoclonal antibody SZ21 was from Immunotech, Coulter Co. Anti-ß3 antibodies AP3, C3a, and E8 were generous gifts of Peter Newman (AP3; Blood Center of Southeastern Wisconsin, Milwaukee) and David Philips (COR Therapeutics Inc, San Francisco, Calif). Horseradish peroxidase–labeled goat anti-mouse IgG and FITC-labeled goat anti-mouse antibody were obtained from Hyclone Laboratories. The
IIbß3 heptapeptide
inhibitor integrilin was the gift of Charles du Mee (COR
Therapeutics, Inc, San Francisco, Calif). Protein concentrations were
measured on an EL 312e Bio-Kinetics Reader (Bio-Tek Instruments Inc).
Platelet counts were obtained in a Coulter counter model ZF
(Coulter Electronics Inc) with a 70-µm-window aperture.
Platelet Aggregation Assay
Experiments performed on the blood of normal human volunteers
had been approved by the Joint Committee for Clinical Investigation of
Johns Hopkins Hospital and Johns Hopkins University School of Medicine,
as well as by the Human Subject Review Committee of Ohio State
University. Platelet aggregation was studied on platelet-rich
plasma (PRP) obtained from the citrated blood of human volunteers under
standardized conditions.21 Platelet counts
were normalized to 3x108
mL-1. PRP samples were preincubated with or
without inhibitors (NAC, apyrase, genistin, or genistein)
for 15 minutes at 37°C in 0.5-cm-diameter cuvettes before being
supplemented with agonists
(H2O2,
NaVO4, ADP, arachidonate, and
thrombin receptor TRAP), and then aggregation was followed for 15 to 30
minutes (single-channel aggregometer from Chrono-Log Corp). Shear was
generated by stirring the PRP (or washed platelets), and the
integrated shear factor versus rpm was
estimated.22 Expressed in percent, aggregation at
30 minutes was recorded and normalized to a standard deflection,
corresponding to light transmission through platelet-poor
plasma.21 For some experiments, platelets
were washed as described21 and then reconstituted
at 3x108 mL-1 in
Tyrode's solution supplemented with fibrinogen (3 g/L).
FITC-Fibrinogen Binding Assay
Activation of the fibrinogen receptor was studied with a flow
cytometric assay by measuring the binding of FITC-bound fibrinogen to
the surface of washed platelets in the presence of
agonists.23 This assay is based on the unique
ability of activated
IIbß3 to bind
fibrinogen in solution on the surface of platelets. Labeling of
fibrinogen with FITC, quantification of fibrinogen in solution, and
determination of the fluorescein to fibrinogen ratio were
performed as reported previously.23 Each sample
(500 µL in a plastic tube) contained washed platelets in
Tyrode's solution (107 cells ·
mL-1), supplemented with FITC-fibrinogen
(0.3 µmol/L). Incubation with FITC-fibrinogen, ADP (20
µmol/L), H2O2 (600
µmol/L), and NaVO4 (10 µmol/L) was
performed for 15 minutes in 0.5-cm-diameter cuvettes (Chrono-Log
aggregometer) at 37°C. Shear was generated by stirring the cells at
1000 rpm for the first 5 minutes of the 15-minute incubation. Samples
were analyzed in a fluorescence-activated cell
sorter (FACScan, Becton Dickinson) calibrated daily for
fluorescence by using Cytofluor fluorescent
beads.23 Data acquisition and processing from
104 cells were carried out on a Hewlett-Packard
computer using FACScan Research software (version B). The median
channel number was used as the measure of platelet
fluorescence intensity, which corresponded to FITC-fibrinogen
binding.23
Flow Cytometric Assay for P-Selectin
We quantified the expression of P-selectin on the surface of
washed platelets (107 platelets ·
mL-1) before and after agonist
stimulation.21 After 15 minutes of exposure to
TRAP (50 µmol/L) or
H2O2±NaVO4,
the platelets were fixed (3.7% formaldehyde in Tyrode's solution)
and then washed with Tyrode's solution. The antibody S12, a monoclonal
directed against the extracellular portion of P-selectin on the surface
