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Blood, 1 August 2002, Vol. 100, No. 3, pp. 917-924
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
NAD(P)H oxidase-dependent platelet superoxide anion release
increases platelet recruitment
Florian Krötz,
Hae Young Sohn,
Torsten Gloe,
Stefan Zahler,
Tobias Riexinger,
Thomas M. Schiele,
Bernhard F. Becker,
Karl Theisen,
Volker Klauss, and
Ulrich Pohl
From the Institute of Physiology and the Department of
Internal Medicine, Ludwig-Maximilians-University Munich, Schillerstr
44, 80336 Munich, Germany, and Ziemssenstr 1, 80336 Munich, Germany.
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Abstract |
Platelets, although not phagocytotic, have been suggested to
release O . Since O-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are
found in several nonphagocytotic tissues, we investigated whether such
an enzyme is the source of platelet-derived O. We
further studied which agonists cause platelet O release and whether platelet-derived O influences
thrombus formation in vitro. Collagen, but not adenosine 5'-diphosphate
(ADP) or thrombin, increased O formation in
washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)-dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47phox and p67phox
and inhibition of platelet O formation by
diphenylene-iodoniumchloride (DPI) and by the specific
peptide-antagonist gp91ds-tat were observed. Whereas
platelet-derived O did not influence initial
aggregation, platelet recruitment to a preformed thrombus following
collagen stimulation was significantly attenuated by superoxide
dismutase (SOD) or DPI. It was also inhibited when ADP released during
aggregation was cleaved by the ectonucleotidase apyrase. ADP in
supernatants of collagen-activated platelets was decreased in the
presence of SOD, resulting in lower ADP concentrations available for
recruitment of further platelets. Exogenous O increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were
stimulated with otherwise subthreshold concentrations of ADP. These
results strongly suggest that collagen activation induces NAD(P)H
oxidase-dependent O release in platelets, which in
turn enhances availability of released ADP, resulting in increased
platelet recruitment.
(Blood. 2002;100:917-924)
© 2002 by The American Society of Hematology.
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Introduction |
Platelet activation is thought to be a key event in
acute vascular thrombosis. Therefore, prevention of enhanced platelet activation is a major target of therapeutic strategies fighting cardiovascular and cerebrovascular diseases.1-3 An
important stimulus for physiologic platelet activation and thrombus
formation is the contact of platelets with components of the
subendothelial matrix, like collagen.4 Although Marcus et
al have shown as early as 1977 that platelets have the ability to
release superoxide anions (O),5 it was
only recently proposed that platelets stimulated by collagen produce
reactive oxygen species (ROS) such as hydrogen peroxide,6
hydroxyl radicals,7 or
O.7,8 While O, a
highly reactive radical, damages cells in high concentrations by
reacting with proteins, lipids, and DNA, in low concentrations its
continuous production, with similarity to second messengers, has been
suggested to indirectly affect signal transduction
processes.9,10 Platelet agonists other than collagen, such
as thrombin or ADP, do not seem to induce ROS formation during
aggregation.8 This difference raises the question whether
O formation could serve a modulating function when
thrombus formation is induced by collagen.
The cellular source of platelet O is unclear.
Growing evidence supports the assumption that platelet activation by
collagen is specifically due to binding to the glycoprotein VI
(GPVI)-receptor,11,12 resulting in a cascade of tyrosine phosphorylation events ultimately leading to activation of
phospholipase C (PLC),13 which is known to
strongly activate protein kinase C (PKC) through production of
diacylglycerol.4 Recently, evidence for the existence of a
neutrophil-type reduced nicotinamide adenine dinucleotide (phosphate)
(NAD(P)H) oxidase in platelets that can be activated by PKC and is
involved in O formation has been
presented,14-16 similar as in other
O-generating systems, like the vascular endothelium.
In endothelial cells, an NAD(P)H oxidase is the main source of
O.17 As O readily
reacts with NO, this has been suggested to result in attenuated
NO-dependent vessel relaxation,18,19 and the role of
O in the regulation of vascular tone has become a
major focus of interest.17 Moreover, antioxidants like
N-acetylcysteine (NAC) have been shown to exert direct
antiaggregatory effects.20 Although these findings raise
the possibility that platelet-derived O is involved
in regulating platelet activation, evidence for a role of
platelet-derived O in platelet function is rare. In
a canine model of coronary arterial thrombosis, thrombus formation was
regulated by intraplatelet redox state.21 Leo and
colleagues have shown that platelets subjected to anoxia/reoxygenation are more reactive, due to an enhanced O generation.14 However, so far it remains unclear whether
an enhanced O production occurs also during direct
platelet activation, such as with collagen, and how this could affect
thrombus growth. Whereas conventional methods of measuring platelet
aggregation in vitro are limited by the amount of cells
used,22 the in vivo situation provides the thrombus with a
continuous further supply of circulating platelets. These additional
platelets, however, are activated not only by the stimulus that
originally initiated thrombus formation, but also by substances like
ADP, thromboxane A2, Ca++, and
others that are released from platelet granules after
activation.4 Therefore, the extent of thrombus formation
in vivo is determined not only by the initial exposure to an
agonist, but also by the release of further stimuli and the recruitment
of further platelets caused by these substances.
In this study we sought to determine which agonists cause platelet
O production, whether a neutrophil-type NAD(P)H
oxidase is the source of this phenomenon, and how platelet function is
affected by platelet release of O. We sought to
discriminate between the aggregation induced by the initial agonist and
the effect of released substances on unstimulated platelets, as the
resulting recruitment represents a crucial element of thrombus growth.
As ADP is one important mediator of recruitment, we investigated
whether O increases ADP-dependent platelet
reactions.23 Availability of this nucleotide is determined
not only by the amount released, but also by ectonucleotidases metabolizing it,24 which, again, are known to be
influenced by ROS in other systems.25 Therefore, we also
investigated whether platelet-derived O can
indirectly influence ADP metabolism, possibly by modulating the
activity of ectonucleotidases.
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Materials and methods |
Platelet and neutrophil preparation
Venous blood was drawn from healthy volunteers who had not taken
any medication for at least 10 days. Informed consent was obtained from
all subjects. Blood that had been anticoagulated by 3.13% sodium
citrate was centrifuged at 150g for 15 minutes. For
experiments investigating platelet function, only the upper third of
the supernatant was used to prevent leukocyte contamination. Washed
platelets (WP) were prepared by another centrifugation step at
600g for 10 minutes and resuspended in calcium-free
modified Tyrode buffer containing 138 mM sodium chloride, 2.7 mM potassium chloride, 12 mM sodium hydrogen carbonate, 400 µM
disodium phosphate, 1 mM magnesium chloride, 5 mM D-glucose,
and 5 mM HEPES (buffer A). WP were used for experiments within 2 hours.
Polymorphonuclear neutrophils (PMNs) for control experiments were
isolated from the buffy coat of blood that had been centrifuged at
150g for 15 minutes according to a magnetic antibody
separation technique as previously described.26 Platelet
and leukocyte counts were obtained using a resistance particle counter
(Coulter Z2, Beckman Coulter, Krefeld, Germany).
