Download Pharmacology of platelet inhibition in humans: implications of the

PLATELETS AND VASCULAR OCCLUSION
Pharmacology of platelet inhibition in humans:
implications of the salicylate-aspirin interaction
GIOVANNI DE GAETANO, M.D., PH.D., CHIARA CERLETTI, PH.D., ELISABETrA DEJANA, PH.D.,
ROBERTO LATINI, M.D.
AND
Downloaded from http://circ.ahajournals.org/ by guest on April 28, 2017
ABSTRACT The current dispute over the effects of "low" vs "high"doses of aspirin should take into
consideration the pharmacokinetics of this drug. In fact, different pharmaceutical formulations of
aspirin may deliver little or no aspirin to the systemic blood. This was the case, for instance, in healthy
volunteers taking 320 mg of compressed aspirin or 800 mg of enteric-coated aspirin. In all instances
thromboxane B2 generation in serum was fully inhibited. Platelet cyclooxygenase might therefore be
effectively acetylated by exposure to aspirin in the portal circulation, whereas vascular cyclooxygenase
could be spared. Thus aspirin formulations ensuring complete first-pass deacetylation should be sought
rather than "low" or "high" doses of unspecified aspirin formulations. Regardless of the type and dose
of aspirin administered, salicylate is formed and accumulates in the circulation. It may antagonize the
effects of aspirin on cyclooxygenase, at least in acute conditions. As an example, after administration
of 1 g of salicylate to healthy volunteers, when plasma levels of the drug were about 75 ,ug/ml, the
effect of 40 mg iv aspirin (given 40 min later) on platelet cyclooxygenase and aggregation was
significantly diminished. In contrast, in patients undergoing saphenectomy, the same dose of salicylate
(1 g) gave plasma drug levels of about 25 ,ug/ml; salicylate was unable to prevent the inhibitory effect
on platelets of 40 mg iv aspirin (given 1 hr later) but did act on vascular prostacyclin. Thus the
combination of salicylate with aspirin at an appropriate dose and blood level ratio may result in almost
complete dissociation of the drug's effect on platelets and vessels in man. Because the studies outlined
in this review address only the short-term outcome of the salicylate-aspirin interaction, the possibility
should be tested that inactivation of cyclooxygenase would occur after long-term administration of
salicylate-aspirin. A pharmacokinetic approach to the "aspirin dilemma" should also consider the
possibility that salicylate and/or its metabolites (e.g., gentisic acid) may interfere with lipoxygenase
activity, synthesis of the prothrombin complex coagulation factors, and fibrinolysis.
Circulation 72, No. 6, 1185-1193, 1985.
Aspirin, salicylate, and inhibition of platelet aggregation
and cyclooxygenase activity. Aspirin inhibits platelet aggregation and the concomitant release reaction.I4 This
exciting discovery, made more than 15 years ago, was
characterized by three main facts: (1) aspirin was active in man at oral doses as low as 150 mg (about 2
mg/kg), (2) its effects were long lasting (at least 24 to
48 hr), and (3) salicylate, the main metabolite of aspirin, was virtually inactive. The last two observations
suggested that the effect of aspirin on platelets was the
result of irreversible acetylation. Consequently, the
salicylate moiety of aspirin was considered irrelevant
for the drug's action on platelets.
From the Laboratory of Cardiovascular Clinical Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy.
Supported by the Italian National Research Council, Progetto Finalizzato "Medicina Preventiva," Sottoprogetto "Malattie Degenerative,"
Contract No. 84.02277.56.
Address for correspondence: Giovanni de Gaetano, M.D., Ph.D. ,Laboratory of Cardiovascular Clinical Pharmacology, Istituto di Ricerche
Farmacologiche Mario Negri, via Eritrea, 62-20157 Milano, Italy.
Vol. 72, No. 6, December 1985
In the early seventies, aspirin and other nonsteroidal
anti-inflammatory drugs (NSAID) were shown to inhibit prostaglandin formation in human platelets.5
Again salicylate was inactive. Acetylation of platelet
proteins by aspirin was subsequently reported.6 Because one of these proteins was a subunit of fatty acid
cyclooxygenase, the role of this enzyme in the aspirin
action was considered to be crucial. Although the nonspecific acetylation of other platelet proteins is currently of unknown biological significance, the inhibition
of cyclooxygenase by aspirin does not appear to explain fully the antithrombotic effects of this drug.]10
Aspirin is quickly deacetylated in vivo to salicylate.