of activated platelets, was used as the primary antibody
(1-hour incubation, 10 µg/mL). After the cells were washed,
platelets were incubated with 10 µg/mL of FITC-labeled goat
anti-mouse secondary antibody (1 hour, 10 µg/mL) and then
analyzed by flow cytometry. Controls included preparations
omitting the first antibody or using anti–phospholipase C
1 as a
negative-control primary antibody.21
Western Blotting of Proteins From Washed Platelets
Washed platelets were extracted at various times after
agonist stimulation in lysis buffer (15 mmol/L HEPES, pH 7.0;
145 mmol/L NaCl; 0.1 mmol/L MgCl2;
10 mmol/L EGTA; 1 mmol/L NaVO4; 0.5%
Triton X-100; 1 mmol/L AEBSF; and 20 µg/mL of protease
inhibitors: chymostatin, leupeptin, antipain, and
pepstatin).24 Normalized samples of washed
platelets (250 µL, 2 mg/mL) were mixed with SDS sample Laemmli
buffer and boiled for 5 minutes, under either reducing (0.35 mol/L DTT)
or nonreducing conditions. Samples (10 µg of protein) were
analyzed by SDS–polyacrylamide gel electrophoresis
(PAGE) and transferred to nitrocellulose membranes. Protein detection
on blocked membranes was carried by using either an
anti-phosphotyrosine monoclonal antibody or
anti-ß3 antibodies and the enhanced
chemiluminescence technique. The antibody used to detect
ß3 was C3a, which recognizes both reduced and
nonreduced ß3.
Immunoprecipitation of Washed-Platelet Proteins
ß3 Immunoprecipitation
The platelets from 1 mL of PRP were washed and resuspended
in Tyrode's solution. After addition of agonists for the indicated
periods of time, platelets were spun in an Eppendorf
centrifuge (model 5415c) for 2 minutes at
104 rpm. After the supernatants were removed, the
cells were extracted on ice in lysis buffer (see above; 0.4 mL),
sonicated for 5 seconds (Branson Sonifier 450 at power level 1), and
normalized for total protein content. Normalized extracts (0.4 mL) were
incubated with 5 µL of E8 plus 5 µL of AP3
anti-ß3 monoclonal antibody or with 5 µL of
nonimmune mouse serum and then supplemented with 100 µL of a 50%
(vol/vol) suspension of protein A–Sepharose beads in water. The
samples were mixed end-over-end at 4°C overnight and then pelleted.
The pellets were washed 3 times with cold PBS. After the last wash, the
beads were boiled for 2 minutes in 200 µL of nonreducing Laemmli
buffer, and the resulting samples were analyzed by Western
blotting. Protein detection on blocked membranes was carried out as
described by using the anti-ß3 antibody SZ21 or
an anti-phosphotyrosine antibody and the enhanced chemiluminescence
technique.
Anti-Phosphotyrosine Immunoprecipitation
The washed platelets from 1 mL of PRP were suspended in
Tyrode's solution and exposed to various agonists for the indicated
periods of time, at the end of which the cells were pelleted by
centrifugation (Eppendorf) for 5 minutes
(6x103 rpm). After the supernatants were
removed, the pellets were extracted on ice in 0.4 mL of lysis buffer
(see above), vortexed, sonicated for 5 seconds at power level 1, and
spun for 10 minutes (104 rpm) at 4°C. The
supernatants were collected and normalized for total protein content,
and then 250 µL of each normalized sample (total protein, 1 mg/mL)
was incubated with 20 µL of either nonimmune agarose beads or agarose
beads covalently coated with anti-phosphotyrosine IgGs overnight, with
end-over-end mixing at 4°C. The beads were then pelleted in an
Eppendorf centrifuge (1.4x104 rpm) and
washed in PBS. After the third wash, the beads were resuspended in
nonreducing Laemmli sample buffer (20 µL) and boiled for 5 minutes.
Disrupted immune complexes were analyzed by SDS–PAGE, and
protein bands were transferred to nitrocellulose membranes. Protein
detection on blocked membranes was carried as described by using an
anti-ß3 antibody (SZ21) or an
anti-phosphotyrosine antibody and the enhanced chemiluminescence
technique.
