Thrombosis Research 110 (2003) 281 – 286

Review Article
Aspirin insensitive eicosanoid biosynthesis in cardiovascular disease
Paola Patrignani *
Department of Medicine and Center of Excellence on Aging, Ce.S.I., ‘‘G. d’Annunzio’’ University, School of Medicine, Chieti, Italy

Abstract

Inhibition of platelet cyclooxygenase (COX)-1 is involved in aspirin cardioprotection observed in clinical trials, but, in some patients,
aspirin is unable to protect from thrombotic complications. An incomplete suppression of platelet thromboxane (TX) A2 biosynthesis has
been assumed to participate in the phenomenon of aspirin resistance, as a consequence of the following possible mechanisms: (i) COX-2
expression in newly formed platelets; (ii) pharmacodynamic interactions between aspirin and coadministered nonsteroidal antiinflammatory
drugs (e.g. ibuprofen); (iii) expression of variant isoforms of COX-1 with reduced sensitivity to irreversible inactivation at Ser529.
Furthermore, aspirin failure may be due to enhanced formation of vasoactive and/or proaggregatory eicosanoids despite an almost complete
suppression of platelet TXA2 biosynthesis by aspirin. Thus, in a subset of patients with unstable angina treated with low-dose aspirin, to
almost completely block platelet COX-1 activity, enhanced TXA2 biosynthesis in vivo has been demonstrated, presumably through an
increased generation of COX-2-dependent PGH2 in plaque monocytes/macrophages or activated vascular cells. The concomitant increased
formation of 8-iso-PGF2a, one of the most abundant F2-isoprostanes in humans, generated by free-radical catalyzed arachidonate
peroxidation, may be involved in aspirin resistance because of its capacity to induce platelet and vascular smooth muscle cell activation
through the interaction with thromboxane receptors (TPs). Finally, enhanced production of vasoactive cysteinyl leukotrienes (cys-LTs) occurs
in unstable angina despite conventional antithrombotic and antianginal treatment. The use of selective pharmacological tools (i.e. highly
selective COX-2 inhibitors, TP antagonists, cys-LT inhibitors and antagonists) will allow to ascertain the contribution of aspirin insensitive
eicosanoid biosynthesis in aspirin cardioprotective failure.
D 2003 Elsevier Ltd. All rights reserved.

Keywords: Aspirin; Thromboxane; F2-isoprostanes; Leukotrienes

The eicosanoids are a family of biologically active
compounds that participate in cell-cell communication, as
a consequence of the activation of membrane associated
receptors that belong to the G-protein-coupled rhodopsin-
type family [1,2]. They are formed from polyunsaturated
fatty acids [principally, arachidonic acid (AA)]. AA is a 20-
carbon essential fatty acid that contains four double bonds
(5,8,11,14-eicosatetraenoic acid), esterified to the phospho-
lipids of cell membranes. Once released through the activity
of phospholipases (PLs), AA is metabolized enzymatically
Abbreviations: AA, arachidonic acid; PG, prostaglandin; PGI2,
prostacyclin; TX, thromboxane; LT, leukotriene; HPETEs, hydroperoxyei-
cosatetraenoic acids; EETs, epoxyeicosatrienoic acids; HETEs, hydroxyei-
cosatetraenoic acids; cys-LTs, cysteinyl leukotrienes; COX, cyclooxygenase;
TXAS, TXA synthase; NSAID, nonsteroidal antiinflammatory drug; IL,
interleukin; TPs, thromboxane receptors; EC, endothelial cell; SMC, smooth
muscle cell; LDL, low density lipoprotein.