It has been suggested that in sites readily accessible to
aspirin, such as circulating platelet cyclooxygenase, it
might be expected to act in its own right as the acetylsalicylate ion and hence to be more potent than salicylate.7 It would also be expected that the salicylate ion,
into which aspirin is converted, would contribute to the
overall activity of aspirin, for instance in actions that
1185
DE GAETANO et al.
need persistent presence of an anti-inflammatory drug
or that are located at poorly accessible sites; this might
apply for example in rheumatic joints.7 Additionally,
salicylate may antagonize some effects of aspirin, such
as its gastrointestinal ulcerogenicity."1
Our aim in this article is to review the available
evidence on (1) the occurrence and the possible clinical
relevance of the interaction between salicylate (or other NSAID) and aspirin on platelet and vascular cyclooxygenase and (2) the existence of additional
mechanism(s) related to salicylate and possibly involved in the antithrombotic effects of aspirin.
Salicylate may antagonize the effects of aspirin on cyclooxygenase
Salicylate-aspirin or other NSAID-aspirin interactions on
animalplatelet cyclooxygenase. Studies in vitro have demDownloaded from http://circ.ahajournals.org/ by guest on April 28, 2017
onstrated that salicylate, which is not inhibitory by
itself, protects cyclooxygenase from aspirin inhibition
in rat'2 and rabbit'3 platelets (figure 1). A similar pharmacologic interaction was apparent in vivo. 14 1' In the
rat, administration of salicylate resulted in dose-related prevention of aspirin's effect on platelet cyclooxygenase activity. This interaction appeared to be competitive and closely dose related for both drugs.'5
Other NSAID structurally related or unrelated to
salicylate, such as diflunisal and sulphinpyrazone, respectively, are able in the rat in vivo to counter the
effect of aspirin on platelets.'5 This effect was apparent
for diflunisal or sulphinpyrazone at doses that did not
inhibit platelet cyclooxygenase. The prevention by indomethacin of the effect of aspirin on platelets was
apparent 24 hr after administration of aspirin. Actually
the effect of indomethacin on platelet cyclooxygenase
is short lasting, whereas that of aspirin lasts at least 24
hr. When the two drugs were given concurrently, the
lasting effect of aspirin was no longer detectable and
inhibition of cyclooxygenase lasted as if indomethacin
alone had been administered. On the other hand, salicylate and diflunisal competitively blunted the inhibitory effect of indomethacin on platelet cyclooxygenase.
All these results could be explained by postulating a
common site of interaction for the different NSAID on
cyclooxygenase.'5 16 The observation that salicylate, a
virtually inactive compound or ineffective doses of
diflunisal and sulphinpyrazone counteract the effect of
aspirin suggests that competition does not occur at the
catalytic site but rather at a supplementary binding
site. Interaction with this putative supplementary site
is necessary but not sufficient for the efficacy of these
drugs as cyclooxygenase inhibitors.'5' 16 Thus salicylate interacts with the binding site of cyclooxygenase
but does not modify its enzymatic activity. However,
neither aspirin nor indomethacin can exert their inhibitory activity on the enzyme when the binding site is
occupied by salicylate.
Further experimental evidence of a supplementary
binding site on cyclooxygenase was provided by the
failure of salicylate to prevent enzyme inhibition by
5,8,11,14-eicosatetraynoic acid (ETYA), a competitive antagonist of arachidonic acid. Moreover, salicylate did not affect either ETYA or nor-dihydroguaiaretic acid inhibition of platelet lipoxygenase.