To define the stoichiometry of the tyrosine-phosphorylated ß3 versus the total ß3, we compared the amount of insoluble ß3 within the anti-phosphotyrosine immunoprecipitate to the amount of ß3 remaining soluble in the supernatant. Densitometric analyses of the precipitated ß3 bands and soluble ß3 bands of 2 separate experiments were used to determine their ratio, after correction for the dilution factor.
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Results |
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H2O2-Induced Aggregation of Platelets Exposed to Orthovanadate and Stirring
We used H2O2 to alter the redox state of human platelets and to study the consequences of such alteration in terms of platelet function. H2O2 (>300 µmol/L) induced aggregation of stirred platelets concurrently exposed to sodium orthovanadate (NaVO4, 1 mmol/L) (Figure 1a
). Previous studies had
indicated that NaVO4 reinforces the effect of
H2O2 on tyrosine kinase
pathways, perhaps by synergistically inhibiting the activity of protein
tyrosine phosphatases within exposed cells.19 In
the absence of NaVO4,
H2O2 (900 µmol/L)
had only limited effect on the aggregation of platelets (Figure 1a and 1b, last bar of the middle panel). In contrast with most agonists
that induce a more rapid aggregation of platelets, a lag phase was
observed between the time of addition of
H2O2 and the initiation of
rapid aggregation (Figure 1a and 1b, left panel). The lag phase seemed
to correspond to a delay in the effect of
H2O2 on platelets,
because preincubation of platelets with
H2O2 and orthovanadate for
30 minutes resulted in rapid aggregation on stirring (not shown). The
length of the lag phase was inversely proportional to the concentration
of H2O2, whereas the extent
of aggregation, once it had occurred, was fairly constant (Figure 1b, middle panel).
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Aggregation induced by H2O2
was highly dependent on stirring (shear) because the speed at which
platelets were stirred had to reach a specific threshold (>300
rpm, corresponding to a calculated integrated shear factor of >80
s-1)22 for aggregation to
take place (Figure 1b
, right panel). In the absence of stirring,
platelet aggregation was not detectable, even in the presence of
900 µmol/L H2O2 and
1 mmol/L NaVO4 (not shown). The presence of
plasma proteins and, perhaps, of antioxidant contained in plasma
affected the ability of
H2O2 to induce platelet
aggregation. Thus, aggregation of washed platelets could be induced
in Tyrode's solution supplemented with a
physiological concentration of fibrinogen. Under
such conditions, lower concentrations of
H2O2 (100 µmol/L)
did suffice to induce maximal aggregation in the presence of
NaVO4 (1 mmol/L) and stirring. In the
absence of fibrinogen, aggregation of platelets in the presence of
H2O2 and
NaVO4 was not detectable (data not shown).
Because fibrinogen was required for aggregation induced by
H2O2 and
NaVO4, next we studied the role of
IIbß3 in this process.
Even in the absence of stirring and with the use of fibrinogen binding
as a probe for IIbß3
activation, ADP (20 µmol/L) was able to induce strong activation
of IIbß3 on the
surface of platelets, with 4x104 to
8.5x104 activated receptor molecules per
cell, depending on the donor under study. In contrast,
H2O2 or
NaVO4 alone and in combination had minimal effect
on receptor activation in the absence of stirring (the
Table). In the presence of shear, while
receptor activation was still near background levels for platelets
exposed to H2O2 or
NaVO4 alone, in the presence of both
H2O2 and
NaVO4, stirring of the platelets induced
>40% of maximal activation of the
IIbß3 receptor (ADP,
20 µmol/L) (the Table). Furthermore, we also confirmed that the
aggregation induced by H2O2
and NaVO4 was mediated by the fibrinogen
receptor, IIbß3 since
we have shown that the specific
IIbß3
inhibitor integrilin inhibited platelet aggregation
induced by H2O2 in a
concentration-dependent fashion and with an IC50
of 0.25 µmol/L, as previously reported for this
inhibitor (Figure 2a).