Measurement of superoxide formation by chemiluminescence
assay
O production was measured by a
chemiluminescence assay using the dye L-012. We have
previously described the high specificity and sensitivity of this assay
for O.27 The reaction volume of 300 µL contained 150 000-300 000/µL WP, calcium chloride (1 mM), and L-012 (100 µM). After measuring the background signal, stimulating substances such as phorbol-12-myristate 13-acetate (PMA, 1 µM), collagen (6 µg/mL), adenosine diphosphate (ADP, 1-50 µM), or
thrombin (0.1-1 U/mL) were added. Photon emission was
expressed as percent increase in relative light units versus control
conditions. Maximum leukocyte contamination in the reaction buffers
was 9/µL.
Measurement of superoxide formation by cytochrome
c assay
In an alternative method to assess platelet O
production, the cytochrome c method was used with minor
modifications as described before.28 WP
(10 × 108) were incubated with cytochrome c
(40 µM), which was dissolved in phosphate-buffered saline
with or without superoxide dismutase (SOD, 250 U/mL) and the respective
stimuli. All stimulations were performed in the presence of 1 mM
calcium chloride. The reduction of cytochrome c was
measured in a spectrophotometer (Ultrospec 2000, Tecan, Grödig,
Austria) at 550 nm over a 30-minute period. The superoxide-dependent
part of cytochrome c reduction was calculated from the
difference between samples incubated with or without SOD
( 550 nM = 21.1 mM 1
cm1).
Measurement of NADH/NAD(P)H oxidase activity
WP were stimulated as indicated. After the reaction was stopped
by adding 2 mM ethyleneglycoltetraacetic acid (EGTA),
platelets were pelleted by centrifugation at 600g for 10 minutes at 4°C and resuspended in lysis buffer containing 1 mM sodium
fluoride, 150 µM
tetra-sodium-diphosphate-10-hydrate, 1 mM
ethylene-diaminetetraacetic acid (EDTA), 400 µM
sodiumorthovanadate, 4 mM disodiumphosphate, 10 µM
phenylmethylsulfonyl fluoride, and 10 µg/mL each of leupeptin, pepstatin, and aprotinin. The platelets were then disrupted by passing
them 5 times through a 29-gauge needle, followed by storage on ice for
24 hours. Disruption was confirmed microscopically. For detection of
O formed by the lysates, the cytochrome c
reduction method was used as described above using 10 µg protein per
experiment. Various concentrations of NAD(P)H or reduced nicotinamide
adenine dinucleotide (NADH) as indicated were used to start
the reaction, and reduction of cytochrome c
was measured for 30 minutes. Protein content was determined by
the method of Bradford.29
Immunoblotting studies
Platelets were washed in the presence of prostacyclin analog
(iloprost, 0.01 µg/mL) and resuspended in buffer A. After
being adjusted to a final volume of 500 000/µL in the presence of
iloprost (0.1 µg/mL) and 1 mM EGTA and subsequent stimulation, lysis
was performed by adding an equal volume of ice-cold lysis buffer
containing Tris, 50 mM; EDTA, 1 mM; sodium orthovanadate, 1 mM; sodium
fluoride, 10 mM; tetra-sodium-diphosphate 10-hydrate, 1.5 mM; disodium
hydrogen phosphate, 4 mM; leupeptin, aprotinin, pepstatin, 1 µM each;
phenylmethylsulfonyl fluoride, 1 mM; Triton-X 100, 1%; sodium dodecyl
sulfate (SDS), 0.1%; and desoxycolic acid, 0.5% (buffer B)
to the platelet suspension. Lysates were centrifuged at
10 000g for 10 minutes to remove debris, and the
supernatants were used for experiments. Proteins were separated by
SDS-polyacrylamide gel electrophoresis following standard procedures
and transferred to a nitrocellulose membrane. After blocking for 30 minutes in blocking buffer (sodium chloride, 200 mM; Tris pH 7.5, 50 mM; bovine serum albumin (BSA), 3%; Tween 20, 0.05%; and
horse serum, 10%; membranes were incubated with the respective primary
antibody in blocking buffer for 1 hour at room temperature. They were
then washed 4 times using TBS-T (Tris base, 50 mM; sodium
chloride, 150 mM; Tween 20, 0.1%) and incubated with horseradish
peroxidase-conjugated secondary antibody for 1 hour at room
temperature. The signal intensities were measured using an enhanced
chemiluminescence (ECL) detection system following the instructions of
the kit used (ECL Western blotting detection reagents, Amersham,
Freiburg, Germany). Bands were recorded with a
videodensitometric system from Bio-Rad (Gel Doc 1000),
Munich, Germany.
Aggregation studies
Platelet aggregation was measured using the turbidimetric method
described by Born.22 WP were adjusted to a concentration between 150 000 and 300 000/µL. Calcium chloride (1 mM) was added 2 minutes before starting the reaction, and aggregation was measured photometrically using a 2-chamber aggregometer (elvi 840, Logos, Milan,
Italy) under continuous stirring at 1000 rpm at
37°C.
Recruitment studies
To determine platelet recruitment, 2 different approaches were
used. One was performed according to a method described by Freedman and
colleagues.30 Aggregation was measured for 7 minutes. Then
an equal portion of untreated WP was added to the tube, which increased
the density of the solution and hence led to a reduction of light
transmission. Aggregation of the newly added platelet-portion in the
presence of an existing thrombus was then measured for 5 minutes and
expressed as percentage of the aggregation that had been reached
initially. In the second assay, recruitment was assessed according to a
method described by Valles et al.31 In this assay,
platelets were stimulated under various conditions as indicated at
37°C without stirring. After stimulation, cells and collagen were
pelleted at 10 000g for 50 seconds, and the cell- and
stimulus-free supernatant was immediately transferred as a stimulus to
another platelet suspension placed in an aggregometer. Concentration in
the final suspensions was adjusted to 200 000/µL. Then aggregation
was measured as described above.
Platelet ADP release
WP were counted and adjusted to a final concentration
of 500 000/µl in buffer A. To prevent aggregation and activation due to exogenously added ADP, iloprost (0.05 µg/mL) was added. Platelets were then stimulated with collagen, and samples of the supernatant were
taken at 7 minutes after stimulation. Supernatant samples were obtained
by centrifugation at 1500g to pellet the platelets and
remove the supernatant, which was immediately mixed with perchloric acid (400 mM) to stop potential enzymatic activities of nucleotidases. After addition of perchloric acid, samples were centrifuged at 10 000g for 5 minutes at 4°C to remove precipitates and
the amount of ADP in the supernatant quantified by high-performance
liquid chromatography (HPLC). As previously described,32
the acidified samples were applied to an EC 250/4 nucleosil
carbohydrate column and eluted with 10 mM
NH4H2PO4 (A; at pH 3.5) or 0.5 mM
NH4H2PO4 (B; at pH 3.0) using a
gradient of 100% of A for 15 minutes, then 30% of B for 1 minute,
40% of B for 4 minutes, and 100% of A at minute 20. Retention time
for ADP was 10.5 minutes.
Materials
Collagen was purchased as fibrils from Nycomed Pharma,
Munich, Germany;
8-amino-5-chloro-7-phenylpyridol(3,4-d)pyridazine-1,4(2H,3H)dione (L-012) was obtained from Aventis, Frankfurt, Germany; and superoxide dismutase from Roche Molecular Biochemicals, Penzberg, Germany. Anti-p47phox, anti-p67phox, and
anti-gp91phox were obtained from Santa Cruz Biotechnology
(Heidelberg, Germany). Prostacyclin analog (iloprost) was purchased
from Schering (Berlin, Germany). Erbstatine analog was from Bachem
(Heidelberg, Germany); the peptides gp91ds-tat and scrambled
gp91ds-tat (scrmb-tat) were kind gifts from Dr
Patrick Pagano (Detroit, MI). EC 250/4 nucleosil carbohydrate columns
were from Macherey-Nagel (Düren, Germany). All other substances
were obtained from Sigma Chemicals, Darmstadt, Germany.