* Dipartimento di Medicina e Scienze dell’Invecchiamento, Sezione di
Farmacologia, Università di Chieti ‘‘G. D’Annunzio’’, Via dei Vestini, 31,
66013 Chieti, Italy. Tel.: +39-871-355-6775; fax: +39-871-355-6718.
E-mail address: ppatrignani@unich.it (P. Patrignani).
into three series of biologically active compounds collec-
tively termed eicosanoids: (1) the prostanoids, i.e. prosta-
glandin (PG) E2, PGF2a, PGD2, prostacyclin (PGI2) and
thromboxane (TX) A2; (2) the leukotrienes (LTs), i.e. LTB4
and cysteinyl LTs (cys-LTs, i.e. LTC4, LTD4 and LTE4) and
the hydroperoxyeicosatetraenoic acids (HPETEs); and (3)
the epoxyeicosatrienoic acids (EETs) and hydroxyeicosate-
traenoic acids (HETEs). Recently, a nonenzymatic pathway
of AA conversion has been discovered, given rise to a novel
series of biologically active compounds that are isomers of
eicosanoids termed isoeicosanoids [3,4].
Several compounds produced by enzymatic and nonen-
zymatic pathways of AA oxidation may influence cardio-
vascular homeostasis and their formation has been found to
be altered in clinical conditions characterized by platelet
activation and vascular dysfunction [5– 12]. Thus, increases
in TXB2, a trigger of platelet aggregation and vascular
smooth muscle cell contraction and proliferation [13,14],
and cysteinyl leukotrienes (cys-LTs), vasoconstrictors of
both coronary and cerebral arteries that can decrease cardiac
output and promote vascular permeability [15], have been

0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0049-3848(03)00382-7
282 P. Patrignani / Thrombosis Research 110 (2003) 281–286

detected in patients with unstable angina [10 –12]. More-
over, enhanced formation of 8-iso-PGF2a, one of the most
abundant F2-isoprostanes in humans, generated by free-
radical catalyzed arachidonate peroxidation, has been
reported in this setting [9].
TXA2 is the main product of AA via the activity of
cyclooxygenase (COX)-1 and COX-2, in platelets and
monocytes, respectively [16 –18]. COX-1 and COX-2 cat-
alyze the same reactions, i.e. the conversion of AA to PGG2
by their cyclooxygenase activity and, then, the reduction of
PGG2 to PGH2 by their peroxidase activity [1]. PGH2 is
isomerized to TXA2 by the activity of TXA synthase
(TXAS) [5]. Apart from platelets that mainly contain
COX-1 [16], most nucleated cells have the gene for COX-
2, which can be expressed in response to inflammatory and
mitogenic stimuli [17,18].
Several lines of evidence suggest that inhibition of
platelet TXA2 production by aspirin explains its cardiopro-
tection observed in clinical trials [19,20]. The best charac-
terized mechanism of action of aspirin is related to its
capacity to inactivate permanently the cyclooxygenase ac-
tivity of COX-1 and COX-2. Aspirin causes a permanent
inactivation of cyclooxygenase activity of COX-isozymes
through the acetylation of a strategically located serine
residue (i.e. Ser529 in the human COX-1 and Ser516 in
the human COX-2) [21,22] (Fig. 1). Aspirin acetylation of
COX-1 results in the blockade of AA oxidation to PGG2.
Differently, acetylated COX-2 is a monoxygenase that
transforms AA into 12R-HETE, thus preventing the forma-
tion of PGG2 (Fig. 1). Because aspirin has a short half-life
(15 –20 min) in the human circulation and is approximately
30 –60-fold more potent in inhibiting platelet COX-1 than
monocyte COX-2 [8], it is ideally suited to act on anucleate
platelets, inducing a permanent defect in TXA2-dependent
platelet function. Moreover, since aspirin probably also
inactivates COX-1 in relatively mature megakaryocytes
and, since only 10% of the platelet pool is replenished each
day, once-a-day dosing of aspirin (75 –100 mg) is able to
maintain virtually complete inhibition of platelet TXA2
production [23]. In contrast, the inhibition of COX-2-de-
pendent pathophysiologic processes (e.g. hyperalgesia and
inflammation) requires larger doses of aspirin (because of
the decreased sensitivity of COX-2 to aspirin) and a much
shorter dosing interval (because nucleated cells rapidly
resynthesize the enzyme) [19].