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To ascertain that the mechanism mediating platelet aggregation by
H2O2 was indeed alteration
of the redox state of platelet proteins, we showed that the
antioxidant molecule NAC was able to block aggregation of platelets
exposed to H2O2, with an
IC50 of 
15 mmol/L (Figure 2b). In
contrast, apyrase (2 U/mL), which would have blocked platelet
aggregation if it were mediated by ADP released from platelet dense
granules on exposure to
H2O2, had only minimal
effect on H2O2-induced
platelet aggregation, as the increase in lag phase induced by
apyrase was barely detectable and aggregation proceeded to completion
(Figure 3). The content of -granules
was not released on exposure of platelets to
H2O2 and
NaVO4. Using the surface expression of P-selectin
as a probe for -granule secretion,21 we found
that the combination of
H2O2 and
NaVO4 had minimal effect on P-selectin expression
on the surface of platelets in the absence of stirring (Figure 4).
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Thus, in the presence of
H2O2 and
NaVO4,
IIbß3 mediates the
aggregation of stirred platelets. Our data suggest that
IIbß3 on the surface
of platelets exposed to redox changes is in a conformation that is
primed for activation by shear (here generated by stirring of the
platelets). Next, we studied the intracellular signaling pathways
that transduced the signal generated by
H2O2, primed the fibrinogen
receptor, and sensitized it to shear.
H2O2 in the Presence of NaVO4
Induces a Strong Tyrosine Kinase Response in Washed
Platelets
H2O2 had been shown to
be a strong agonist of tyrosine kinases (or an antagonist
of protein tyrosine phosphatases) in smooth muscle cells and
endothelial cells.19 Moreover,
NaVO4 is an established inhibitor of
tyrosine protein phosphatases. Their combined effect on platelet
aggregation points toward a mechanism of platelet activation that
involves tyrosine phosphorylation of platelet
substrates. Therefore, we tested the possibility that
H2O2 can induce
platelet protein phosphorylation on tyrosine and
that such an effect was required for
H2O2-induced platelet
aggregation. Neither H2O2
nor NaVO4 alone was able to induce marked
phosphorylation of platelet substrates. However,
the combination of H2O2 and
NaVO4 was able to induce, even in the absence of
other agonists or shear and in a concentration-dependent fashion, the
phosphorylation on tyrosine of multiple substrates in
platelets (Figure 5a), and at least
some of this phosphorylation reaction could be blocked
by NAC (see tyrosine phosphorylation of
ß3).
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We next hypothesized that tyrosine phosphorylation of
platelet substrates was required for platelet aggregation
induced by H2O2 and
NaVO4. To test this hypothesis, we first studied
the relationship between the time course of the tyrosine
phosphorylation reaction and that of the aggregation of
platelets exposed to
H2O2 and
NaVO4. Platelets were disrupted in lysis
buffer at serial times after addition of
H2O2 and
NaVO4, and normalized amounts of extracted
proteins were analyzed on Western blots by using an
anti-phosphotyrosine antibody as the probe. We observed that the timing
of the aggregation was tightly aligned with the timing of the tyrosine
phosphorylation reaction triggered by
H2O2 and
NaVO4 (cf Figure 5b with Figure 1). Indeed, the
aggregation of stirred platelets was triggered specifically when
the tyrosine phosphorylation of platelet substrates
was nearly maximal.
We next tested the ability of the tyrosine kinase inhibitor
genistein to inhibit platelet aggregation induced by
H2O2. At concentrations
sufficient to block tyrosine phosphorylation of
platelet substrates,20 platelet
aggregation was inhibited by genistein, while the control isoflavone
genistin had no effect on either tyrosine
phosphorylation or platelet aggregation at
concentrations matching those of genistein (Figure 2c). Hence, the
tyrosine phosphorylation of platelet substrates
seems to play an important role in mediating redox signals and is
required for platelet aggregation induced by
H2O2 and
NaVO4.
The ß3 Subunit Is Phosphorylated on
Tyrosine in Platelets Exposed to
H2O2
ß3, the regulatory subunit of
IIbß3, contains in its
short cytoplasmic domain a sequence that corresponds to a consensus
target peptide for tyrosine kinases.25 Therefore,
we tested the possibility that tyrosine phosphorylation
of ß3 in platelets exposed to
H2O2 may represent
an important step in priming the fibrinogen receptor for activation
induced by shear (stirring of platelets, in this case).