On the basis of these and other data presented in
detail elsewhere, 17 a functional model of pharmacologic inhibition of cyclooxygenase by aspirin and other
NSAID can be proposed (figure 2). This model would
explain the selectivity of NSAID for the cyclooxygen-
z
0
(/)
(I
i
LI)
z
OX
A
AA
AA
a.-
-J
(D
CONTROL
SALICYLATE
ASA
SAL I C YLATE +ASA
FIGURE 1. Prevention by salicylate of aspirin inhibition of arachidonic acid-induced platelet aggregation. Rat platelet
aggregation was induced by threshold concentrations of arachidonic acid (AA 0.5 mM). Aspirin (100 jM) preincubation (1 min)
with PRP completely prevented aggregation, whereas sodium salicylate (1 mM) 1 min before was ineffective. The combination
of both compounds resulted in an almost normal aggregation. ASA = aspirin; PRP = platelet-rich plasma.
1186
CIRCULATION
PLATELETS AND VASCULAR OCCLUSION
ARACHIDONIC
ACID
SALICYLATE
BINDING
SITE
CATALYTIC SITES
CYCLO-OXYGENASE
L IPOXYGENASE
FIGURE 2. Functional model of cyclooxygenase and lipoxygenase inhibition. Salicylate, aspirin, and the other NSAIDs bind to
a binding site related to but distinct from the catalytic site of cyclooxygenase. This binding is necessary but not sufficient for
enzyme inhibition. 5,8,11,14-Eicosatetraynoic acid (ETYA) competes with arachidonic acid for the catalytic sites of both
cyclooxygenase and lipoxygenase. Thus salicylate does not interfere with the effect of ETYA.
Downloaded from http://circ.ahajournals.org/ by guest on April 28, 2017
ase pathway of arachidonic acid metabolism in relation
to lipoxygenase. I6J 17 The intensity of interaction with
the supplementary site and the ensuing modifications
of the catalytic site would determine these compounds'
potency as cyclooxygenase inhibitors. This model
might also account for the finding that salicylate, aspirin, and indomethacin equipotently inhibit the lipoxygenase pathway.'8 Recently, additional evidence for
two distinct enzymatic sites on rabbit platelet cyclooxygenase has been presented.19
Salicylate-aspirin interaction on animal vascular cyclooxy-
genase. The interaction between salicylate and aspirin
is not restricted to platelet cyclooxygenase.'2 In fact,
salicylate administered to rats may prevent the inhibitory activity of aspirin on vascular cyclooxygenase.20
This effect was even more evident than that on platelets. Indeed the doses of salicylate needed to completely counteract the effect of the same dose of aspirin on
vascular cyclooxygenase were lower than those on
platelets. As a direct consequence of this observation,
the administration of salicylate and aspirin at appropriate dose ratios could achieve complete inhibition of
platelet cyclooxygenase without affecting the vascular
enzyme.
Whether the different efficacy of salicylate in preventing aspirin effect derives from a different sensitivity to or access of the drug at various cellular levels
remains to be established.
Salicylate-aspirin interaction at other cellular levels in animals. In the guinea pig, salicylate, inactive by itself,
prevents the protective effect of aspirin in arachidonic
acid-induced bronchoconstriction in vivo.21 Because
this arachidonate effect was not mediated by platelet
aggregation but most likely by endoperoxide and
thromboxane A2 (TxA2) production by the lung, the
Vol. 72, No. 6, December 1985
salicylate-aspirin interaction could occur in the lungs.
It has been reported that salicylate diminishes the
gastric lesions induced by aspirin and other NSAID in
rats." This effect seems to be secondary to a depression of prostaglandin synthesis in the stomach, and the
counteracting effect of salicylate was correlated to its
ability to protect gastric cyclooxygenase from NSAID
inhibition.1"1 22
Salicylate-aspirin interaction on human platelet cyclooxy-
genase. All the above data have been obtained in vitro
or in vivo in animal studies. Interference by salicylate
with aspirin inhibition of human platelet cyclooxygenase activity has also been observed.2-25 The question
remains as to whether a similar interaction occurs between salicylate and other NSAID on cyclooxygenase
in vivo in man and as to its clinical relevance.