Although aspirin is antiinflammatory due to inhibition of
COX-2 at higher doses, inhibition of platelet COX-1 at low
doses is sufficient to explain the cardioprotection observed
in clinical trials [19,20]. In fact, overview analysis of
indirect comparisons in clinical trials indicates that the
reduction in the incidence of vascular events in high-risk
patients with high doses of aspirin (500 –1500 mg/day) does
not exceed that attained with lower doses (75 –150 mg/day)
[20]. However, in some patients, aspirin is unable to protect
from thrombotic complications; this phenomenon is known
as ‘‘aspirin resistance’’.
An incomplete suppression of platelet TXA2 biosynthesis
has been assumed to participate in the phenomenon of
aspirin resistance, as a consequence of the following possi-
ble mechanisms: (i) COX-2 expression in newly formed
platelets [24]; (ii) pharmacodynamic interactions between
aspirin and coadministered nonsteroidal antiinflammatory
drugs (e.g. ibuprofen) [25]; (iii) expression of variant iso-
forms of COX-1 with reduced sensitivity to irreversible
inactivation at Ser529 (an hypothesis to be tested).
Platelet COX-2 expression has been detected by immu-
nostaining, in patients with platelet regeneration because of
immune thrombocytopenia or peripheral blood stem cell
transplantation [24]. It can be assumed that an incomplete
suppression of thromboxane biosynthesis by aspirin may

Fig. 1. Aspirin (acetyl salicylic acid) acetylates serine 529 of human COX-1 and serine 516 of COX-2. Aspirin is the only nonsteroidal antiinflammatory drug
(NSAID) known to react covalently (and time-dependently) with the cyclooxygenase channel of COX-1 and COX-2 causing a selective acetylation of a
specific serine residue (Ser529 for human COX-1 and Ser516 for human COX-2). Aspirin acetylation of COX-1 results in the blockade of AA oxidation to
PGG2. Differently, acetylated COX-2 is a monoxygenase that transform AA into 12R-HETE, thus preventing the formation of PGG2.
 P. Patrignani / Thrombosis Research 110 (2003) 281–286
occur in platelet expressing COX-2. Whether this phenom-
enon is operative in acute coronary syndromes and it may
participate in clinical aspirin failure remains to be studied.
Pharmacological and epidemiological studies have
shown that the coadministration of ibuprofen (a nonselec-
tive nonsteroidal antiinflammatory drug, NSAID) can lead
to the attenuation of the antiplatelet effect of aspirin [25,26].
In fact, the stronger binding affinity of the nonaspirin
NSAID to COX-1 channel may preclude aspirin from
permanently modifying platelet COX-1. In contrast, this
pharmacodynamic interaction does not occur with rofe-
coxib, a highly selective COX-2 inhibitor, diclofenac or
acetaminophen presumably because of their limited or low
affinity with platelet COX-1 [25].
Finally, it is theoretically possible that polymorphisms
and/or mutations in the COX-1 gene affecting Ser529 may
represent the structural basis for aspirin resistance in some
patients. Recently, nine single-nucleotide polymorphisms in
COX-1 of healthy subjects have been identified [27]. How-
ever, subjects, who were heterozygous for the A-842G/
C50T haplotype, showed a significant greater inhibition of
PGH2 formation by aspirin as compared with common allele
homozygotes.
Clinical aspirin failures in acute coronary syndromes
may be also due to enhanced formation of vasoactive and/
or proaggregatory eicosanoids despite an almost complete
suppression of platelet TXA2 biosynthesis by aspirin. Thus,
I will review the studies demonstrating enhanced formation
of TXA2, F2-isoprostanes and cys-LTs, in this setting.
1. Aspirin-insensitive thromboxane biosynthesis
Studies performed in vitro have demonstrated that aspi-
rin-treated platelets restore their capacity to synthesize
Fig. 2. Aspirin-treated platelets metabolize PGH2 derived from vascular cells and macrophages into TXA2. COX-2 induction in activated endothelial cells,
smooth muscle cells and macrophages may contribute to TXA2 biosynthesis by providing PGH2 to the TXA synthase (TXAS) of aspirinated platelets.