Statistical analysis
For descriptive purposes, all data are expressed as
means ± SEM. Data were analyzed using one-way ANOVA or
Student t test. For paired recruitment experiments,
paired t tests were performed. Differences were considered
significant when the error probability level was
P < .05.
| |
Results |
Superoxide formation in platelets is stimulus specific
Stimulation with collagen (6 µg/mL) or phorbol ester
(PMA, 1 µM) led to a significant, long-lasting 2.4-fold and 5.8-fold increase in O production compared to control
platelets (n = 24, P < .05, n = 21, and
P < .01, respectively, 10 minutes after stimulation). In
contrast, ADP in a concentration range from 1 to 50 µM as well as
thrombin (0.1-1 U/mL) did not enhance platelet O
formation significantly (Figure 1A). Both
SOD and NAC abolished the collagen- or PMA-induced O
signal (Figure 2A), whereas boiled SOD
was without effect. Collagen-induced O production
was furthermore completely prevented by the specific PKC-inhibitor
chelerythrine (50 µM, n = 10, P < .01, Figure 1B) or
the tyrosine kinase inhibitor erbstatine analog (1 µM, n = 4,
P < .05, not shown). To control our findings, platelet
O production was also assessed using the cytochrome
c reduction method (Figure 2C). In these experiments,
collagen also induced a 2.1-fold increase in O
production over control conditions (0.0323 ± 0.008 vs 0.0155 ± 0.004 nmol/min/105 cells, n = 6,
P < .05). To exclude that neutrophils contaminating the
solution accounted for the observed O release,
control experiments with increasing concentrations of neutrophils
(10-100/µL) in buffer alone or in platelet suspensions were
performed. Neutrophils alone or stimulated with collagen did not show
an increase of L-012 chemiluminescence (L-012-CL). These neutrophils,
however, showed a substantial O production
when stimulated with the calcium ionophore A23187 (1 µM,
Table 1).
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| Figure 1.
Collagen induces platelet
O production.
(A) Effects of various stimuli on platelet superoxide
(O) production. Collagen (6 µg/mL, n = 24) and
phorbol-12-myristate 13-acetate (PMA, 1 µM, n = 21), but not ADP
(5-50 µM, n = 9) or thrombin (0.1-1 U/mL, n = 12),
increased O formation by platelets (10 minutes after
stimulation). O was measured by L-012
chemiluminescence. (B) Influence on NAD(P)H oxidase and PKC inhibitors
on collagen-induced (6 µg/mL) O formation.
Kinetics of collagen-induced O production and its
inhibition by DPI (100 µM, n = 6) and chelerythrine (50 µM,
n = 10). Data are shown as mean ± SEM; * (#), ** (##)
significantly different at P < .05, .01 vs stimulus and
DPI (*) or chelerythrine (#).
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| Figure 2.
Inhibition of platelet O
release.
(A) Effect of O scavengers on platelet release of
O. N-acetylcysteine (NAC, 1 mM, n = 9)
or superoxide dismutase (SOD, 250 U/mL, n = 9) significantly
decreased O release after PKC stimulation using PMA,
whereas boiled SOD was without effect (not shown). When collagen was
used as a stimulus, similar results were obtained (6 µg/µL, n = 7
each, 10 minutes after stimulation). (B) Specific NAD(P)H oxidase
inhibitors prevent platelet O production. A peptide
specifically inhibiting the interaction between gp91phox
(or its analogs) and p47phox (gp91ds-tat, 100 µM) but not its scrambled analog (scrmbl-tat, 100 µM)
inhibits platelet O production as measured by L-012
chemiluminescence (n = 9, at 10 minutes). (C) Amount of
O production as measured by cytochrome c
reduction. Platelet suspensions were stimulated in cytochrome
c dissolved in PBS, after preincubation with the respective
inhibitors when indicated (n = 6, at 10 minutes). Data are shown as
mean ± SEM; *, ** significantly different versus control at
P < .05 and .01, respectively; #, ##
P < .05 and .01 versus stimulus.
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Table 1.
Neutrophils do not contribute to superoxide
(O) production of platelet suspensions
stimulated with collagen
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NAD(P)H oxidase-dependent superoxide formation in
platelets
Next, we sought to investigate the enzymatic source of
collagen-induced O production in platelets. We used
various inhibitors of possible enzymatic sources. Rotenone (10-50 µM), indomethacin (1-10 µM), or oxypurinol (100-300 µM) were
without effect on collagen-induced O production (not
shown), making mitochondrial electron transport, cyclooxygenase, or
xanthine oxidase improbable sources. In contrast, DPI (100 µM), an
unspecific inhibitor of NAD(P)H oxidase, completely abolished
O formation caused by collagen (n = 6,
P < .01) and PMA (n = 7, P < .0001,
Figure 1B), as did the specific inhibitor of NAD(P)H oxidase, the
chimeric peptide gp91ds-tat. When preincubated for 30 minutes, gp91ds-tat (100 µM) completely abolished
collagen-induced O production as measured by
L-012-CL (n = 6, P < .05, Figure 2B) or cytochrome
c reduction (Figure 2C). In both cases, the same concentration of the scrambled peptide (scrmb) was without effect (n = 6).
To further determine whether O production in
platelets was NAD(P)H oxidase dependent, we measured O formation in lysates of untreated or
collagen-stimulated platelets in the presence or absence of various
concentrations of NADH or NAD(P)H. Platelet superoxide production in
the presence of NADH had a dose-dependent effect, with highest
levels observed at 500 µM (Figure 3A).
NAD(P)H also increased the signal, although to a lesser extent than
NADH. Treatment with collagen before lysis increased
O production in the presence of both substrates
(n = 3, Figure 3A). PMA also increased it to 0.64 ± 0.12 nmol/mg protein/min (P < .01, n = 13, not shown),
whereas pretreatment with ADP (50 µM) as a control did not
significantly enhance it (not shown).
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| Figure 3.
NADH/NAD(P)H oxidase activity and expression.
(A) NADH/NAD(P)H oxidase activity in stimulated platelets.
Addition of NADH led to a dose-dependent increase in
O production in lysates of platelets (n = 3). When
platelets had been stimulated with collagen, it was further increased
compared to lysates of control platelets. NAD(P)H as a cosubstrate
caused less O production (n = 3). (B) NAD(P)H
oxidase subunit expression in platelets. Immunoblots of platelet
protein expression (PLT) of the NAD(P)H oxidase subunits
p47phox and p67phox compared to neutrophil
protein (PMN) as a positive control. As described in "Materials and
methods," 40 µg of protein lysed was subjected to
SDS-polyacrylamide-gel-electrophoresis, blotted on
nitrocellulose membranes, and visualized using polyclonal antibodies
against the indicated proteins.
