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Oxygen Free Radicals and Platelet Activation

Article  in  Free Radical Biology and Medicine · February 1997

DOI: 10.1016/S0891-5849(96)00488-1

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 Free Radical Biology & Medicine, Vol. 22, No. 6, pp. 999–1006, 1997
Copyright q 1997 Elsevier Science Inc.
Printed in the USA. All rights reserved
0891-5849/97 $17.00 / .00

PII S0891-5849(96)00488-1

Original Contribution
OXYGEN FREE RADICALS AND PLATELET ACTIVATION

Luigi Iuliano,* Angela Rita Colavita,* Roberto Leo,*

Domenico Praticò,† and Francesco Violi*
*Institute of Clinical Medicine I, University La Sapienza, Rome, Italy; and †Center for Experimental Therapeutics, University of
Pennsylvania School of Medicine, Philadelphia, PA, USA

(Received 29 May 1996; Revised 4 September 1996; Accepted 4 September 1996)

Abstract—This article reviews our current understanding of the role of oxygen free radicals in platelet activation.
Several studies have indicated that platelets, in analogy to other circulating blood cells, are able to produce oxygen
free radicals, which are likely to play an important role in the mechanism of platelet activation and aggregation.
Platelet activation has been obtained with very low, physiologically relevant concentrations of radicals generated
chemically, by leukocytes, and by hemoglobin derived from membrane leakage of erythrocytes. Knowledge of the
role of reactive species in platelet physiology is relevant because platelets are brought into close contact with other
cells capable of producing free radicals, such as neutrophils, macrophages, and endothelial cells, during the formation
of thrombus. The physiopatological importance of these findings is high because it is now emerging that free radicals
may have a role in the mechanism of atherosclerosis and its thrombotic complications, where the causative role of
platelets is well documented. This background suggests therapeutic interventions with antioxidants as antiplatelet
agents to improve the pharmacological effect of classical antiplatelet drug such as aspirin. Copyright q 1997
Elsevier Science Inc.
Keywords—Free Radicals, Platelets, Atherosclerosis, Antioxidants

INTRODUCTION lationship between OFR and platelet function. Due to

Oxygen free radicals (OFR), which include the super-
oxide anion (O 2 0i) and the hydroxyl radical (HOi), and
other oxygen species, like H2O2 and the singlet oxygen
(1 DgO 2), are highly reactive substances that react with
lipids, proteins and DNA, provoking irreversible
changes of their biomolecular structure. There is a
growing body of evidence that OFR play an essential
role in the control of cell functions. They are interme-
diate metabolites in several enzymic reactions, they are
involved in the post-translational protein turnover, and
play a role in the control of signal transduction. Many
components of the vascular system, such as leukocytes,
monocytes and endothelial cells are able to release OFR
upon appropriate stimulation. It is less well known that
also platelets can produce OFR; the physiological role
of this phenomenon has long remained obscure. In the
last 10 years several studies have investigated the re-
Address correspondence to: Prof. Francesco Violi, Institute of
Clinical Medicine I, University La Sapienza, 00185 Rome, Italy.
the growing body of evidence supporting the patho-
physiological role of platelets in cardiovascular dis-
ease, knowledge of this interaction may be useful in the
further understanding of the mechanism(s) accounting
for vascular complications.
This review will focus on the relationship between
OFR and platelet function and on the possible interplay
of OFR, platelets and cardiovascular disease.
Production of OFR by platelets
Among the initial studies on platelets and OFR is
that of Marcus et al.1 that demonstrated O 2 0i produc-
tion by platelets using the ferricytochrome c and nitro-
blue tetrazolium assays. In this study, superoxide gen-
eration was not blocked by aspirin, indicating produc-
tion by a membrane-associated system different from
the cyclooxygenase enzyme. However, baseline super-
oxide production was measurable in resting platelets,
but these levels did not change following platelet ac-

