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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 484-490
Hydrogen Peroxide Is Involved in Collagen-Induced Platelet Activation
By
Pasquale Pignatelli,
Fabio M. Pulcinelli,
Luisa Lenti,
Pier Paolo
Gazzaniga, and
Francesco Violi
From the Department of Experimental Medicine and Pathology and
Institute of 1st Clinical Medicine, University La Sapienza, Rome,
Italy.
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ABSTRACT |
In this study, we investigated whether (1) collagen-induced platelet
aggregation is associated with a burst of H2O2,
(2) this oxidant species is involved in the activation of platelets,
and (3) the pathways of platelet activation are stimulated by
H2O2. Collagen-induced platelet aggregation was
associated with production of H2O2, which was
abolished by catalase, an enzyme that destroys H2O2. H2O2 production
was not observed when ADP or thrombin were used as agonists. Catalase
inhibited dose-dependently thromboxane A2 production,
release of arachidonic acid from platelet membrane, and Inositol
1,4,5P3 (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This
study suggests that collagen-induced platelet aggregation is associated
with a burst of H2O2 that acts as a second
messenger by stimulating the arachidonic acid metabolism and
phospholipase C pathway.
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INTRODUCTION |
COLLAGEN IS AN important platelet agonist
that is thought to be involved in the early stages of platelet
activation during both hemostasis and thrombosis. The first step in the
interaction of platelets to collagen is adhesion through specific
platelet receptors, most probably the integrin glycoprotein
GPIa/IIa.1-3 After adhesion, a signal is transmitted into
the cells that produces other signaling events that eventually lead to
increases in cytosolic Ca2+, activation of protein kinase
C, activation of phospholipase A2, and platelet secretion.
The products of these events, ADP, and thromboxane A2
(TxA2) act to recruit more platelets by a process of
aggregation. The exact mechanism of the signal transduction by collagen
is not completely clear. However, several investigators4-6 have shown that collagen activates platelets via a tyrosine
kinase-dependent phosphorylation of PLC 2. Current evidence indicates
that the receptor for signal transduction is a 62-kD protein named
GP VI.7,8
H2O2 generated by superoxide-dismutase (SOD)
seems to be directly involved in the collagen-induced activation of
platelets in experiments in which subthreshold concentrations of
collagen and SOD caused platelet aggregation9; catalase,
which destroys H2O2, fully prevented
SOD-dependent aggregation.9
The role of H2O2 and oxygen free radicals in
the mechanism of collagen-induced platelet aggregation was also
outlined by stimulation of platelets with either threshold or high
concentrations of collagen. Del Principe et al10 showed
that collagen-induced platelet aggregation is associated with
H2O2 release and that catalase inhibits both H2O2 formation and platelet aggregation.
Furthermore, in aspirin (ASA)-treated platelets, aggregation induced by
high concentrations of collagen was inhibited by several antioxidants
such as salicylic acid, vitamin E, and dipyridamole.11
These data suggest that H2O2 and oxygen free
radicals have a role in the activation of platelets mediated by
collagen, but the underlying mechanism has not been fully understood.
It has been suggested that oxidant species may behave as second
messengers in stimulating cyclooxygenase-dependent and independent
pathways of platelet activation, but experimental evidence in support
of this suggestion is still lacking.11 The aim of the
present study was to investigate the role of
H2O2 on the mechanism of platelet activation by
collagen. To this purpose, we analyzed the separate effects of catalase
and ASA plus catalase on collagen-induced platelet responses such as
H2O2 production, aggregation, secretion,
phospholipase C (PLC) activation, calcium mobilization,
TxA2 production, and arachidonic acid release. The results
support the suggestion that H2O2 formation may
have a role in stimulating cyclooxygenase-dependent and independent
pathways of platelet aggregation.
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MATERIALS AND METHODS |
Materials.
32Pi was from Amersham (Arlington Heights, IL).