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As NAD(P)H oxidases are composed of at least 4 subunits in neutrophils
and other vascular cells,17 of which the small g-protein rac, p22phox, and p67phox have previously been
shown to be expressed in platelets,16,33 we performed
immunoblotting studies investigating the presence of the remaining
unidentified subunits p47phox, gp91phox, and
p67phox. Although we could demonstrate protein
expression of p47phox and of p67phox in all of
our platelet preparations (n = 5), we could not detect gp91phox using commercially available antibodies (Figure
3B).
Aggregation of platelets is differentially affected by
superoxide
Aggregation induced by various doses of collagen was not
influenced by endogenous O, since SOD (500 U/mL) did
not significantly affect the extent of aggregation (Table
2). Likewise, treatment with the
inhibitor of NAD(P)H oxidase, DPI (30 µM), had no significant effect
on aggregation. SOD or DPI also had no effect when aggregation was
induced by various doses of ADP. In contrast, the platelets were
reactive to NO-synthase inhibition. When collagen-stimulated platelets were incubated with the NO-synthase inhibitor
N-nitro-L-arginine (L-NA, 30 µM),
aggregation was significantly enhanced (Table 2).
Preincubation of platelets with exogenous O
using the O-generating system xanthine (300 µM)/xanthine oxidase (2 mU/mL, 30 seconds) showed different results
depending on the stimulus. While aggregation in response to collagen
was not altered, O markedly enhanced the sensitivity
for ADP. At a concentration of 1 µM, ADP induced only a reversible
aggregation under control conditions, while it induced
irreversible aggregation in the presence of O
(n = 9, Figure 4B). Xanthine/xanthine oxidase (X/XO) alone did not induce aggregation during the period observed in these experiments (not shown).
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| Figure 4.
ADP-dependent recruitment following collagen
stimulation.
(A) Tracing of parallel recruitment experiments showing
dependence of recruitment on ADP release following collagen
stimulation. Aggregation was induced by 6 µg/mL collagen fibrils. At
7 minutes following stimulation, additional platelets were
added simultaneously with 1 U/mL apyrase or the same volume of vehicle.
Apyrase, which cleaves ADP, inhibited recruitment of further platelets
(graph representative of 9 similar experiments). (B) Enhancement of
ADP-dependent platelet aggregation by reactive oxygen species. Tracing
of a parallel experiment showing enhanced aggregation of a subthreshold
concentration of ADP (1 µM) in the presence of exogenously created
O using xanthine (300 µM, X) and xanthine oxidase
(2 mU/mL, XO, graph representative of 9 similar experiments). (C) ADP
in platelet supernatants. ADP measured in the supernatants of
collagen-stimulated platelets is decreased in the presence of SOD (500 U/mL) and increased in the presence of X/XO (concentrations as above).
In supernatants of unstimulated platelets, there was little release of
ADP, which was not significantly increased by treatment with X/XO alone
(n = 11). **Significantly different versus control at
P < .01; #, P < .05 versus collagen.
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Platelet recruitment is ADP dependent and enhanced by
superoxide
As O production by platelets was observed to be
sustained for up to 30 minutes (Figure 1B), we further studied the role
of its release on the recruitment of platelets that were brought in
contact with the thrombus 7 minutes after initial stimulation of
aggregation. At this time, significant amounts of O
had been observed to be released by the collagen-stimulated thrombus
(Figure 1A-B). To investigate the effect of O
generated by collagen-stimulated platelets, we performed recruitment
experiments using either the O scavenger SOD (500 U/mL) or DPI (100 µM; representative traces are shown in Figure 5A). Only following collagen stimulation
(6 µg/mL) did SOD and DPI decrease platelet recruitment by
19% and 16%, respectively, whereas recruitment following thrombin- or
ADP-induced aggregation was not affected by SOD or DPI (n = 10 each,
P < .01 for collagen vs collagen + SOD or collagen
vs collagen + DPI, Figure 5A-B). We could also confirm the
observation that platelet recruitment is dependent on platelet NO
production by preincubating platelets with L-NA (30 µM)
for 15 minutes. These platelets showed not only an increased velocity
of aggregation, but also a 13% increase in recruitment
(P < .001, Figure 5B), as measured by the method of
Freedman et al.30 Furthermore, the extent of recruitment following ADP-induced aggregation was significantly less than that
following collagen stimulation (6 µg/mL, n = 10,
P < .05), in spite of the fact that high doses of ADP (50 µM) were used.
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| Figure 5.
Influence of antioxidants on recruitment
following collagen stimulation.
(A) Representative tracings of platelet recruitment experiments.
Recruitment induced by various agonists showed different reactions to
scavenging of O and inhibition of NAD(P)H oxidase.
Seven minutes after platelets had been stimulated (white arrow) in the
presence of either vehicle or SOD (500 U/mL), an equal amount
of untreated platelets was added (black arrow) and recruitment of
additional platelets to the preformed thrombus measured for another 5 minutes. Aggregation of recruited platelets resembled that of
ADP-induced aggregation, indicating that ADP release from platelets
during aggregation is the main stimulus acting on recruitable
platelets. (B) Left: Aggregation of recruited platelets at 5 minutes
after addition to a preformed thrombus as percent of control
(recruitment following collagen stimulation). SOD (500 U/mL) and DPI
(100 µM) significantly decreased recruitment, whereas L-NA
(30 µM) enhanced it (n = 10 each). Right: SOD only decreased
recruitment when stimulation was due to an agonist that induces
platelet O production like collagen, but not when it
was due to thrombin or ADP (n = 10 each). Boiled SOD was without
effect (not shown). **Significantly different versus control at
P < .01.
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To control these findings, an alternative model of measuring platelet
recruitment was performed as described by Valles et al.31
Supernatants of platelet suspensions stimulated by collagen in the
presence of SOD induced a decreased aggregation as compared to
supernatants of platelets exposed to collagen alone ( 17.2% ± 4.9%
for SOD, n = 7, P < .05; and 22.5% ± 5.9% for DPI,
n = 7, P < .05, Figure
6B). Again, this was not the case when
ADP or ADP in the presence of SOD was used as the initial stimulus (n = 7). Aggregation induced by the supernatants of
collagen-stimulated platelets showed different kinetics and a faster
onset than that directly induced by collagen (Figure 6A, gray tracing).
These tracings rather resembled tracings of aggregation induced by ADP. Therefore, we tested whether the recruitment observed after stimulation with collagen was due to ADP release. Apyrase, an ectonucleotidase that
hydrolyzes ADP to adenosine monophosphate (AMP) and free phosphate, was used to address this issue. Apyrase (1 U/mL) completely abolished platelet recruitment (n = 9, Figure 4A) in both models of
platelet recruitment, indicating that release of ADP was indispensable for recruitment following collagen stimulation.
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| Figure 6.
Aggregation induced by supernatants of
collagen-stimulated platelets.
(A) Recruitment measured as aggregation induced by supernatants of
activated platelets. Aggregation tracings of WP stimulated with
supernatants of differentially activated platelets. WP were stimulated
with collagen (6 µg/mL) in the presence or absence of SOD (500 U/mL)
and pelleted by centrifugation. The supernatants were collected and
immediately transferred to an aggregation cuvette with unstimulated
platelets to measure aggregation of these untreated WP. As a background
tracing collagen-induced aggregation (6 µg/mL) is shown (gray). (B)
Recruitment is inhibited by SOD or DPI. Recruitment measured as
aggregation induced by supernatants obtained as described above was
significantly inhibited by SOD (500 U/mL) when collagen was used as a
stimulus. Boiled SOD was without effect. Similar results were obtained
when inhibiting NAD(P)H oxidase using DPI (100 µM, n = 7).