999

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1000 L. IULIANO et al.
tivation and aggregation. This is contrast to leukocytes
where activation by strong agonists results in a marked
increase in superoxide production. The superoxide rad-
icals thus produced are incorporated into the phago-
cytic vacuole, which is important for bacterial killing.
Later work has shown that platelet activation is asso-
ciated with an increase in superoxide production,2 – 6
even if not in a burst-like manner, with a possible role
for killing parasites.2 Further studies reported that dur-
ing aggregation other oxygen reactive species are re-
leased by platelets. Hydrogen peroxide, delivered dur-
ing platelet activation, was detected by fluorescent
methods, using scopoletin3 or 2,7-dichlorofluorescein
as a probe6 and by the use of catalase, which is able to
inhibit several platelet responses to agonists.7 It is also
evident that some of the hydrogen peroxide produced
by platelets is converted into the very reactive hydroxyl
radical through Fenton-like chemistry, as platelet ag-
gregation can be inhibited by HOi scavengers.8 – 10 In
addition, products resulting from the reaction of HOi,
formed during platelet activation, and the scavengers,
which inhibit the platelet response, were demonstrated.
Platelet-generated salicylate-HOi adducts, 2,3 and 2,5
dihydroxybenzoic acids, were isolated by high-perfor-
mance liquid chromatography,10 and the DMPO-HOi
adduct was detected by ESR.9
Carbon centered radicals as intermediate species of
eicosanoids metabolism have been detected, for ex-
ample by ESR, but it is not known whether their role
is confined to the mechanism of the lipooxygenase and
PGH synthase enzymes, or if they escape from the spe-
cific enzymatic cascade system and exist as free
species.
Finally, among free radical species is to be men-
tioned the bioregulator nitric oxide (NOi),11 which ex-
erts well characterized effects on platelets. A nitric ox-
ide synthase has been purified to homogeneity from
human platelet cytosolic fractions,12 However, this spe-
cies is outside the scope of this review as the host of
functions and effects of NOi have been extensively de-
scribed elsewhere.13,14
Mechanism of OFR release by platelets
OFR production by platelets can be ascribed to sev-
eral pathways, including membrane oxidases, nitric ox-
ide synthase, and the arachidonic acid pathway.
An oxidase similar to that working in phagocytic
cells, which catalyzes the production of superoxide us-
ing NADPH as electron donor, was also proposed as a
putative source of superoxide in platelets. However, to
the best of our knowledge, the enzyme has not been
purified from platelets and indirect evidence of its ex-
istence is based on the use of the non-specific inhibitor
/ 2b27 2406 Mp 1000 Tuesday Jan 07 12:02 PM EL–FRB (vol.22#6 ’97) 2406
diphenyliodonium.15 Studies by Salvemini et al.16
showed that diphenyliodonium inhibited the aggrega-
tion of platelets stimulated with thrombin, ADP or col-
lagen. Evidence for the existence of a distinct oxidase
in the platelet membrane, which produces hydrogen
peroxide, was provided by Finazzi-Agrò et al.5 They
reported that this enzyme, which requires NADH as
cofactor, could be activated after challenging platelets
with particulate membrane-perturbing agents. It is even
possible that some hydrogen peroxide is derived from
dismutation of the platelet-derived superoxide, and
support for this proposal exists in the literature. Our
group5 demonstrated that the addition of SOD to ar-
achidonic acid- or collagen-primed platelets induces
aggregation, thromboxane A2 production, and degran-
ulation, through dismutation of the platelet-derived
O 2 0i to H2O2, which is a potent activator of primed
platelets.
A potential additional source of O 2 0i/H2 O 2 could be
the nitric oxide synthase enzyme, as it was demon-
strated that the combination of L-arginine deficiency
and NADPH sufficiency predisposes nitric oxide syn-
thase to produce superoxide or hydrogen peroxide.17
Previous studies provided evidence that free radicals
are formed during the eicosanoid metabolism at the
level of the PGH synthase enzyme. PGH synthase ex-
hibits both cyclooxygenase and hydroperoxidase activ-
ities, which are associated with the protein and occur
at two reciprocal sites. The cyclooxygenase (bis-oxy-
genase) activity catalyzes PGG2 formation and the per-
oxidase activity reduces the 15-hydroperoxy group of
PGG2 to PGH2 utilizing an array of electron donors.
These reducing substrates are cooxidized during the
peroxidase reaction with formation of radical interme-
diates and leading to the possibility of initiation of
chain reactions due to radicals escaping from the active