283
TXA2 if they are incubated with thrombin-stimulated endo-
thelial cells [28]. Since the extent of TXA2 production was
proportional to the amount of endothelial COX-2 expres-
sion, it has been suggested that COX-2-dependent PGH2
can be utilized by the TXAS of aspirinated platelets to
produce TXA2 (Fig. 2). Differently from platelets, that are
persistently inactivated by aspirin, endothelial cells can
rapidly (2– 4 h) recover the aspirin-dependent irreversible
inhibition of COX-2 activity after treatment with phorbol
esters or interleukin (IL)-1a due to de novo synthesis of
COX-2 [28]. These in vitro findings suggest that after a
single daily administration of the short-lived aspirin, extra-
platelet TXA2 biosynthesis can be restored through the
induction of COX-2 in nucleated cells (i.e. vascular cells,
monocytes/macrophages) in response to a local inflamma-
tory milieu (Fig. 2). This phenomenon may be operative in
vivo. In fact, in a subset of patients with unstable angina
treated with low dose aspirin to almost completely block
platelet COX-1 activity, enhanced TXA2 biosynthesis in
vivo, as reflected by the urinary excretion of 11-dehydro-
TXB2 and 2,3-dinor-TXB2, two major urinary metabolites
of TXA2, has been demonstrated [6,10]. The involvement of
extraplatelet sources, possibly expressing COX-2, has been
suggested by the finding that indobufen, an antiplatelet drug
that largely inhibits both platelet COX-1 and monocyte
COX-2 at therapeutic plasma concentrations, causes a more
profound reduction of TXA2 biosynthesis than low dose
aspirin, a selective inhibitor of platelet COX-1, in this
setting [8]. Thus, COX-2 induction in plaque monocyte/
macrophages or activated vascular cells may contribute to
aspirin-insensitive TXA2 biosynthesis in patients with acute
coronary syndromes by generating PGH2 as a substrate for
the TXAS of the same cell or by providing PGH2 to the
TXAS of aspirinated platelets (Fig. 2). The contribution of
aspirin-insensitive TXA2 biosynthesis to the pathophysiol-
284 P. Patrignani / Thrombosis Research 110 (2003) 281–286
ogy of acute coronary syndromes is supported by the
finding that baseline urinary excretion of 11-dehydro-
TXB2 predicts the future risk of myocardial infarction or
cardiovascular death in a subgroup of high-risk aspirin-
treated patients participating in the HOPE trial [29].
2. Aspirin-insensitive formation of 8-iso-PGF2a
Isoprostanes are a family of prostaglandin-like com-
pounds formed non-enzymatically through free radical cat-
alyzed attack on esterefied arachidonate followed by
enzymatic release from cellular or lipoprotein phospholipids
[3,4]. They circulate in plasma at low concentrations and are
excreted in urine. The non-invasive measurement of F2-
isoprostane may represent a useful tool for identifying
populations with enhanced rates of lipid peroxidation.
Isoprostanes are such close relatives of the prostaglan-
dins that they exert their actions by capturing the receptors
for prostaglandins. 8-iso-PGF2a (also referred to as iPF2a-
III) is an abundant F2-isoprostane formed in vivo in man.
This compound is of particular interest because it induces
vasoconstriction and modulates the function of human
platelets through the interaction with TXA2/PGH2 receptors
(TP). Praticò et al. [30] have reported that 8-iso-PGF2a
synergizes with subthreshold concentrations of pro-aggre-
gatory stimuli to evoke a full platelet aggregation (Fig. 3).
Interestingly, enhanced formation of F2-isoprostanes has
been reported in a variety of syndromes associated with
increased platelet activation. Thus, it has been proposed
that 8-iso-PGF2a may represent the signal transduction
mechanism of oxidative stress-dependent platelet activation
(Fig. 3).