[3H]-Ins 1,3,4 P3 and
[3H]-arachidonic acid (3H-AA) were from NEN
Life Science Products (Boston, MA). Fura 2-AM and
2 7-dichlorofluorescein diacetate (DCFH-DA) were from
Molecular Probes (Eugene, OR). Sepharose 2B was from Pharmacia
(Uppsala, Sweden). Collagen, type 1, was from Semmelweis (Mascia
Brunelli, Milan, Italy). High-performance liquid chromatography (HPLC)
columns, Partisil 10 SAX were from Whatman (Haverhill,
MA). Luciferin and Luciferase were from Chrono-Log
(Havertown, PA). Bovine serum albumin, HEPES, ASA, catalase (bovine
liver, tymol-free), fibrinogen, inorganic-pyrophosphatase,
acid/citrate/dextrose, digitonin, EGTA, EDTA, Tris, perchloric acid,
formaldehyde, CaCl2, indomethacin, ammoniun formate, maleic
acid, Lucigenin (bis-methylacridinium nitrate), bovine erythrocyte SOD,
creatine phosphate (CP), creatine phosphokinase (CPK), and
ferricytochrome c (c-7752) were from Sigma Chemicals Co (St
Louis, MO).
Inactive catalase was denaturated by treatment with
3-amino-1,2,4-triazole according to Darr and Fridrovich.12
Diphenylene iodonium (DPI) was purchased from Aldrich (Milwaukee, WI).
Platelet preparation.
Human blood was obtained from drug-free, healthy volunteers and
anticoagulated with acid/citrate/dextrose.13 Platelet-rich plasma (PRP), which was obtained by centrifugation (15 minutes at
180g), was recentrifuged (800g for 20 minutes) to
concentrate the platelets, and the pellet was resuspended in 0.5 vol of
autologous platelet-poor plasma.
The platelet suspensions were incubated for 1 hour at 37°C with 3 µmol/L Fura 2-AM or with 40 µmol/L DCFH-DA, or 32Pi 2 mCi/mL of cell suspension, or 3H-AA 2 µCi/mL of cell
suspension.
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| Fig 1.
Cytofluorimetric evaluation of
H2O2 production in DCFH-DA-loaded platelets.
(A) Fluorescence peak of unstimulated platelets. (B) Fluorescence peak
of unstimulated platelets with 250 U/mL of catalase (absence of
fluorescence). (C) Shift of fluorescence after incubation with
H2O2 (1 mmol/L). (D) Shift induced by collagen 50 µg/mL. (E) Effects of the same dose of collagen in GFP treated with catalase (500 U/mL). (F) Effects of the same dose of collagen in
GFP treated with catalase (1,000 U/mL). Results are representative of
five separate experiments.
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| Fig 2.
(A) DCF fluorescence in collagen-stimulated platelets
(100%) and in collagen-stimulated platelets added with scalar
concentrations (250, 500, and 1,000 U/mL) of catalase. (B)
Cytofluorimetric evaluation of oxidation of DCFH-DA-loaded platelets
to fluorescent DCF induced by scalar doses of collagen. Data are
expressed as the mean fluorescence channel ± SEM of five separate
experiments.
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Platelets were separated from plasma proteins and from excess of Fura
2-AM and 32Pi by gel filtration on Sepharose 2B using
Ca2+ free Tyrode's buffer containing 0.2% bovine serum
albumin, 5 mmol/L glucose, and 10 mmol/L HEPES, pH 7.35. After gel
filtration, the cell suspension (gel-filtered platelets [GFP]) was
adjusted to a final concentration of 2 × 108
cells/mL.
The ASA-treated platelets were obtained by incubating GFP with ASA (100 µmol/L for 10 minutes at 37°C). Catalase was added in different
concentrations (250 to 1,000 U/mL) 5 minutes before the activation of
GFP with collagen. The ratio between the volumes of catalase and
platelet suspension was 5 to 20 µL/mL.
CP/CPK (20 mmol/L and 50 U/mL, respectively) was added 1 minute before
platelet stimulation.
As control, in all experiments, GFP were incubated with the dilution
medium.
Platelet aggregation.