site into the free solution.18 – 21 An additional source of
free radicals during the conversion of arachidonic acid
is the lipooxygenase enzyme which catalyzes the for-
mation of arachidonyl hydroperoxides through the re-
dox cycling of a non-heme iron atom in the active site.
Occasional release of peroxyl radicals from the enzyme
active site was documented by ESR.22
Role of OFR on platelet function
The effect of OFR in platelet function has been ex-
amined with chemical- and cellular-generated fluxes of
free radicals.
Handin et al.23 incubated platelets with an O 2 0i gen-
erating system, i.e. xanthine and xanthine oxidase, and
demonstrated that O 2 0i causes platelet aggregation and
induces serotonin release. While SOD prevented su-
peroxide-induced platelet activation, catalase and man-
 Free radicals and platelets
nitol did not, indicating that at least in this system
O 2 0i was the oxygen species involved in platelet acti-
vation. Studies aimed at investigating the role of H2O2
in platelet physiology provided conflicting data, as it
was reported that H2O2 either inhibits or enhance7,24 – 27
platelet aggregation. This paradox can be resolved by
the fact that inhibitory effects were only observed using
high concentrations of H2O2. Ohyashiki et al.27 reported
inhibition of ADP-induced platelet aggregation with 2–
10 mM H2O2. This concentration, which can seriously
damage the structure of platelet membrane and the fine
tuned exposition of receptors, is far from what one
would expect in physiological conditions. For example,
H2O2 released by activated leukocytes may reach local
levels of 10–12 mM28 Accumulating evidence indicates
that H2O2 potently favours activation of platelets
primed with arachidonic acid or collagen, with Kapp in
the submicromolar range.29,30 Consistent with this, our
group31 demonstrated the activation of platelets in
whole blood by polymorphonuclear leukocyte-derived
hydrogen peroxide. Recently, we9 reported that acti-
vation of platelets exposed to H2O2 is mediated by hy-
droxyl radicals (HOi) formed in an extracellular Fen-
ton-like reaction, and detected directly by ESR
measurements of the spin-trapped HOi adduct. The role
of HOi was confirmed in experiments with radicals gen-
erated exogenously by EDTA-bound iron, and it was
proposed that OFR act as ‘second messengers’ during
the platelet activation process. The biological impor-
tance of these studies was corroborated by experiments
in which the relationship between erythrocytes and
platelet function was investigated. Thus, it was shown
that nanomolar levels of hemoglobin released from
damaged red blood cells can induce platelet activation,
with a molecular mechanism involving a Hb-derived
free radical species detectable by ESR, but still
unknown.32 The general question of which pathway
is involved in platelet activation triggered by free
radicals is unresolved. However, it seems likely that
PLA2 is a crucial component since PLA2 inhibitors
prevent activation of platelets by OFR.9,29 In accor-
dance with this proposal is also the susceptibility of
platelet PLA2 to lipid peroxides generated in the
membrane by Fe///tert-butyl hydroperoxide33 or
during LDL oxidation.34
Taken together, these data indicate that platelets are
activated upon exposure to OFR-generating systems,
including those derived from polymorphonuclear leu-
kocytes and red blood cells. Interestingly, it has been
demonstrated that platelets are in close contact with
leukocytes and erythrocytes in clinical settings associ-
ated with vascular disease, such as unstable angina or
myocardial infarction.37 The presence of OFR in the
surrounding medium is therefore an important stimulus
/ 2b27 2406 Mp 1001 Tuesday Jan 07 12:02 PM EL–FRB (vol.22#6 ’97) 2406
1001
for platelet aggregation and may contribute to thrombus
formation (Fig. 1).
Interaction between OFR and platelet signal
activation pathways
Several pathways can be taken into account in ex-
plaining the mechanism by which OFR induce platelet
activation (Fig. 2). Free arachidonate is the precursor
of the main platelet activator thromboxane A2. The
availability of free arachidonate is regulated by two
mechanisms: i) the release from phospholipids, through
the action of phospholipases; ii) the reincorporation
into phospholipids by the enzyme arachidonyl-CoA
synthetase. This reacylating enzyme was reported by
Hornberger et al.36 to be inhibited by H2O2, with a con-
sequent rise in free arachidonate that is converted into
thromboxane A2. Many authors reported that OFR ac-
tivate phospholipase A2 through stimulation of several
signal pathways.9,29,33,37 We demonstrated that incuba-
tion of platelets with a potent and selective inhibitor of
the Na//H/ antiporter inhibited platelet aggregation in-