Immunoblots of immunoprecipitated
tyrosine-phosphorylated proteins corresponding to
extracts of platelets exposed to
H2O2 and
NaVO4 demonstrated a 95-kDa protein (nonreduced
conditions) that reacted strongly with anti-ß3
(Figures 6 and 7). The fraction of
ß3 that was phosphorylated on
tyrosine in platelets exposed to
H2O2 and
NaVO4 was measured to be 9.8±1.9% (mean±SEM)
of the total by densitometric analysis of Western blots. The
anti-phosphotyrosine–immunoprecipitated band corresponding to
ß3 was nearly undetectable on Western blots of
extracts from unstimulated platelets, platelets exposed to ADP
(20 µmol/L, ±NaVO4), and extracts of
platelets exposed to
H2O2 or
NaVO4 alone (Figure 6a) or on blots of proteins
precipitated with nonimmune beads (not shown).
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Because ß3 could have coprecipitated with the
anti-phosphotyrosine beads as a result of its binding to a tyrosine
phosphorylated–protein ligand, we also
immunoprecipitated ß3 with 2 murine monoclonal
antibodies (E8 and AP3 used concurrently) and developed the Western
blots with anti-ß3 (SZ21) and also
anti-phosphotyrosine (Figure 6b). Although the intensity of the band
corresponding to ß3 was the same in all lanes
detected with anti-ß3 (SZ21), the
anti-phosphotyrosine antibody detected a band at the specific molecular
weight of nonreduced ß3 in extracts exposed to
both NaVO4 and
H2O2 only (Figure 6b). In
experiments in which the antibodies E8 and AP3 were replaced by
nonimmune serum, no band was detected at the molecular weight of
ß3 (not shown). Owing to the compact secondary
structure of nonreduced ß3 imposed by several
disulfide bridges, reduced ß3 migrates as if it
were a protein larger than nonreduced
ß3.25 When proteins from
platelets exposed to agonists were studied on Western blots, the
differential migration of ß3, depending on the
redox conditions (reduced versus nonreduced), was clearly detectable,
supporting the validity of our method for detecting
ß3 (Figure 6c). Moreover, under both reduced
and nonreduced conditions, a tyrosine-phosphorylated
protein band migrated to the precise position of
ß3.
Next, we studied the time course of tyrosine
phosphorylation of ß3 in
platelets exposed to
H2O2 and
NaVO4 and detected the
phosphorylation of the ß3 band
peaking at a time when aggregation of platelets was initiated (cf
Figure 7a and 7b versus Figure 1a).
Addition of NAC at a concentration that blocks platelet aggregation
(10 mmol/L, Figure 2b) prevented the tyrosine
phosphorylation of ß3 (Figure 7c) as well as platelet aggregation. Together, these data suggest
that tyrosine phosphorylation of
ß3 in response to
H2O2 and
NaVO4 might contribute to the primed state of the
fibrinogen receptor.
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We conclude that H2O2, especially in the presence of NaVO4, induces the aggregation of stirred platelets. Such aggregation requires the binding of
IIbß3 to
its extracellular adhesive molecule ligand(s) and the
phosphorylation on tyrosine of protein substrates and
is associated with the phosphorylation on tyrosine of
ß3. The phosphorylation of
ß3 on tyrosine has been shown to participate in
outside-in integrin signal transduction after platelet
aggregation.25 Thus, in platelets stimulated
by thrombin, the phosphorylation of
ß3 was detectable only after engagement of
IIbß3, because
inhibition of aggregation by a cyclic Arg-Gly-Asp peptide
antagonist prevented the phosphorylation of
ß3.25 The
phosphorylation of ß3 is
remarkably transient with agonists like thrombin, even in the presence
of NaVO4, and detection of
ß3 phosphorylation requires
immediate boiling of the extracts to inhibit
phosphatases.25 Therefore, it was expected that
ADP (20 µmol/L) was unable to induce tyrosine
phosphorylation of ß3 under our
conditions, even in the presence of NaVO4. However, when platelets were activated by the combination of H2O2 and NaVO4, the tyrosine phosphorylation of ß3 was readily detectable, even in the absence of fibrinogen binding and aggregation, and might mediate the priming of the receptor for its responsiveness to stirring. Further work will be needed to provide a specific mechanism explaining why our conditions are able produce the aggregation-independent phosphorylation of ß3. We anticipate that such a mechanism might involve the ability of H2O2 and NaVO4 to synergistically inhibit the activity of platelet protein tyrosine phosphatases.26 It has been hypothesized that, in response to classic platelet agonists, the tyrosine phosphorylation of ß3 is too transient to be detected because of the concurrent activation of protein tyrosine phosphatases. In most systems, protein tyrosine phosphatases are known to have the potential to overwhelm the activity of tyrosine kinases,27 and therefore, it might require the activity of extremely potent protein tyrosine phosphatase inhibitors (such as the synergistic effect of H2O2 and NaVO4) to reveal the phosphorylation on tyrosine of a substrate like ß3.