*Significantly different versus control (stimulus without SOD or DPI)
at P < .05.
|
|
Oxidative influence on ADP released after collagen
stimulation
We had observed that collagen stimulation led to a long-lasting
production of O and that ADP release was a crucial
element of platelet recruitment following collagen stimulation.
Furthermore, platelets that had been pretreated with X/XO had shown an
increased aggregation response to ADP. We speculated that an altered
activity of a platelet ectonucleotidase could have been involved.
Inactivation of ectonucleotidases resulting in increased concentrations
of ADP has been described before.25 Therefore, we
investigated whether there is evidence for altered amounts of ADP
available to recruit further platelets under different circumstances,
by measuring amounts of ADP released from platelets. Supernatants of
unstimulated platelets contained little ADP (0.06 ± 0.01 µmol/min/100 000 cells, n = 11), which was not significantly altered when experiments were performed in the presence of exogenous ROS using the X/XO system (0.11 ± 0.02 µmol/min/100 000
cells, n = 5). In supernatants of collagen-stimulated platelets,
however, there were high amounts of ADP (0.69 ± 0.05 µmol/min/100 000 cells, n = 11, n < .01). They were even
increased (to 0.94 ± 0.05 µmol/min/100 000 cells, n = 5,
P < .05), when collagen stimulation was performed in the
presence of exogenous ROS produced through the X/XO system (X:
300µM, XO: 2mU). Scavenging O derived
from collagen-stimulated platelets by SOD (500 U/mL) reduced amounts of
ADP in the supernatants to 0.49 ± 0.06 µM/min/100 000 cells
(n = 11, P < .05; Figure 4C).
| |
Discussion |
This study demonstrates that collagen induces an enhanced
release of O from platelets through activation of an
NAD(P)H oxidase. The released O modulates platelet
function in an autocrine manner by increasing ADP-dependent recruitment
of further platelets to a forming thrombus, whereas the initial
aggregation is not affected by O.
While in accordance with other observations, all untreated platelets
showed a basal O release5; only
collagen stimulated the platelets to increase O release substantially. This response was specific for
collagen,8,34 since neither ADP nor thrombin increased
platelet O release although they also induced a
strong aggregation. Collagen-induced platelet signaling is thought to
be mainly mediated by binding to the surface receptor glycoprotein VI
(GPVI),12 whereas ADP acts via several purinergic
receptors35 and thrombin via protease-activated receptors36 and, to a lesser degree, via
GPIb.37 GPVI binding induces activation and
phosphorylation of PLC through a pathway involving numerous tyrosine
phosphorylation steps.13 As collagen-dependent platelet
O production was prevented by a blocker of tyrosine
kinases and also by inhibition of PKC, collagen-specific tyrosine
phosphorylation events eventually leading to PKC activation might
explain that superoxide production is specific for collagen. However,
collagen activation of PKC alone cannot fully explain this, as thrombin
is also known to induce PKC activation.36 Recent findings
showing that LPS-stimulated platelet O release is
abolished by an inhibitor of PKC,38 however, support
involvement of PKC. The collagen-induced O
production was furthermore inhibited by the flavoenzyme inhibitor DPI,
consistent with an NAD(P)H oxidase being the source of
O. Such O-generating NAD(P)H
oxidases have been identified in a variety of tissues, including
leukocytes and endothelial cells.17,39-41 NAD(P)H oxidase
is known to be a multicomponent protein complex assembled by the
cellular subunits p47phox, p67phox, and the
membrane-bound proteins p22phox and gp91phox,
which together with the small GTPDase rac1/2 associate to form the
active enzyme complex.17,41 In fact, rac is known to be activated in platelets upon collagen stimulation.33 The
presence of the subunits p22phox and p67phox as
parts of a putative platelet NAD(P)H oxidase has also recently been
demonstrated.16 Indeed, we could confirm existence of
platelet p67phox of a neutrophil-type NAD(P)H oxidase at
the protein level. In addition, we were also able to show the presence
of p47phox in platelets for the first time. However, we did
not succeed in detecting the gp91phox protein. It could be
that platelets express a different isoform of this important subunit,
which could not be detected by the antibody used. Recently a peptide
(gp91ds-tat) has been designed to specifically inhibit the
interaction of the NAD(P)H oxidase subunit p47phox with the
large membrane spanning subunit gp91phox. This peptide
inhibited the interaction of p47phox with
gp91phox by binding to the docking sequence (ds) of
p47phox at gp91phox.42 In our
experiments, it completely abolished platelet O production in response to collagen. Its effect on platelet
O production is not in contradiction to the missing
proof of gp91phox as the docking sequence for
p47phox at gp91phox shows high homology between
gp91phox and its so-far characterized isoforms nox1 and
nox4 (renox).39,43 Existence of a platelet NAD(P)H oxidase
is further supported by the finding that addition of NADH or NAD(P)H to
platelet lysates increased O production
significantly and particularly so when the platelets had been
pretreated with collagen. Preference of NADH as a substrate can also be
seen in endothelial NAD(P)H oxidases,40 giving additional
evidence for an isoform of gp91phox being present in platelets.
When characterizing the role of a NAD(P)H oxidase in forming
O, it is important to rule out a contribution of
leukocytes to the O production observed. In our
experiments, the highest leukocyte contamination amounted to 9 cells/µL. Control experiments using 10 to 100 leukocytes/µL in
either platelet buffer or in the presence of platelets revealed that in
this concentration the leukocytes, in contrast to platelets, did not
induce O production in response to collagen. They
were, however, fully functioning, since their response to A23187 showed
the expected effects.
SOD in amounts sufficient to scavenge all O
released by platelets did not affect aggregation of platelets in
response to collagen stimulation. This is in accordance with previous
studies.44-46 There are, nevertheless, several
controversial reports on effects of exogenous ROS on platelet function.
Although there is one report of exogenous hydrogen peroxide
(H2O2) impairing aggregation in response to
ADP,47 the high amounts of H2O2
needed for this observation are unlikely to exist in vivo. On the other hand, several reports support an enhancing role of ROS on platelet activity. Handin et al found that X/XO aggregated washed platelets and
caused [14C]-serotonin release.45
O derived from pyrogallol increased aggregation and
adhesion,48 whereas inhibition of the
O-producing enzyme NAD(P)H oxidase by DPI inhibited
aggregation.49 It has also been reported that platelets
exposed to anoxia/reoxygenation generate O and
hydroxyl radicals, being the cause of enhanced spontaneous
aggregation.14 However, all these studies failed to show
an effect of platelet-derived O on thrombus
formation directly. Our studies show a more differential role of
O in the course of aggregation and thrombus growth.
While the initial formation of a thrombus caused by collagen
stimulation was not sensitive to O, the recruitment
of further platelets for thrombus growth clearly was. Moreover,
platelet-derived O formation was involved in the
recruitment of platelets to a preformed thrombus only when a stimulus
that increased platelet O release was the initial
stimulus for thrombus formation. When thrombus formation was induced by
ADP or thrombin, which we show not to increase O
formation in platelets, recruitment was not affected by the
O scavenger SOD. The finding that recruitment
following collagen stimulation could be abolished by apyrase suggests
that recruitment is independent of a direct effect of collagen.