duced by OFR.9 Phospholipase A2 is also activated by
Ca// mobilization but until now there is no clear evi-
dence that OFR stimulate Ca// mobilization in plate-
lets. While Mirabelli et al.38 showed an increase in cy-
tosolic Ca// concentration upon platelet exposure to
oxidative stress, others9 failed to demonstrate that OFR
induce changes in intraplatelet Ca//. Positive results
must be interpreted with caution when a high flux of
OFR is used, since artifactual signals could be gener-
ated. In fact, H2O2 concentrations higher than 100
mM may induce membrane leakage of intracellular con-
tent with concomitant delivery of FURA-2 in the ex-
tracellular space, giving rise to a signal by extracellular
calcium (unpublished results from our laboratory).
The stimulation of phospholipase A2 may also occur
indirectly through the activation of the protein tyrosine
kinase system by OFR. Phosphorylation of protein ty-
rosine kinases (PTK) has been shown to play a critical
role in the induction of cellular responses to extracel-
lular stimuli in a wide variety of cell types.39 In plate-
lets, many diverse agonists induce multiple waves of
tyrosine phosphorylation, suggesting potential roles for
such protein modifications throughout the platelet ac-
tivation process. PTK inhibitors, such as tyrphostins,
genistein and erbstatin have been shown to block sev-
eral platelet events including IIb/IIIa activation and ag-
gregation.40 Inhibition of protein tyrosine phosphatases,
enzymes which inhibit the activity of PTK, causes a
dramatic increase in tyrosine phosphorylation and a
concurrent platelet aggregation. Several recent reports
clearly indicate a role for OFR in regulating PTK ac-
tivity. It has been demonstrated that H2O2 inhibits ty-
1002
Fig. 1. Free radicals generated by leukocytes and by hemoglobin released from red blood cell lysis contribute to platelet aggregation
and thrombus formation at the level of vascular wall.
rosine phosphatases leading to the activation of PTK.41
UVB irradiation produces free radicals and increases
prostaglandin biosynthesis through a signal transduc-
tion pathway leading to activation of phospholipase
A2.37 In studies defining the regulation of cytosolic
phospholipase A2 activation, mitogen activated protein
(MAP) kinase was reported to play a role. Phospholi-
pase A2 has been shown to increase its activity 2–6
fold after phosphorylation operated by MAP kinase.42,43
MAP kinase is phosphorylated and activated by a PTK-
dependent pathway.44
In brief, oxidative stress activates tyrosine kinases
associated with the plasma membrane, this induces ty-
rosine phosphorylation which in turn phosphorylates
MAP kinase. This kinase subsequently phosphorylates
and activates cytosolic PLA2 and produces an increase
in substrate (arachidonic acid) for the enzyme prosta-
glandin endoperoxide synthase (Fig. 2).
Chao et al.45 have demonstrated that PTK phos-
phorylation can also be regulated by NADPH-depen-
dent processes such as the generation of OFR. In fact,
PTK phosphorylation is sensitive to both exogenous
and endogenous oxiding agents and antioxidants.46 The
phosphotyrosine accumulation may result from either
oxidant-induced activation of tyrosine kinases, from in-
hibition of tyrosine phosphatases, or from a combina-
/ 2b27 2406 Mp 1002 Tuesday Jan 07 12:02 PM EL–FRB (vol.22#6 ’97) 2406
L. IULIANO et al.
tion of these effects. It should be emphasized that the
signal transduction mechanisms involved are probably
much more complicated than outlined above, in anal-
ogy with the situation found in other cell types.
OFR may also stimulate arachidonate metabolism
acting at different levels; for instance, by increasing the
activity of the cyclooxygenase enzyme. It has been re-
ported that H2O2 and other hydroperoxides are able to
stimulate this enzyme, so inducing an increased pro-
duction of thromboxane A2.47 Consistent with this, as-
pirin inhibits aggregation of primed platelets stimulated
by H2O2.48 But, OFR can also activate platelets at a
level independent from the eicosanoids pathway, as it
resulted from experiments in which several antioxi-
dants were shown to reduce cyclooxygenase-indepen-
dent platelet aggregation, which was observed in
aspirin treated platelets challenged with high con-
centrations of agonists. Thus, vitamin E, the HOi scav-
enger salicylate.49 and the antioxidant dipyridamole,
which scavenges O 2 0i, HOi and peroxyl radicals,50 – 52
significantly inhibited aggregation of aspirin-treated
platelets.53 – 55
Finally, OFR might affect platelet function by re-
acting with nitric oxide (NO*), a vasodilating substance
released by endothelium in response to stimulation with
a variety of endogenous substances. NO* is also pro-
 Free radicals and platelets 1003