The specific effect of ROS and, in particular, H2O2 on platelet responsiveness appears to be complex. In the presence of physiologically relevant concentrations of NO, H2O2 increases the inhibitory potency of NO toward platelets by 10- to 100-fold.8 Inhibition of platelets by NO/H2O2 is independent of the formation of peroxynitrite and appears to be mediated by the enhanced formation of cGMP, leading to activation of cGMP kinase.8 Moreover, Ambrosio et al9 showed that exposure of platelets to H2O2 reduces their aggregation response to ADP (by 48%), collagen (by 71%), or thromboxane (by 50%). Inhibition of platelet aggregation is associated with a marked increase in cGMP in the exposed cells and could be blocked by the guanylate cyclase inhibitor LY-83583.8 9
However, Pignatelli et al28 have shown that H2O2 might serve as a second messenger in the activation process of platelets exposed to collagen. In a dog model of cyclic flow variation in stenosed and endothelium-injured coronary vessels, ROS and, in particular, H2O2 increased platelet aggregation.29 30 Platelets have been known to produce superoxide under certain conditions.31 More recently, platelets directly exposed to anoxia and reoxygenation were shown to produce superoxide and hydroxyl radicals and to be activated in a autocrine/paracrine fashion by these ROS through the stimulation of arachidonic acid metabolism.7 Furthermore, aggregation of platelets induced by arachidonate was potentiated by the presence of H2O2, and low amounts of H2O2 were required for aggregation induced by subthreshold concentrations of arachidonate, since catalase abolished platelet aggregation stimulated by arachidonic acid under these conditions.32 33 In contrast, catalase had no effect on ADP-, collagen-, or thromboxane-induced aggregation. Yet catalase, in collagen-stimulated platelets, did prevent thromboxane A2 production and the release of arachidonate.28
Together, these data suggest that H2O2 might play an important role in the platelet response to vessel injury.7 28 29 30 In the presence of a functional endothelium that produces NO, low levels of H2O2 potentiate the stabilizing effect of NO on resting circulating platelets. However, vascular injuries that have damaged endothelial cells lead to a loss of NO production by these cells and to increased free-radical production by anchored phagocytes and perhaps also by activated (dysfunctional) endothelial cells.34 In the presence of arachidonic acid produced by the activation of phospholipase A2,7 ROS can then catalyze the aggregability of exposed platelets. On clustering, platelets could occlude the affected blood vessel. Then, triggered by hypoxia and reoxygenation, platelets could generate radicals, which in turn would induce (in concert with arachidonate) further platelet aggregation, resulting in amplification of the thrombotic process.