In vitro platelet aggregation was evaluated according to
Born14 in a four-sample Aggrecorder II Menarini (Florence,
Italy) at 37°C using siliconized glass cuvets under continuous
stirring at 1,000 rpm. Fibrinogen (1 mg/mL) was added before the
agonist.
The threshold concentration (TC) of agonist was defined as the lowest
dose that induced more than 50% of aggregation.
ATP release.
Collagen-induced platelet secretion was evaluated by measuring the
release of ATP. The activation of platelets (see above) was stopped
after 2 minutes with formaldehyde/EDTA, as according to Costa and
Murphy.15 After centrifugation at 10,000g for 30 seconds, the ATP concentration in the supernatant was measured using an
LKB 1251 luminometer (Pharmacia) after the addition of Luciferin (40 mg/mL) and Luciferase (880 U/mL). The results were expressed as the
percentage of ATP released relative to the total ATP present in lysed
cells.16
Production of TXB2.
The activation of platelets (see above) was stopped after 3 minutes
with indomethacin (14 µmol/L). TXA2 production was
determined using TXB2 enzyme-linked immunosorbent assay
kits (Boehringer Mannheim GmbH, Mannheim, Germany).
Platelet cytosolic Ca2+ concentrations.
Calcium measurements were made using the fluorescent indicator dye
Fura-2. The fluorescence changes were then monitored with an SFM 25 fluorimeter (Kontron Instruments AG, Zurig) set at 340 nm
excitation and 510 nm emission. To convert fluorescence measurements into Ca2+ concentrations, the Fmin was
determined after the addition of digitonin (50 µmol/L) in the
presence of EGTA (2 mmol/L) and Tris base (20 mmol/L); the
Fmax was measured by the addition of excess CaCl2 (10 mmol/L). The calcium concentration was calculated
using these values and a kd of 224 nmol/L, according to
Grynkiewicz et al,17 after correction for extracellular
dye.
PLC activation.
Because the activation of PLC produces inositol-1,4,5 P3
from phosphatidyl-inositol-4,5-bis-phosphate (PIP2) and Ins
1,4,5 P3 is converted within 30 to 60 seconds into
inositol-1,3,4,5 P4 (Ins 1,3,4,5 P4) that is
rapidly degraded into the more stable inositol-1,3,4 P3
(Ins 1,3,4 P3),18 we have studied the Ins 1,3,4 P3 production 1 minute after the platelet stimulation with collagen or thrombin. The collagen activation of the
[32P]-labeled platelets resuspended in phosphate free
Tyrode's buffer was stopped by means of perchloric acid (0.44 N). The
neutralized platelet extracts (1 × 109 cells/mL) were
treated overnight with Zn2+-pyrophosphatase at 20 U/mL in
the presence of Tris-maleic buffer 0.1 mol/L, pH 6.5, and then passed
on an HPLC column that was eluted with a 50-minute linear gradient of
water as first buffer and ammonium formate at 1.5 mol/L, pH 3.75, as
final buffer. Inositol peaks were detected using a dual-channel
(3H-32P) HPLC radioactivity detector FLO-ONE
A100 (Radiomatic; Camberra Co, Tampa, FL) using [3H]-Ins
1,3,4 P3 as pure standard.18
Tritiated AA release.
The tritiated AA release was studied by prelabeling platelets with
tritiated AA. Platelets resuspended in plasma were incubated with 2 µCi/mL tritiated AA for 1 hour at 37°C, then washed twice to
remove the remaining free arachidonate, and finally resuspended in
Tyrode's buffer. Samples of tritiated AA-labeled platelets were
preincubated for 3 minutes at 37°C before stimulation. After 1 minute, the reaction was stopped by adding a solution containing 5 mmol/L EDTA, 5 mmol/L theophylline, and 0.2 µg/mL prostaglandin E1 (PGE1). After centrifugation for 3 minutes
at 5,000g, the percentage of 3H-AA released into
the supernatant was determined by liquid scintillation counting of 100 µL aliquots in 2 mL of aqueous fluid. Each experiment was performed
in duplicate.