Fig. 2. OFR and platelet signal activation pathways. (/) denotes activation and (0) denotes inhibition. PTK: protein tyrosine
kinase; MAP: mitogen activated protein; cPLA2: cytosolic phospholipase A2; P: phosphate residue; PTP: protein tyrosine phos-
phatase; Na//H/: sodium-hydrogen antiporter; AA: arachidonic acid.

duced by platelets in which it inhibits aggregation by
increasing cGMP.11 – 14 The biological activity of NO*
is limited by the concurrent presence of O 2 0i, which
reacts rapidly with NO* in aqueous solution (k Å
3.7 1 107 M01 s01)56 to form the peroxynitrite anion
(OONO-), which in turn decomposes to HOi at neu-
tral pH.57
Taken together, these findings suggest that OFR be-
have as stimulator of platelet aggregation by acting at
different levels of the signal activation pathways, i.e.
by inhibiting NOi and by stimulating arachidonic acid
metabolism, Ca// influx and tyrosine kinases (Fig. 2).
Platelet function and antioxidants
The role of OFR in the mechanism of platelet acti-
vation has been assessed indirectly by using antioxi-
dants, in particular by examining the role of vitamin E
as modulator of platelet function.
Dietary manipulation in animals demonstrated that
vitamin E deficiency is associated with increased plate-
let aggregation and synthesis of arachidonic acid me-
tabolites thromboxane B2 and 12-hydroxyeicosatetrae-
noic acid.58 – 60 In addition, rats, fed a vitamin E-free
diet, were reported to exhibit increased activity of phos-
pholipase A2.61
Vitamin E supplementation in man has produced
equivocal results regarding platelet effects. Some au-
thors failed to affect substantially platelet aggregation
and arachidonic acid metabolism after vitamin supple-
mentation to healthy subjects, using high doses (400–
1500 international units/day)62 or low doses (200 in-
ternational units/day)63 vitamin E.
No clear cut evidence has emerged from in vitro
studies about the inhibitory effect of vitamin E on plate-
let function. Some reports of platelet responses may
have limited in vivo relevance due to the use of rela-
tively high concentrations (about 1 mM) of vitamin