Here, we have shown that
H2O2 in the absence of
established platelet agonists but in synergy with shear and the
tyrosine protein phosphatase inhibitor
NaVO419 can induce platelet
aggregation. H2O2-induced
aggregation under our conditions is independent of ADP release from
dense granules and is mediated by
IIbß3 binding to
fibrinogen or to von Willebrand factor (vWF), as it can be
blocked with the highly selective inhibitor
integrilin.35 Tyrosine kinase pathways seem to be
involved in the aggregation reaction induced by
H2O2 and
NaVO4, since a very strong
phosphorylation reaction is observed in platelets
exposed to H2O2 and
NaVO4, which culminates exactly at a time when
rapid aggregation is initiated.36 37 38 Moreover,
aggregation can be blocked by the rather specific tyrosine kinase
inhibitor genistein.20 Previous
studies have reported on the tyrosine phosphorylation
of phospholipase C in platelets exposed to vanadate and
H2O236 and
have suggested that p72syk might by an important tyrosine kinase in
this process.37
Here, we show tyrosine phosphorylation of the
ß3 subunit of integrin
IIbß3 in platelets
exposed to H2O2 and
NaVO4. Tyrosine phosphorylation
of ß3 peaks in intensity at the onset of
aggregation and might play an important role in priming the receptor to
become activated on exposure to shear. Tyrosine
phosphorylation of the ß3
subunit has been shown unequivocally to occur on thrombin-induced
aggregation of platelets, but not when cells were exposed to an
inhibitor of aggregation, indicating that engagement of the
receptor was required.25 A conserved consensus
motif surrounding tyrosine 747 is shared with other receptors
(epidermal growth factor and insulin receptors) known to be
phosphorylated on tyrosine on ligand binding. Moreover,
the peptide sequence surrounding tyrosine 747, once
phosphorylated on tyrosine, is also known to bind
signaling molecules of receptor tyrosine kinase
pathways.25
Using H2O2 and NaVO4, we were able to demonstrate the tyrosine phosphorylation of ß3, even with washed platelets that were not able to aggregate, and with a timing that parallels closely the course of priming of the fibrinogen receptor for shear-induced aggregability. While we are in the process of studying further the mechanism that unveiled the tyrosine phosphorylation of ß3 under our conditions, we assume that it is related to the potent inhibition of protein tyrosine phosphatases. To this extent, pervanadate, the complex of vanadate with H2O2, has been shown to be a particularly potent inhibitor of phosphatases.38 Alternatively, the combination of H2O2 and NaVO4 might stimulate specific tyrosine kinases not recruited by other platelet agonists in the absence of aggregation.
Finally, we would like to address the potential physiological relevance of platelet activation by the combination of oxidants and shear. In acute coronary syndromes such as unstable angina and myocardial infarction, platelets are known to be exposed to ROS produced by phagocytes, endothelial cells, or even platelets themselves. In fact, clusters of platelets adhering to neutrophils have been detected in the blood of patients with unstable angina.3 4 5 The precise concentration of ROS present at the interface between platelets and neutrophils, or between platelets and activated endothelial cells, is unknown but could be quite high. Moreover, the lesions that trigger acute coronary syndromes correspond to a narrowing of a coronary vessel, with or without ulceration or rupture of the atherosclerotic plaque. At such sites, wall shear stress can be markedly increased (hundreds of dynes per square centimeter).39 40
Goto and colleagues41 have shown that under
shearing flow conditions, platelet aggregation requires both vWF
binding to glycoprotein Ib and
IIbß3 binding to its
adhesive ligands. The activation of platelets can be induced by
high shear stress alone, a process that is initiated by
glycoprotein Ib interaction with
vWF but results in aggregation only if the latter can bind concurrently
to IIbß3. In contrast,
platelets exposed to high shear rate after activation by exogenous
agonists such as ADP and epinephrine can aggregate when
fibrinogen is the ligand for
IIbß3, yet vWF binding
to glycoprotein Ib also needs to
occur.41 High shear was shown to increase
tyrosine phosphorylation of proteins of 130, 100, 85,
74, 70, 64, 58, and 40 kDa within seconds after the beginning of
exposure of platelets to high shear force.42
It is tempting to speculate that both the binding of
glycoprotein Ib to vWF and the
priming of the receptor through a pervanadate-mediated molecular
reaction can contribute in synergy to activation of
IIbß3, thus providing
a key step in the initiation of platelet aggregation. Thus, it is
likely that the combination of ROS and shear contributes significantly
to the activation of platelets, specifically at the site of
unstable coronary lesions.
|
Acknowledgments |
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This study was supported by NIH grant HL52315, the Bremer Foundation, and AHA Established Investigator award (P.J.G.-C.).
Received August 27, 1997; accepted April 22, 1998.
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References |
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