Furthermore, kinetics of collagen-induced aggregation and of
aggregation induced by supernatants of collagen-stimulated platelets
showed different behavior, giving additional evidence for a released
substrate rather than the original stimulus being responsible for
recruitment of further platelets. It favors the concept of an effect of
platelet-derived O on platelet function, which is
independent of O merely scavenging platelet-derived NO. However, a contribution of O scavenging NO and
thereby influencing platelet recruitment cannot be ruled out.
It is well known that release of ADP from platelet-dense granules is a
major factor for platelet recruitment.50 When washed platelets pretreated with exogenously generated O were stimulated with ADP, otherwise subthreshold concentrations of ADP
were sufficient to induce irreversible aggregation, suggesting ADP-dependent effects could be modulated by O. Direct comparison of the quantities of O produced by
platelets, neutrophils, and the O-generating system
X/XO revealed that the amount of O in
supernatants of platelets was less than that of the other systems when appropriate stimuli were used (data not shown). Thus, the importance of this "priming" of ADP-dependent aggregation might be
even more pronounced in the presence of adequately activated neutrophils.
By directly measuring the amount of ADP released from resting or
collagen-activated platelets, we provide a possible explanation for the
effects of collagen-induced O production on platelet
recruitment. Under basal conditions, there was no substantial release
of ADP. Collagen induced a strong release of ADP, but the amounts of
ADP in supernatants of collagen-stimulated platelets were decreased
when O was scavenged by SOD. Moreover, amounts of
ADP were even increased when stimulation was performed in the presence
of exogenous radicals produced by X/XO. As X/XO added to platelets that
were not stimulated with collagen did not induce significant ADP
increase, the increase in collagen-stimulated platelets could not have
been due to an increased release induced by X/XO. While these data
cannot exclude that O alters affinity of ADP to one of the purinergic ADP receptors identified on platelets,35
a more likely mechanism is that O increases the
availability of ADP by inactivating a platelet ectonucleotidase (Ecto-ATPDase). ATPDases hydrolyze ATP to ADP and ADP to AMP, which no
longer induces aggregation. When located on the outer membrane surface,
they can be called ecto-ATPDases. Indeed, in endothelial cells, an
ecto-ATPDase that has been shown to be identical to
CD39,51 is inhibited in an autocrine manner by ROS
released during activation with TNF .25 Platelets have
been shown to also express CD39, which also contains ecto-ATPDase
activity,52 but further characterization of this
nucleotidase is so far lacking. Hence, oxidative inactivation of
platelet CD39 or another platelet ATPDase would fully explain our
findings, although we cannot give direct evidence for this. We
therefore suggest that the influence of platelet-derived
O on platelet recruitment might be due to oxidative
modification of a platelet ATPDase during thrombus formation.
The release of O from platelets upon exposure
to collagen might be an efficient, physiological method of
amplification of thrombus formation in situations where prolonged
thrombus formation takes place. Enhanced platelet activation, as it
may, for example, occur in acute coronary
syndromes,1,53 might be influenced by platelet
O production, which in turn may critically depend on
the initial mode of platelet activation. Also in other pathophysiologic
situations, like hyperglycemia, enhanced collagen-induced platelet
activation seemed to be due to platelet overproduction of
O.54 Furthermore, platelets from
diabetic patients have been shown to release increased amounts of
O, which in turn increased ADP-dependent
Ca++-signaling.55 Therefore, enhanced
platelet O formation might be one mechanism
explaining increased susceptibility of certain risk groups for arterial
thrombosis. Further studies are needed to identify patient
populations with increased platelet O formation.
We propose that platelet recruitment due to alterations in
oxidative state might contribute to collagen-dependent platelet activation. It can be speculated that increased platelet recruitment due to platelet O release could shift the balance
between thrombotic and antithrombotic influences, resulting in enhanced
susceptibility to acute thrombotic events. Then, assessment of the
activity of a platelet NAD(P)H oxidase and of collagen-specific
signaling pathways might help to identify patients at risk and to
develop new therapeutic strategies.
| |
Acknowledgments |
We thank Dr Patrick Pagano for providing
gp91ds-tat and its scrambled analog. We also thank
Professor B. Engelmann, S. Zieseniss, and I. Mueller for
support and expert discussion; and D. Kiesl for technical assistance.
| |
Footnotes |
Submitted September 4, 2001; accepted April 1, 2002.
Supported by a grant from the Friedrich-Baur-Stiftung of the
Ludwigs- Maximilians-University.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Florian Krötz, Institut für
Physiologie, Schillerstr 44, 80336 Munich, Germany; e-mail:
fkroetz{at}lmu.de.
| |
References |
1.
Fuster V, Badimon L, Badimon JJ, Chesebro JH.
The pathogenesis of coronary artery disease and the acute coronary syndromes.
N Engl J Med.
1992;326:310-318[Medline]
[Order article via Infotrieve].
2.
Brott T, Bogousslavsky J.
Treatment of acute ischemic stroke.
N Engl J Med.
2000;343:710-722[Free Full Text].
3.
Ross R.
The pathogenesis of atherosclerosis: a perspective for the 1990s.
Nature.
1993;362:801-809[CrossRef][Medline]
[Order article via Infotrieve].
4.
Siess W.
Molecular mechanisms of platelet activation.
Physiol Rev.
1989;69:58-178[Free Full Text].
5.
Marcus AJ, Silk ST, Safier LB, Ullman HL.
Superoxide production and reducing activity in human platelets.
J Clin Invest.
1977;59:149-158[Medline]
[Order article via Infotrieve].
6.
Pignatelli P, Pulcinelli FM, Lenti L, Gazzaniga PP, Violi F.
Hydrogen peroxide is involved in collagen-induced platelet activation.
Blood.
1998;91:484-490[Abstract/Free Full Text].
7.
Pratico D, Pasin M, Barry OP, et al.
Iron-dependent human platelet activation and hydroxyl radical formation: involvement of protein kinase C.
Circulation.
1999;99:3118-3124[Abstract/Free Full Text].
8.
Caccese D, Pratico D, Ghiselli A, et al.
Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation: role of arachidonic acid metabolism.
Thromb Haemost.
2000;83:485-490[Medline]
[Order article via Infotrieve].
9.
Wolin MS.
Interactions of oxidants with vascular signaling systems.
Arterioscler Thromb Vasc Biol.
2000;20:1430-1442[Abstract/Free Full Text].
10.
Droge W.
Free radicals in the physiological control of cell function.
Physiol Rev.
2002;82:47-95[Abstract/Free Full Text].
11.
Jandrot-Perrus M, Busfield S, Lagrue AH, et al.
Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.
Blood.
2000;96:1798-1807[Abstract/Free Full Text].
12.
Nieswandt B, Brakebusch C, Bergmeier W, et al.
Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen.
EMBO J.
2001;20:2120-2130[CrossRef][Medline]
[Order article via Infotrieve].
13.
Pasquet JM, Quek L, Pasquet S, et al.
Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI.
J Biol Chem.
2000;275:28526-28531[Abstract/Free Full Text].
14.
Leo R, Pratico D, Iuliano L, et al.
Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated.
Circulation.
1997;95:885-891[Abstract/Free Full Text].
15.
Tajima M, Sakagami H.