In a recent study in human volunteers we observed
that orally administered indomethacin (50 mg) prevented the long-lasting effect of a subsequent dose of
aspirin (500 mg) on platelet cyclooxygenase activity
and on platelet aggregation.25 Similarly, ingestion of
ibuprofen prevented the aspirin effect.26 More recently
we investigated whether salicylate and aspirin interact
on platelets in man at doses and plasma levels of clinical relevance.27 Six volunteers, given oral doses of
sodium salicylate (250 or 1000 mg) presented peak
plasma salicylate levels averaging 20 and 75 ,ug/ml,
respectively. Neither platelet aggregation nor serum
immunoreactive thromboxane B2 (TxB2) formation
was modified by either salicylate treatment. Forty minutes later, all volunteers received 40 mg iv aspirin, the
lowest single dose that suppressed arachidonate-induced platelet aggregation and TxB2 generation. Aspirin plasma levels were not affected by previous salicylate ingestion. Inhibition by aspirin of both platelet
1187
DE GAETANO et al.
aggregation and TxB2 generation was significantly
prevented by the larger salicylate dose, giving plasma
salicylate levels similar to those after an oral dose of
800 mg of enteric-coated aspirin. Pretreatment with
250 mg of salicylate, giving plasma salicylate levels
similar to those obtained with an oral dose of 320 mg of
compressed aspirin, resulted in proportionally diminished prevention of aspirin's effect on platelet aggregation but did not significantly modify its effect on serum
TxB2 generation.
Salicylate-aspirin interaction in man as a possible pharma-
TABLE 1
Effect of aspirin and salicylate on immunoreactive serum TxB2 and
vascular 6-keto-PGF,a generation in 21 patients undergoing
saphenectomy
cologic solution to the "aspirin dilemma." When it was
Ap < .01 compared with the other two groups (Duncan's test). 6Keto-PGF,a was measured by specific radioimmunoassay in the supernatant of epigastric segments of saphenous vein incubated for 5 min
with arachidonic acid (50 ttM), as described.3'
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shown that aspirin, while inhibiting TxA2 in platelets,
equally inhibited antiaggregatory prostacyclin (PGI2)
in vascular cells, the "aspirin dilemma" emerged.28 29
Simultaneous inhibition of PGl2 and TxB2 synthesis
was considered a possible reason for the disappointing
results of clinical trials on the drug's antithrombotic
effect.8
In an effort to solve the "aspirin dilemma," studies
were devised to determine the aspirin dose that would
inhibit TxA2 production but would not interfere with
PGI2 synthesis in vascular cells. Platelet cyclooxygenase was believed to be more sensitive than vascular
cyclooxygenase to aspirin inhibition. If it had been, the
"aspirin dilemma" could have been solved easily by
using low doses of the drug. Unfortunately, studies
with endothelial cells in culture, laboratory animals,
and man have not completely dissociated the inhibitory
effect of aspirin on platelets from that on vascular cells
(for review see refs. 8 and 30).
We have already mentioned that in the rat, salicylate
was more effective in counteracting the inhibitory activity of aspirin on vascular cyclooxygenase than that
on platelet cyclooxygenase.20 We have recently completed a study3' in patients undergoing saphenectomy,
aimed at verifying a similar differential salicylate-aspirin interaction in man. Twenty-one female patients,
admitted to the Milan- University Institute of Vascular
Surgery for removal of varicose veins, entered this
study. Seven took no medications, and six were given
40 mg iv aspirin 1 hr before operation. The remaining
eight patients took 1000 mg sodium salicylate orally 1
hr before aspirin. During surgery, segments of epigastric vein in close proximity to the saphenous vein were
removed for measurements of prostacyclin production.
At the same time, venous blood was collected for measurements of serum TxB2 synthesis.
As reported in table 1, TxB2 generation was completely inhibited in all patients given aspirin alone,
whereas prostacyclin generation was reduced by more
than 50%. In the salicylate-aspirin group, TxB2 gen1188
Vascular
Treatment
Control
Aspirin (40 mg iv)
Salicylate (1 g po) + aspirin
nd
=
6-keto-PGF,,
n
Serum TxB2
(pmol/ml)
7
6
8
44.8+4
nd
4.3+1
2.73±0.2
1.18 ± 0.2A
2.11 ±0.2
(pmollmg)
not detectable (<0.075 pmol/ml).
eration remained undetectable in three patients and
reached about 10% of control values in the other five.
In contrast, six patients generated prostacyclin levels
within the control range and only two appeared to be
partially inhibited. Mean salicylate blood levels at the
moment of aspirin administration were 25.9 + 5
jug/ml.