Detection of superoxide anion.
The chemiluminescence (CL) of lucigenin was detected with an LKB 1251 luminometer (see above), as previously described.19
Briefly, CL was detected in GFP at a fixed concentration of 3 × 108 cells/mL at 37°C. Each sample, added with 0.25 mmol/L lucigenin, 1 mmol/L CaCl2, and 150 mg/dL fibrinogen,
was stimulated with 1 µg/mL collagen and the CL, obtained at
intervals of 1 to 3 minutes, was measured. Samples
containing lucigenin plus components (with the exception of platelets)
were counted, and these blank values were subtracted from the CL
signals obtained from collagen-stimulated platelets. CL was expressed
as nanomoles of O2 per milliliter per
minute. In some experiments, 300 U/mL SOD, or 50 µmol/L DPI, or 100 µmol/L ASA was added to the platelet suspension before collagen
stimulation.
Flow cytometric analysis.
PRP, which was obtained by centrifugation (15 minutes at 180g),
was recentrifuged (800g for 20 minutes) to concentrate the platelets. The pellet was resuspended in phosphate-buffered saline, 8 µL/mL of 5 mmol/L DCFH-DA was added, and, after 15 minutes of incubation, the platelet suspension was washed twice and resuspended in
Tyrode's buffer at the final concentration of 2 × 108 cells/mL. Platelet preparation was activated with
collagen alone or in the presence of catalase and stopped with 2 mmol/L
EGTA after 1 minute of reaction.
All samples were analyzed on a Coulter Epics (Hialeah, FL) flow
cytometer equipped with an argon laser (480 nm emission). The
instrument was set up to measure logarithmic forward light scatter
(LFS), which is a measure of particle size; logarithmic 90° light
scatter (LSS), which is a measure of cell granularity; and green (DCF)
510 to 550 nm fluorescence (LFL1). Fluorescent parameters (in arbitrary
units [AU]) were collected using three-decade logarithmic
amplification.
Statistical analysis.
Data are reported as the mean ± SEM. The comparison between
variables was analyzed using the Student's t-test for unpaired data. Significance was accepted as the P < .05 level.
| |
RESULTS |
Flow cytometric analysis.
This method uses the properties of DCFH-DA,20-22 which
rapidly diffuses across cell membranes and is then trapped within the cell by a deacetylation reaction. In the presence of hydrogen peroxide,
this compound is oxidized to DCF, which is highly
fluorescent.23
Figure 1 shows the DCF green fluorescence distribution
of unactivated platelets (Fig 1A; mean fluorescence, 20 ± 2), which was fully prevented by 250 U/mL of catalase (Fig 1B). The largest increase of DCF fluorescence occurred with 1 mmol/L
H2O2, which caused a mean fluorescence channel
shift of 5 times (mean fluorescence, 97 ± 7; P < .02 v control; Fig 1C). Collagen-induced platelet activation
doubled the shift of DCF fluorescence in comparison to control (mean
fluorescence, 44 ± 16; P < .04 v control; Fig 1D). Catalase inhibited the shift of DCF fluorescence induced by
collagen dependently upon its concentration (mean fluorescence with 500 U/mL = 18 ± 3, P < .05 v collagen; mean
fluorescence with 1,000 U/mL = 12 ± 2, P < .05 v collagen; Fig 1E and F and Fig 2A).
Figure 2B reports a dose-response curve showing that the increase of
H2O2 is dependent on collagen concentration.
Comparing the curve with the mean fluorescence observed while adding 1 mmol/L H2O2 to the platelet suspension, the
amount of H2O2 released by collagen-stimulated
platelets seems to be lower than 1 mmol/L. When using ADP or thrombin
as platelet agonists, no increase in DCF fluorescence was observed (not
shown).
Platelet aggregation.