Cipollone et al. [9] have found that in patients with
unstable angina treated with low dose aspirin to almost
completely suppress platelet COX-1 activity, urinary 8-iso-
PGF2a excretion was significantly higher (339 F 122 pg/mg
creatinine, mean F S.D., n = 20) than in matched patients
Fig. 3. F2-isoprostanes may represent the signal transduction mechanism of
oxidative stress-dependent platelet activation. 8-iso-PGF2a, one of the most
abundant F2-isoprostanes in humans, synergizes with sub-threshold
concentrations of pro-aggregatory stimuli to evoke a full platelet
aggregation.
with stable angina (236 F 83 pg/mg creatinine, n = 20,
P = 0.001) and in healthy subjects (192 F 71 pg/mg creati-
nine, n = 40, P < 0.0001). Similarly, patients with unstable
angina had significantly higher 11-dehydro-TXB2 excre-
tions than did patients with stable angina (532 F 227 vs.
194 F 89 pg/mg creatinine, n = 20, P < 0.0001). A statisti-
cally significant correlation was found between 8-iso-PGF2a
and 11-dehydro-TXB2 excretion in patients with unstable
angina (q = 0.721, P < 0.0001), but not in patients with
stable angina or healthy subjects. Moreover, a statistically
significant inverse correlation was found between plasma
levels of vitamin E and urinary 8-iso-PGF2a (q = 0.710,
P = 0.004) and 11-dehydro-TXB2 (q = 0.696, P = 0.006) in
patients with unstable angina. Urinary 8-iso-PGF2a did not
increase as a result of myocardial ischemia. In fact, in
patients with stable angina, a similar urinary excretion of
this isoprostane was found before [234 F 128 pg/mg creat-
inine (n = 48)] and after effort-induced myocardial ischemia
[219 F 111 (n = 24) pg/mg creatinine ( P = NS)].
The results of this study suggest that increased non-
enzymatic formation of F2-isoprostanes may provide an
important biochemical link between an altered oxidant/
antioxidant balance and aspirin-insensitive thromboxane
biosynthesis in patients with unstable angina.
3. Enhanced production of vasoactive cys-LTs in unstable
angina despite aspirin and antianginal treatment
LTs are potent lipid mediators that have an important role
in asthma and other disease processes characterized by
inflammation, cellular proliferation and altered vasocon-
striction, as well as in homeostasis [31]. LT biosynthesis
occurs predominantly in myeloid cells and is triggered by
different stimuli, including antigens, microbes, cytokines,
immune-complexes.
AA is converted by 5-lipoxygenase (5-LO) to 5-HPETE,
which is unstable and is transformed both enzymatically, via
the HPETE peroxidase, and non-enzymatically into 5-
HETE. 5-LO has also another enzymatic activity that
catalyzes the conversion of 5-HPETE to the epoxide
LTA4. LTA4 is transformed into two classes of biological
compounds, LTB4 and cysteinyl LTs (LTC4, LTD4 and
LTE4) by the activity of LTA4 hydrolase and LTC4 synthase,
respectively.
Pharmacological studies have shown that cys-LTs acti-
vate at least two receptors, designated CysLT1 and CysLT2.
CysLT1 receptor activation by LTD4 causes contraction and
proliferation of smooth muscle, oedema, eosinophil migra-
tion and damage of the mucus layer of the lung [15].
Recently, the molecular cloning and characterization of
human CysLT2 has been reported [32]. CysLT2 mRNA
has been detected in lung macrophages and airway smooth
muscle cells, cardiac purkinje cells, adrenal medulla cells,
peripheral blood leukocytes and brain. The prevalence of
CysLT2 receptor on purkinje cells in the hearth and its
 P. Patrignani / Thrombosis Research 110 (2003) 281–286
activation in vascular tissues suggest that in physiologic
conditions, cys-LTs may regulate cardiac contractility and
maintain vascular tone via the CysLT2 receptor. Enhanced
cys-LT biosynthesis has been detected during the acute
phase of unstable angina and during PTCA [11,12].