This observation confirms in man the results obtained in the rat. In this animal as well as in the present
study in man, vascular cells showed lower sensitivity
to aspirin than platelets. Salicylate resulted in both
instances in an amplification of the difference in response to aspirin of platelets and vascular cells. The
mechanism of this property of salicylate (e.g., different access of the drug at various cellular levels) remains to be defined. This observation, however, may
constitute an original approach on which to base the
clinical use of aspirin in a supposedly beneficial antithrombotic direction.
When analyzed with respect to the study in healthy
male volunteers, this study in female patients undergoing saphenectomy merits some comments. As summarized in table 2, the same salicylate-aspirin dose ratio
(1000 mg oral vs 40 mg iv) resulted in significant
prevention of the aspirin effect on platelet TxB2 generation in the group of volunteers but not in patients.
The most likely explanation is that at the moment of
aspirin administration, the plasma levels of salicylate,
measured 1 hr after ingestion (abut 25 ,g/ml), were
markedly lower than those measured 40 min after ingestion in volunteers (about 75 gg/ml). In fact, salicylate levels in patients were similar to those found in
volunteers 40 min after 250 mg of salicylate. In the
latter condition, as well as in patients, no significant
prevention of aspirin's effect on TxB2 synthesis could
be detected. Possibly, gastrointestinal absorption of
salicylate was reduced in patients by anesthesia.
CIRCULATION
PLATELETS AND VASCULAR OCCLUSION
TABLE 2
Salicylate-aspirin interaction at platelet and vascular level and
plasma salicylate levels in healthy volunteers and in patients undergoing saphenectomy
Subjects
Volunteers
Volunteers
Saphenectomy
patients
Salicylate
levels
SAL/ASA
interaction
TxB2 PGI2
Salicylate
(mg, oral)
Interval
(min)
250
1000
40
40
22.3 ± 1.7
76.3 ± 5.1
No
Yes
1000
60
25.9 ± 5.0
No
(,uglml)
Yes
SAL = salicylate; ASA = aspirin.
Plasma salicylate levels were measured immediately before aspirin
(40 mg iv). For other details see table 1.
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Independently of the reasons why the same dose of
salicylate gave rise to different plasma levels of the
drug in the two groups of subjects, it appears that the
plasma levels of salicylate rather than the amount of
the drug administered are relevant for an optimal interaction with aspirin.
In summary, we have discussed the hypothesis that
platelet and/or vascular cyclooxygenase might be
spared acetylation by aspirin if the plasma levels of
salicylate are carefully balanced with the plasma levels
of aspirin. This hypothesis has been tested experimentally both in vitro and ex vivo in short-term animal and
human studies. Protection of cyclooxygenase with salicylate acutely prevented irreversible inactivation by
aspirin; experimental evidence that salicylate protects
cyclooxygenase from inactivation by aspirin after
long-term administration of both compounds is still
lacking. Cyclooxygenase protection by salicylate
might slow the rate of its inactivation by aspirin but not
prevent it completely; thus the cyclooxygenase population might be finally inactivated during long-term
salicylate-aspirin therapy, despite a careful balance between salicylate and aspirin levels. On the basis of
these considerations we addressed our attention to
another possible approach to solve the "aspirin
dilemma."
pirin after either 320 mg of compressed aspirin or 800
mg of enteric-coated aspirin were not statistically different (2.9 ± 0.06 and 3.3 ± 0.3 gg/ml, respectively). Serum TxB2 generation was almost completely
inhibited 1 hr after either aspirin formulation.
Of even greater interest was the observation that
similar full TxB2 inhibition was obtained in five volunteers in whose peripheral circulation aspirin levels
were not measurable (figure 3). Three subjects had
received a compressed aspirin and two an enteric-coated aspirin. No significant differences in salicylate area
under the plasma concentration vs time curve could be
found between subjects with detectable or undetectable aspirin plasma levels after either formulation (table
3). On account of the potential for erratic absorption
pattern of aspirin, detectable plasma levels of the unchanged drug may depend on fortuitous blood collection times.32 In our study, however, this was unlikely
because as many as eight blood samples were collected
within the first hour after compressed aspirin and six
100-
320
mg
COMPRESSED
ASA DETECTABLE
oJ
ASA N. D.