In every GFP preparation, we searched for the lowest collagen
concentration (TC) able to induce more than 50% of aggregation. In all
cases, the effect of this concentration was completely inhibited by
preincubation of GFP with ASA; to restore aggregation, ASA-treated
platelets were stimulated with fourfold to eightfold TC of collagen
(Fig 3B).
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| Fig 3.
Catalase inhibits collagen-induced platelet aggregation
both in untreated and ASA-treated platelets. (A): (a) TC of collagen; (b, c, and d) TC collagen plus catalase (250, 500, and 1,000 U/mL respectively). (B): (a) same as in (A); (b) ASA (100 µmol/L)-treated platelets stimulated with TC collagen; (c) ASA-treated platelets stimulated by eightfold TC collagen; (d) same as in (c) plus catalase at 250 U/mL. Similar results have been obtained in five separate experiments. The threshold concentration (TC) in this experiment was 2 µg/mL.
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Catalase inhibited dose-dependently the aggregation induced by TC of
collagen (Fig 3A); moreover, it inhibited the aggregation of
ASA-treated GFP induced by fourfold to eightfold TC of
collagen (Fig 3B).
Catalase had no effect when other agonists such as ADP (2 µmol/L) or
thrombin (0.1 U/mL) were used (Table 1).
No effect was observed when denatured catalase was added to GFP
stimulated by collagen (data not shown).
ATP release.
In degranulation studies, we used the same TC of collagen tested for
aggregation studies. ATP release induced by TC collagen was inhibited
by catalase in a dose-dependent fashion
(Fig 4A).
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| Fig 4.
Catalase inhibits collagen-induced ATP secretion both in
untreated and aspirin-treated platelets. (A) TC of collagen; TC
collagen plus catalase (250, 500, and 1,000 U/mL). (B) ATP release in
platelets stimulated with TC of collagen in ASA (100 µmol/L)-treated
platelets stimulated with TC and fourfold to eightfold TC of collagen
added with or without catalase (250 U/mL). Data are expressed as the mean ± SEM of five separate experiments (*P < .01;
**P < .001).
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ATP release induced by TC collagen was completely inhibited by
preincubation of GFP with ASA. Fourfold to eightfold TC of collagen was
able to restore platelet degranulation to levels similar to those
obtained in control platelets (Fig 4B).
Catalase was able to inhibit the secretion of ASA-treated platelets
stimulated by fourfold to eightfold TC of collagen.
Platelet TXB2 formation.
Collagen-induced TXB2 formation was dose-dependently
inhibited by catalase. This effect seems to be specific for collagen, because no change was observed with 0.1 U/mL of thrombin
(Fig 5). Thus, even with the higher
concentration of catalase (1,000 U/mL), no significant changes of
TXA2 production were found in thrombin-stimulated
platelets.
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| Fig 5.
Catalase inhibits TXB2 production in collagen
but not thrombin-stimulated platelets. Platelet TXB2
production in collagen (10 µg/mL)-stimulated platelets added with and
without 250 to 1,000 U/mL of catalase and in thrombin (0.1 U/mL)-stimulated platelets incubated with and without 1,000 U/mL of
catalase. Data are expressed as the mean ± SEM of five separate
experiments (*P < .05; **P < .01).
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Changes in intracellular calcium concentration.
The changes in intracellular calcium concentration induced by collagen
in control GFP or in GFP treated with ASA, catalase, or catalase plus
ASA are reported in Fig 6. In samples
stimulated with collagen (10 µg/mL), catalase dose-dependently
inhibited intracellular calcium mobilization (Fig 6A). In ASA-treated
platelets, the intracellular calcium mobilization induced by collagen
(10 µg/mL) was reduced by about 70% but was restored by increasing collagen concentrations (20 and 40 µg/mL; Fig 6B). The addition of
catalase to ASA-treated platelets again reduced intracellular calcium
mobilization; this effect was evident even with very high concentration
of collagen (40 µg/mL; Fig 6B).
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| Fig 6.