Cipollone et al. [10] have recently evidenced that LTC4
biosynthesis, as reflected by urinary LTE4 excretion, was
significantly higher in patients with unstable angina
(57 F 47 pg/mg creatinine, n = 48) as compared with
patients with stable angina (32 F 12 pg/mg creatinine,
n = 48, P < 0.02), patients with non-ischemic chest pain
(21 F 10 pg/mg creatinine, n = 12, P = 0.002) and healthy
subjects (28 F 19 pg/mg creatinine, n = 12, P < 0.02). TXA2
biosynthesis, as reflected by urinary 11-dehydro-TXB2
excretion, averaged 365 F 475 pg/mg creatinine (n = 48) in
the unstable angina patients. Metabolite excretion was
significantly higher as compared to stable angina patients
on chronic aspirin therapy (153 F 103 pg/mg creatinine,
n = 32, P < 0.05), aspirin-treated patients with non-ischemic
chest pain (78 F 41 pg/mg creatinine, n = 12, P < 0.0001)
and healthy subjects after three-day aspirin administration
(26 F 19 pg/mg creatinine, n = 12, P < 0.0001). Urinary 11-
dehydro-TXB2 was also significantly higher in stable angina
as compared to both non-ischemic chest pain ( P < 0.02) and
healthy volunteers ( P < 0.0001). Eicosanoid metabolite ex-
cretion did not increase as a result of myocardial ischemia,
as reflected by LTE4 and 11-dehydro-TXB2 values of
33 F 12 and 250 F 233 pg/mg creatinine (n = 48), respec-
tively, before the exercise stress test, and of 36 F 14 and
269 F 138 pg/mg creatinine (n = 24) after effort-induced
myocardial ischemia ( P = NS). In patients with unstable
angina, enhanced urinary LTE4 excretion was significantly
reduced by the treatment with the synthetic glucocorticoid
6-methylprednisolone (6-MP; 1 mg/kg BID for 2 days). In
fact, 6-MP caused a time-dependent decrease of LTE4
excretion from a value of 90 F 55 pg/mg creatinine, before
6-MP infusion, to 15 F 9 pg/mg creatinine, in the last urine
collection following the 4th steroid infusion. Thus, 6-MP
administration was associated with 76% suppression in LTE4
from the first through the last day of study (68 F 50 vs.
16 F 7 pg/mg creatinine, n = 24, P < 0.0001). In contrast,
placebo administration was not associated with changes in
LTE4 from the first through the last day of the study
(53 F 43 vs. 49 F 38 pg/mg creatinine).
4. Conclusions
Increased production of vasoactive eicosanoids may
occur in unstable angina patients treated with low dose
aspirin to almost completely suppress platelet TXA2 bio-
synthesis. Enhanced TXA2 biosynthesis in this setting may
occur through an increased generation of COX-2-dependent
PGH2 in plaque monocytes/macrophages or activated vas-
cular cells. The concomitant increased formation of 8-iso-
PGF2a, through the non-enzymatic oxidation of AA cata-
285
lysed by free radicals, may be involved in aspirin resistance
because of its capacity to induce platelet and vascular
smooth muscle cell activation. Finally, enhanced production
of vasoactive cys-LTs occurs in unstable angina despite
conventional antithrombotic and antianginal treatment.
The use of selective pharmacological tools (i.e. highly
selective COX-2 inhibitors, TP antagonists, cys-LT inhib-
itors and antagonists) in this setting will allow to ascertain
the possible contribution of aspirin insensitive eicosanoid
biosynthesis to aspirin cardioprotective failure.
Acknowledgements
I wish to acknowledge Drs. Francesco Cipollone,
Giovanni Ciabattoni, Maria Gina Sciulli, Giulia Renda and
Stefania Tacconelli (all from ‘‘G. d’Annunzio’’ University
of Chieti) and Dr. Carlo Patrono (‘‘La Sapienza’’ University
of Rome) for the design and performance of the studies that
I reviewed.
Supported by a grant from the Italian Ministry of
University and Research (MURST) to the Center of
Excellence on Aging, ‘‘G. d’Annunzio’’ University of Chieti.
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