0
z
os50
0
0
-_-L
10min
0m
10
lh
30m
800 mg
24h
4h
ENTERIC-COATED
ASA
DETECTABLE
AS A
N.D0.
_J
z
o
so0
Aspirin formulations that deliver only salicylate to the
systemic circulation: a pharmacokinetic approach to the
"aspirin dilemma." The importance of salicylate plasma
levels mentioned in the preceding section suggests that
a better knowledge of the pharmacokinetics of aspirin
might help solve the "aspirin dilemma." Little attention has been paid to aspirin pharmacokinetics with
regard to the different pharmaceutical formulations
used in thrombosis prevention trials. This approach
seemed of particular interest in view of observations in
healthy volunteers27 that the peak plasma levels of asVol. 72, No. 6, December 1985
10min
FIGURE 3. Serum
320 mg of
h
TxB2
2 4h
measured at different times after
compressed (upper panel)
or
ingestion
of
800 mg of enteric-coated (lower
panel) aspirin in subjects with detectable or undetectable aspirin levels.
Figures are percent values of predrug serum TxB2 in each subject and
SEM. Three subjects had detectable and three undetectable aspirin
plasma levels after 320 mg aspirin, while four had detectable and two
undetectable aspirin plasma levels after 800 mg enteric-coated aspirin.
In this last group the mean and the two single values are reported,
respectively. ASA
=
aspirin; ND
=
not detectable.
1189
DE GAETANO et al.
TABLE 3
Pharmacokinetics of aspirin after ingestion of two diferent drug
formulations in six healthy volunteers
AUC (0o,
Aspirin
(nmol/ml/hr)
preparation
(oral dose)
n
Aspirin
Salicylate
Compressed
(320 mg)
3
3
4
2
17.3±1.4
undetectable
31.9 ± 3.3
undetectable
544 ±-42
830 ± 40
2680 +153
2190-2851
Enteric-coated
(800 mg)
AUC = area under the plasma concentrations between 0 and m.
Detection limit of aspirin = 0.5 nmol/ml.
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during the first 3 hr after enteric-coated aspirin. The
time of peak aspirin concentration in subjects in whom
it was detectable was 33 min for compressed and 83
min for enteric-coated aspirin.27
Experiments in vitro showed that incubation of human whole blood from three different donors with 0.5
,.tM aspirin (corresponding to the detection limit of the
high-performance liquid chromatographic assay method used) for 1 hr at 370 C did not result in any significant reduction of serum TxB2 generation. As shown in
figure 4, concentrations of aspirin at least 50 times this
detection limit were required to prevent TxB2 generation by more than 90%.
It has long been known that aspirin undergoes extensive first-pass deacetylation within the enterohepatic
circulation.33 Whether this first-pass effect is dosedependent is not yet known. Thus inhibition of serum
TxB2 generation in the absence of measurable aspirin
in systemic peripheral blood might result from acetylation of platelet cyclooxygenase by exposure of circulating cells to the drug in the portal circulation. Cyclooxygenase in the peripheral vasculature could thus
come into contact only with inactive salicylate, with
the possible exception of the mesenteric and portal
100*
*0
5 0
vessels exposed to aspirin before the first-pass occurs.
Thus aspirin formulations ensuring extensive firstpass deacetylation rather than merely "low" or "high"
doses of unspecified aspirin formulations should be
sought as a pharmacologic goal for solving the "aspirin
dilemma." The current dispute on the difference in
sensitivity to "low" doses of aspirin between platelet
and vascular cyclooxygenase may well arise from the
wide variability of aspirin pharmacokinetics. Indeed,
as in our experiments, the same dose or formulation
may or may not deliver measurable amounts of aspirin
to the systemic circulation; the inhibitory effect on
platelet cyclooxygenase would be achieved in any
case, but vascular PGI2 synthesis would only be spared
in the absence of detectable peripheral aspirin levels.