Catalase inhibits collagen-induced calcium mobilization
both in untreated and ASA-treated platelets. (A) Calcium mobilization in collagen-stimulated platelets added with or without 250, 500, or
1,000 U/mL catalase. Data are expressed as the mean ± SEM of five
separate experiments (*P < .05; **P < .01). (B)
Calcium mobilization in ASA (100 µmol/L)-treated platelets with or
without catalase (250 U/mL) stimulated with scalar concentrations of
collagen. Data are expressed as the mean ± SEM of five separate
experiments (*P < .05; **P < .01). (C) Cytosolic
calcium concentration induced by collagen (40 µg/mL) was reduced by
catalase (250 U/mL) in platelets pretreated with aspirin (100 µmol/L)
and the ADP scavenger system CP/CPK. Data are expressed as the mean ± SEM of five separate experiments (*P < .01).
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The inhibition of calcium mobilization by catalase was also observed
when aspirinated platelets were treated with the ADP scavenger system
CP/CPK (Fig 6C).
PLC activation.
The 32P-labeled-Ins 1,3,4 P3 (IP3) production
by collagen-stimulated platelets added with and without catalase, ASA,
or ASA plus catalase is shown in Fig 7. As
previously described,4,18,24 we had to use higher
concentrations of collagen (50 µg/mL) and thrombin (1 U/mL) to
measure IP3. Collagen-induced IP3 formation was inhibited by catalase
in a dose-dependent fashion (Fig 7A). Thrombin-induced PLC activation
was not affected by catalase (Fig 7A). In ASA-treated platelets
stimulated with 50 µg/mL collagen, catalase elicited a further
reduction of IP3 formation in comparison with platelets treated with
aspirin alone. This effect was evident when aspirin-treated platelets
were also incubated with CP/CPK (Fig 7B).
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| Fig 7.
Catalase inhibits 32P-Ins 1,3,4 P3 (IP3)
production. (A) Catalase (250 to 500 to 1,000 U/mL) inhibited
32P-Ins 1,3,4 P3 (IP3) production by collagen (50 µg/mL)
but was ineffective if thrombin (1 U/mL) was used as an agonist. Data are expressed as the mean ± SEM of five separate experiments
(*P < .05). (B) In ASA (100 µmol/L)-treated platelets, 250 U/mL catalase inhibited 32P-Ins 1,3,4 P3 (IP3) production
by collagen (50 µg/mL). Similar findings were obtained in samples
added also with the ADP scavenger system CP/CPK. Data are expressed as
the mean ± SEM of five separate experiments (*P < .05;
**P < .01).
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Tritiated AA release.
Platelets stimulated by collagen released 3H-AA, which was
inhibited by catalase depending on the concentration used. Conversely, aspirin did not affect 3H-AA release
(Table 2).
Superoxide production.
The O2 release measured by lucigenin CL
increased time dependently upon platelet stimulation with collagen and
was reduced 63% by SOD (n = 5; P < .01;
Fig 8A), 40% by DPI (n = 5; P < .05), and 63% by ASA (n = 5; P < .01; Fig 8B).
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| Fig 8.
O2 production in platelets
stimulated with collagen. (A) Time course of
O2 production estimated by lucigenin
chemoluminescence in platelets stimulated with 1 µg/mL collagen. Data
are expressed as the mean ± SEM of five separate
experiments. (B) Bar groups showing O2
production estimated by lucigenin 3 minutes after stimulation with 1 µg/mL collagen in platelets incubated with or without 50 µmol/L DPI
or 100 µmol/L ASA. Data are expressed as the mean ± SEM of five
separate experiments (*P < .05; **P < .01).