Tetrahydrobiopterin impairs the action of endothelial nitric oxide via superoxide derived from platelets.
Br J Pharmacol.
2000;131:958-964[CrossRef][Medline]
[Order article via Infotrieve].
16.
Seno T, Inoue N, Gao D, et al.
Involvement of nadh/nadph oxidase in human platelet ros production.
Thromb Res.
2001;103:399-409[CrossRef][Medline]
[Order article via Infotrieve].
17.
Griendling KK, Sorescu D, Ushio-Fukai M.
NAD(P)H oxidase: role in cardiovascular biology and disease.
Circ Res.
2000;86:494-501[Abstract/Free Full Text].
18.
Cai H, Harrison DG.
Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress.
Circ Res.
2000;87:840-844[Abstract/Free Full Text].
19.
Rubanyi GM, Vanhoutte PM.
Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor.
Am J Physiol.
1986;250:H822-H827[Medline]
[Order article via Infotrieve].
20.
Anfossi G, Russo I, Massucco P, Mattiello L, Cavalot F, Trovati M.
N-acetyl-L-cysteine exerts direct anti-aggregating effect on human platelets.
Eur J Clin Invest.
2001;31:452-461[CrossRef][Medline]
[Order article via Infotrieve].
21.
Kanaya S, Ikeda H, Haramaki N, Murohara T, Imaizumi T.
Intraplatelet tetrahydrobiopterin plays an important role in regulating canine coronary arterial thrombosis by modulating intraplatelet nitric oxide and superoxide generation.
Circulation.
2001;104:2478-2484[Abstract/Free Full Text].
22.
Born GV.
Aggregation of blood platelets by adenosine diphosphate and its reversal.
Nature.
1962;194:927-929[Medline]
[Order article via Infotrieve].
23.
Born GV.
Adenosine diphosphate as a mediator of platelet aggregation in vivo: an editorial view.
Circulation.
1985;72:741-746[Free Full Text].
24.
Zimmermann H.
Extracellular metabolism of ATP and other nucleotides.
Naunyn Schmiedebergs Arch Pharmacol.
2000;362:299-309[CrossRef][Medline]
[Order article via Infotrieve].
25.
Robson SC, Kaczmarek E, Siegel JB, et al.
Loss of ATP diphosphohydrolase activity with endothelial cell activation.
J Exp Med.
1997;185:153-163[Abstract/Free Full Text].
26.
Zahler S, Kowalski C, Brosig A, Kupatt C, Becker BF, Gerlach E.
The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method.
J Immunol Methods.
1997;200:173-179[CrossRef][Medline]
[Order article via Infotrieve].
27.
Sohn HY, Gloe T, Keller M, Schoenafinger K, Pohl U.
Sensitive superoxide detection in vascular cells by the new chemiluminescence dye L-012.
J Vasc Res.
1999;36:456-464[CrossRef][Medline]
[Order article via Infotrieve].
28.
Sohn HY, Keller M, Gloe T, Morawietz H, Rueckschloss U, Pohl U.
The small G-protein Rac mediates depolarization induced superoxide formation in human endothelial cells.
J Biol Chem.
2000;275:18745-18750[Abstract/Free Full Text].
29.
Bradford MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem.
1976;72:248-254[CrossRef][Medline]
[Order article via Infotrieve].
30.
Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD.
Nitric oxide released from activated platelets inhibits platelet recruitment.
J Clin Invest.
1997;100:350-356[Medline]
[Order article via Infotrieve].
31.
Valles J, Santos MT, Aznar J, et al.
Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment.
Blood.
1991;78:154-162[Abstract/Free Full Text].
32.
Becker BF, Reinholz N, Ozcelik T, Leipert B, Gerlach E.
Uric acid as radical scavenger and antioxidant in the heart.
Pflugers Arch.
1989;415:127-135[CrossRef][Medline]
[Order article via Infotrieve].
33.
Soulet C, Gendreau S, Missy K, Benard V, Plantavid M, Payrastre B.
Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets.
FEBS Lett.
2001;507:253-258[CrossRef][Medline]
[Order article via Infotrieve].
34.
Finazzi AA, Menichelli A, Persiani M, Biancini G, Del Principe D.
Hydrogen peroxide release from human blood platelets.
Biochim Biophys Acta.
1982;718:21-25[Medline]
[Order article via Infotrieve].
35.
Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP.
Molecular basis for ADP-induced platelet activation, I; evidence for three distinct ADP receptors on human platelets.
J Biol Chem.
1998;273:2024-2029[Abstract/Free Full Text].
36.
Coughlin SR.
Protease-activated receptors in vascular biology.
Thromb Haemost.
2001;86:298-307[Medline]
[Order article via Infotrieve].
37.
Dormann D, Clemetson KJ, Kehrel BE.
The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity.
Blood.
2000;96:2469-2478[Abstract/Free Full Text].
38.
Zielinski T, Wachowicz B, Saluk-Juszczak J, Kaca W.
The generation of superoxide anion in blood platelets in response to different forms of proteus mirabilis lipopolysaccharide: effects of staurosporin, wortmannin, and indomethacin.
Thromb Res.
2001;103:149-155[CrossRef][Medline]
[Order article via Infotrieve].
39.
Geiszt M, Kopp JB, Varnai P, Leto TL.
Identification of renox, an NAD(P)H oxidase in kidney.
Proc Natl Acad Sci U S A.
2000;97:8010-8014[Abstract/Free Full Text].
40.
Bayraktutan U, Blayney L, Shah AM.
Molecular characterization and localization of the NAD(P)H oxidase components gp91-phox and p22-phox in endothelial cells.
Arterioscler Thromb Vasc Biol.
2000;20:1903-1911[Abstract/Free Full Text].
41.
Babior BM.
NAD(P)H oxidase: an update.
Blood.
1999;93:1464-1476[Free Full Text].
42.
Rey FE, Cifuentes ME, Kiarash A, Quinn MT, Pagano PJ.
Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O(2)(-) and systolic blood pressure in mice.
Circ Res.
2001;89:408-414[Abstract/Free Full Text].
43.
Suh YA, Arnold RS, Lassegue B, et al.
Cell transformation by the superoxide-generating oxidase Mox1.
Nature.
1999;401:79-82[CrossRef][Medline]
[Order article via Infotrieve].
44.
Salvemini D, de Nucci G, Vane JR.
Superoxide dismutase cooperates with prostacyclin to inhibit platelet aggregation: a comparative study in washed platelets and platelet rich plasma.
Thromb Haemost.
1991;65:421-424[Medline]
[Order article via Infotrieve].
45.
Handin RI, Karabin R, Boxer GJ.
Enhancement of platelet function by superoxide anion.
J Clin Invest.
1977;59:959-965[Medline]
[Order article via Infotrieve].
46.
Radomski MW, Palmer RM, Moncada S.
An L-arginine/nitric oxide pathway present in human platelets regulates aggregation.
Proc Natl Acad Sci U S A.
1990;87:5193-5197[Abstract/Free Full Text].
47.
Ambrosio G, Golino P, Pascucci I, et al.
Modulation of platelet function by reactive oxygen metabolites.
Am J Physiol.
1994;267:H308-H318[Medline]
[Order article via Infotrieve].
48.
Salvemini D, de Nucci G, Sneddon JM, Vane JR.
Superoxide anions enhance platelet adhesion and aggregation.