/ 2b27 2406 Mp 1003 Tuesday Jan 07 12:02 PM EL–FRB (vol.22#6 ’97) 2406
1004 L. IULIANO et al.
E.64,65 Blood vitamin E concentration ranges from 10
to 60 mM,66 and can be increased by 2.5–3 times after
diet supplementation with 900 IU of alpha-(RRR)-to-
copherol for one week.67 We demonstrated that 50–100
mM vitamin E significantly reduced cyclooxygenase-
independent platelet aggregation.53 Some of the re-
ported effects derived from the use of high concentra-
tions of vitamin E might be attributed to vitamin E
quinone, that inhibits platelet function more effectively
than vitamin E by a magnitude of 5–10-fold.68 Inhi-
bition of platelet aggregation by ascorbic acid,69 and by
synthetic antioxidants such as butylated hydroxyani-
sole65 and butylated hydroxytoluene,70 was also re-
ported. Therefore, further studies are necessary to as-
sess how antioxidants affect platelet function in vivo.
Dose-response studies as well as analysis of the rela-
tionship between plasma and platelet concentration of
antioxidants and platelet function are still lacking.
These studies should define if and to what extent an-
tioxidant supplementation affects platelet function, and
the optimal dosage necessary to reach such an effect.
Finally, it is interesting to note that the well known
drug dipyridamole, used in coronary artery disease for
its antiplatelet and vasodilating activity,71 was recently
shown to possess antioxidant activity, including super-
oxide and hydroxyl radical scavenging, and chain
breaking activity.50 – 52 Considering that the antiplatelet
activity of dipyridamole has never been clearly eluci-
dated, and in the light of the activating effect of OFR
upon platelets, it is conceivable that dipyridamole ex-
erts the antiplatelet activity essentially through an an-
tioxidant mechanism.
Oxygen free radicals and platelet function: are there
any clinical implications?
Few clinical studies have carefully investigated the
potential relationship between OFR, platelet function
and cardiovascular disease in vivo. Some indirect evi-
dence suggesting that OFR might be involved in the
activation of platelets comes from studies performed in
diabetic patients. Increased platelet activation in vivo
was demonstrated by Davı̀ et al.72 who showed that
patients with diabetes mellitus have high urinary ex-
cretion of 11-dehydro-tromboxane B2, the stable me-
tabolite of tromboxane A2 which is a potent stimulator
of platelet aggregation. The mechanism accounting for
platelet activation in diabetes mellitus is yet unclear but
it is conceivable that this could be favoured by an ox-
idant-antioxidant imbalance, as diabetic patients have
a reduced content of vitamin E in the platelet
membrane.73 Taking into account the role of antioxi-
dant on platelet function, it cannot be ruled out that
platelet hyperaggregation may also be related to in-
/ 2b27 2406 Mp 1004 Tuesday Jan 07 12:02 PM EL–FRB (vol.22#6 ’97) 2406
creased production of OFR in patients with diabetes
mellitus. Consistently, Colette et al.74 demonstrated that
dietary vitamin E supplementation reduces platelet hy-
peractivity and aggregation in insulin-dependent dia-
betic patients, and has a beneficial effect in the treat-
ment of thromboembolic complications, particularly in
conjunction with an inhibitor of platelet aggregation.
Further evidence on the role of OFR-mediated plate-
let activation in cardiovascular disease comes from the
experimental model of cyclic flow variation in vessels,
which is mediated by platelet aggregation. In a canine
model with cyclic flow variation in stenosed and en-
dothelium- injured coronary arteries, Yao et al.75 ex-
amined the role of OFR on platelet aggregation in vivo.
In this model treatment with either antioxidants, recom-
binant human manganese- and copper-zinc-superoxide
dismutase, or catalase eliminated platelet aggregation-
associated coronary cyclic flow variation in 63%, 62%,
and 64% of the animals, respectively. Ikieda et al.,76
reported comparable results, and in addition showed
that the infusion of xanthine-xanthine oxidase or hy-
drogen peroxide significantly increased the frequency
of cyclic flow variations.
These results provide evidence that OFR play an
important role in inducing platelet aggregation in vivo,
and indicate that this effect may be of relevance in fa-
voring thrombotic events. Thus, the use of antioxidants
could be useful to reduce platelet aggregation and may
represent a new therapeutic approach to prevent throm-
bosis in vivo.
CONCLUSIONS
The demonstration of a close relationship between
OFR and platelet function and the possibility to regu-
late the interaction by antioxidants, should be consid-
ered as an important background to explore further the
role of OFR in clinical settings in which platelet hy-
perfunction has a role in the progression of vascular
disease. The study of that interaction between OFR and
platelets will be useful in understanding the atheroscle-
rotic process, in which there is an imbalance between
oxidative processes and antioxidant defence with a shift
towards an increase of oxidative process. Furthermore,
there is much evidence indicating that the use of anti-
oxidant retards the progression of atherosclerosis and
ameliorates endothelial dysfunction. Further studies are
necessary to establish whether this is solely due to a
direct effect of the antioxidants on endothelium or also
includes platelet functional changes.
Acknowledgements — This work was supported by the Italian Na-
tional Research Council (Consiglio Nazionale delle Ricerche grant #
06152, 95.02298.04, to L.I.).
 Free radicals and platelets
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ABBREVIATIONS
OFR—oxygen free radicals
DMPO—5,5-dimethyL-1-pyrroline-N-oxide
ESR—electron spin resonance
PLA2 —phospholipase A2
PTK—protein tyrosine kinase
MAP—mitogen activated protein