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| |
DISCUSSION |
This study shows that collagen-induced platelet activation is
associated with a burst of H2O2 that was
dependent on the concentration of collagen used. Platelet production of
H2O2 was shown by DCFH oxidation and its
disappearance on platelet preincubation with catalase. To assess
whether this oxidant species has a role on platelet function, we
investigated whether catalase, which destroys H2O2, affects platelet aggregation and specific
pathways of platelet activation. First of all, we showed that catalase
inhibited platelet aggregation. Such an effect was not aspecific,
because catalase elicited platelet inhibition in a dose-dependent
fashion and was ineffective when denatured. The role of
H2O2 on platelet function was closely related
to the agonist collagen, because other agonists such as ADP and
thrombin did not provoke platelet H2O2
formation; furthermore, catalase had no effect on platelet activation
induced by ADP or thrombin. We then investigated whether
H2O2 could behave as a second messenger by
stimulating specific intracellular pathways such as arachidonic acid
metabolism and PLC activation. Collagen-induced platelet
TXA2 formation was significantly inhibited by catalase, suggesting that H2O2 interferes with
arachidonic acid metabolism. Previous studies have shown that peroxides
are an important stimulus for the activation of cyclooxygenase
enzyme25; therefore, H2O2 could
amplify platelet response to collagen by stimulating this enzyme.
Alternatively, H2O2 could favor arachidonic acid release by platelet membrane, because oxidant species have been
shown to stimulate phospholipase A2 enzyme.26
Our findings are consistent with this suggestion, because catalase
inhibited dose-dependently the arachidonic acid release by collagen.
The amplification of platelet response by H2O2
was not restricted to the activation of arachidonic acid metabolism,
because catalase was able to inhibit Ca2+ mobilization in
both ASA-treated and non-ASA-treated platelets, suggesting that
H2O2 could also stimulate PLC activation. Our findings are consistent with the hypothesis that
H2O2 stimulates PLC, because catalase
dose-dependently inhibited IP3 formation; however, our findings do not
explain the molecular mechanism underlying this effect.
Previous study showed that collagen-induced PLC activation is dependent
on tyrosine kinase activation4 and that
H2O2 plays a role in the activation of tyrosine
kinase.27 Hence, it could be postulated that the activation
of phospholipase C2 by H2O2 could be
mediated by the stimulation of tyrosine kinase, but we do not have
conclusive data to support this hypothesis.
Several sources of oxidant species have been suggested to be activated
upon platelet stimulation, but definitive evidence is still lacking.
Activation of arachidonic acid metabolism could be theoretically an
important source, because, in other cell lines, it has been shown that
oxidant species are produced through the stimulation of this
pathway.11 Preliminary data from our group showed that
O2 results from the activation of
arachidonic acid pathways because arachidonic acid-stimulated platelets
produced this oxidant species and aspirin partly prevented
it.19 Our findings are consistent with this suggestion,
because the release of O2 by
collagen-stimulated platelets was reduced 63% by aspirin. However, it
is likely that other sources of O2 are
present in platelets, because catalase interfered with platelet function also when cyclooxygenase pathway was inhibited, suggesting that H2O2 may be produced through alternative
pathways. Even if it has been shown by other
investigators28 and ourselves19 that DPI, an
inhibitor of NADPH oxidase, inhibits platelet function and
O2 platelet release, its lack of
specificity does not allow to fully conclude that platelets produce
O2 through the activation of NADPH
oxidase. Whatever the source of O2, the
release of this oxidant species by collagen-stimulated platelets is an
important step for the formation of H2O2,
because O2 is enzymatically dismutated
to H2O2 by SOD, an enzyme that is present in
platelet cytosol.29
In conclusion, this study shows that H2O2 is
produced by collagen-stimulated platelets and acts as second messenger
by activating arachidonic acid metabolism and PLC pathway. Our findings
provide a rationale to further analyze the relationship between
antioxidants and platelet function and to investigate whether the
antiatherosclerosis effect so far reported for this drug category is
also related to some interference with platelet function.
| |
FOOTNOTES |
Submitted March 24, 1997;
accepted September 8, 1997.
Supported in part by Grant C.N.R.-FATMA, subproject 8-contract
C.T.95.00834.PF41.
Address reprint requests to Pier Paolo Gazzaniga, MD, Dipartimento di
Medicina Sperimentale e Patologia, Università degli Studi di Roma
"La Sapienza," Viale Regina Elena 324, 00161 Roma, Italy.
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.
| |
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Abstract
Introduction
Abstract