Br J Pharmacol.
1989;97:1145-1150[Medline]
[Order article via Infotrieve].
49.
Salvemini D, Radziszewski W, Mollace V, Moore A, Willoughby D, Vane J.
Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation.
Eur J Pharmacol.
1991;199:15-18[CrossRef][Medline]
[Order article via Infotrieve].
50.
Born GV.
Adenosine diphosphate as a mediator of platelet aggregation in vivo.
Adv Exp Med Biol.
1985;192:399-409[Medline]
[Order article via Infotrieve].
51.
Marcus AJ, Broekman MJ, Drosopoulos JH, et al.
The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.
J Clin Invest.
1997;99:1351-1360[Medline]
[Order article via Infotrieve].
52.
Koziak K, Sevigny J, Robson SC, Siegel JB, Kaczmarek E.
Analysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothelial cells, platelets and leukocytes.
Thromb Haemost.
1999;82:1538-1544[Medline]
[Order article via Infotrieve].
53.
Neumann FJ, Gawaz M, Ott I, May A, Mossmer G, Schomig A.
Prospective evaluation of hemostatic predictors of subacute stent thrombosis after coronary Palmaz-Schatz stenting.
J Am Coll Cardiol.
1996;27:15-21[Abstract].
54.
Yamagishi SI, Edelstein D, Du XL, Brownlee M.
Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction.
Diabetes.
2001;50:1491-1494[Abstract/Free Full Text].
55.
Schaeffer G, Wascher TC, Kostner GM, Graier WF.
Alterations in platelet Ca2+ signalling in diabetic patients is due to increased formation of superoxide anions and reduced nitric oxide production.
Diabetologia.
1999;42:167-176[CrossRef][Medline]
[Order article via Infotrieve].
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|
|
R. Carnevale, P. Pignatelli, L. Lenti, B. Buchetti, V. Sanguigni, S. Di Santo, and F. Violi
LDL are oxidatively modified by platelets via GP91phox and accumulate in human monocytes
FASEB J,
March 1, 2007;
21(3):
927 - 934.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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|
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K. Bedard and K.-H. Krause
The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology
Physiol Rev,
January 1, 2007;
87(1):
245 - 313.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Krotz, M. Keller, S. Derflinger, H. Schmid, T. Gloe, F. Bassermann, J. Duyster, C. D. Cohen, C. Schuhmann, V. Klauss, et al.
Mycophenolate Acid Inhibits Endothelial NAD(P)H Oxidase Activity and Superoxide Formation by a Rac1-Dependent Mechanism
Hypertension,
January 1, 2007;
49(1):
201 - 208.
[Abstract]
[Full Text]
[PDF]
|
|
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|
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|
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R. N. Carter, G. Tolhurst, G. Walmsley, M. Vizuete-Forster, N. Miller, and M. P. Mahaut-Smith
Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte
J. Physiol.,
October 1, 2006;
576(1):
151 - 162.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Pignatelli, S. Di Santo, B. Buchetti, V. Sanguigni, A. Brunelli, and F. Violi
Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment
FASEB J,
June 1, 2006;
20(8):
1082 - 1089.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Marcondes, M. H. M. Cardoso, R. P. Morganti, S. M. Thomazzi, S. Lilla, F. Murad, G. De Nucci, and E. Antunes
Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: A role for {alpha}-actinin nitration
PNAS,
February 28, 2006;
103(9):
3434 - 3439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chakrabarti, O. Vitseva, D. Iyu, S. Varghese, and J. E. Freedman
The Effect of Dipyridamole on Vascular Cell-Derived Reactive Oxygen Species
J. Pharmacol. Exp. Ther.,
November 1, 2005;
315(2):
494 - 500.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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A. J. Begonja, S. Gambaryan, J. Geiger, B. Aktas, M. Pozgajova, B. Nieswandt, and U. Walter
Platelet NAD(P)H-oxidase-generated ROS production regulates {alpha}IIb{beta}3-integrin activation independent of the NO/cGMP pathway
Blood,
October 15, 2005;
106(8):
2757 - 2760.
[Abstract]
[Full Text]
[PDF]
|
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F. Krotz, B. Engelbrecht, M. A. Buerkle, F. Bassermann, H. Bridell, T. Gloe, J. Duyster, U. Pohl, and H.-Y. Sohn
The Tyrosine Phosphatase, SHP-1, Is a Negative Regulator of Endothelial Superoxide Formation
J. Am. Coll. Cardiol.,
May 17, 2005;
45(10):
1700 - 1706.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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O. Vitseva, D. A. Flockhart, Y. Jin, S. Varghese, and J. E. Freedman
The Effects of Tamoxifen and Its Metabolites on Platelet Function and Release of Reactive Oxygen Intermediates
J. Pharmacol. Exp. Ther.,
March 1, 2005;
312(3):
1144 - 1150.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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F. Krotz, H.-Y. Sohn, and U. Pohl
Reactive Oxygen Species: Players in the Platelet Game
Arterioscler Thromb Vasc Biol,
November 1, 2004;
24(11):
1988 - 1996.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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M. A. Buerkle, S. Lehrer, H.-Y. Sohn, P. Conzen, U. Pohl, and F. Krotz
Selective Inhibition of Cyclooxygenase-2 Enhances Platelet Adhesion in Hamster Arterioles In Vivo
Circulation,
October 5, 2004;
110(14):
2053 - 2059.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Stocker and J. F. Keaney Jr.
Role of Oxidative Modifications in Atherosclerosis
Physiol Rev,
October 1, 2004;
84(4):
1381 - 1478.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Pignatelli, V. Sanguigni, L. Lenti, D. Ferro, A. Finocchi, P. Rossi, and F. Violi
gp91phox-Dependent Expression of Platelet CD40 Ligand
Circulation,
September 7, 2004;
110(10):
1326 - 1329.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. W. Essex, M. Li, R. D. Feinman, and A. Miller
Platelet surface glutathione reductase-like activity
Blood,
September 1, 2004;
104(5):
1383 - 1385.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Krotz, T. Riexinger, M. A. Buerkle, K. Nithipatikom, T. Gloe, H.-Y. Sohn, W. B. Campbell, and U. Pohl
Membrane Potential-Dependent Inhibition of Platelet Adhesion to Endothelial Cells by Epoxyeicosatrienoic Acids
Arterioscler Thromb Vasc Biol,
March 1, 2004;
24(3):
595 - 600.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
N. Tanaka, T. Sato, H. Fujita, and I. Morita
Constitutive Expression and Involvement of Cyclooxygenase-2 in Human Megakaryocytopoiesis
Arterioscler Thromb Vasc Biol,
March 1, 2004;
24(3):
607 - 612.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
H.-Y. Sohn, F. Krotz, T. Gloe, M. Keller, K. Theisen, V. Klauss, and U. Pohl
Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine
Cardiovasc Res,
June 1, 2003;
58(3):
638 - 646.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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F. Krotz, H. Y. Sohn, M. Keller, T. Gloe, S. S. Bolz, B. F. Becker, and U. Pohl
Depolarization of Endothelial Cells Enhances Platelet Aggregation Through Oxidative Inactivation of Endothelial NTPDase
Arterioscler Thromb Vasc Biol,
December 1, 2002;
22(12):
2003 - 2009.
[Abstract]
[Full Text]
[PDF]
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|
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Top
Abstract
Introduction