Similar conclusions have recently been reached by other
investigators.", 35
Salicylate may contribute to the overall activity of aspirin
in the prevention of vascular occlusion. Although inhibition of prostanoid synthesis has become an attractive,
unifying explanation of the anti-inflammatory activity
of aspirin-like drugs, this mechanism does not explain
all aspects of the anti-inflammatory action of salicylates, especially in conditions in which aspirin and
salicylate are of approximately equal potency.7 It has
therefore been suggested that the anti-inflammatory
activity of aspirin-like drugs probably does not arise
from a single mechanism but may be mediated through
an additional or alternative mechanism, which becomes operative only at doses in excess of those required to suppress cyclooxygenase. The possibility has
also been discussed that aspirin has additional components to its mechanism of action not possessed by other
NSAIDs, such as flufenamic acid or indomethacin.
The major lines of research into alternative mechanisms that might explain the therapeutic action of sa-
licylate have been reviewed extensively.7
Because several pathways may be involved in the
initiation and growth of thrombi, one would expect
aspirin to be inhibitory only in conditions in which
prostanoid biosynthesis (cyclic endoperoxides and
TxA2) plays a major role in thrombus formation.
Effects of aspirin and salicylate on blood coagulation and
z
0 -x
v
.X
r
0.5
1
-
2.5
5
10
25
50
100
[AiM] Aspi rin
FIGURE 4. Effect of different aspirin concentrations on TxB2 generation in human serum in vitro. Blood was collected without anticoagulant
from the antecubital vein of healthy subjects and incubated in glass tube
at 370 C for 1 hr in the presence of aspirin. TxB2 generation was
measured in serum. The values (mean from four experiments) are percent inhibition of control values.
1190
fibrinolysis. A recent study9 in rabbits with in-dwelling
aortic catheters strongly suggests that aspirin at doses
that inhibit only platelet cyclooxygenase activity is not
a satisfactory inhibitor of arterial thrombosis when
there is a significant fibrin component. At very high
doses aspirin inhibited thrombosis, but this effect was
not mediated through inhibition of platelet function. In
fact salicylate was more effective than aspirin. The
tendency for high doses of aspirin or salicylate to inCIRCULATION
PLATELETS AND VASCULAR OCCLUSION
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hibit thrombus formation appears related to a prolongation of the one-stage prothrombin time and to activation of whole blood fibrinolytic activity; which of these
pathways is most important in reducing thrombus formation remains to be established.
This experimental finding is in keeping with some
observations in man. There are reports that doses of 2
to 3 g of aspirin per day in man prolong prothrombin
time36 37 and increase whole blood fibrinolytic activity.3 -0 Moroz39 reported that aspirin and sodium salicylate (1.8 g) were equipotent in stimulating fibrinolytic
activity through the protease action of leukocytes.
The beneficial effect of high doses of aspirin in
preventing venous thrombosis and pulmonary embolism after total knee replacement41 may be due not just
to the effect of aspirin on platelets but also to its effect
(and/or of the salicylate) on blood coagulation and
fibrinolysis. However, in a recent study on healthy
human volunteers, aspirin (650 mg given orally both
18 and 2 hr before blood collection) inhibited the rise
in vascular plasminogen activity after venous occlusion.42 Similarly a decrease in fibrinolysis induced by
both cellular and plasmatic factors after ingestion of 1
g of aspirin per day for 4 days had been reported.43
Recently Roncaglioni et al.4 have shown in rats that
salicylate, like warfarin, with which it may have some
common chemical features (figure 5), reduced the
plasma concentration of the prothrombin complex
clotting factors in vivo. It also caused an accumulation
of microsomal substrates for vitamin K-dependent
carboxylase in the liver and lung. These effects of
salicylate were dose-dependent (between 4 x 50 and 4
x 200 mg/kg) and were counteracted by simultaneous
administration of vitamin K (20 mg/kg). The mechanism of salicylate-induced hypoprothrombinemia in
the rat and in the rabbit has been extensively investigated by several groups.45-48 Because the effect of sa-
OH
0
0
licylate was additive to that of warfarin,44 relatively
low doses of aspirin or salicylate should interfere with
oral anticoagulant therapy. Aspirin-induced fluctuation in clotting factor levels of as little as 5% to 10%
of normal might have serious consequences in patients
undergoing anticoagulant treatment, although it might
not even be measurable in healthy volunteers.
These considerations should be borne in mind when
analyzing the results of clinical trials combining aspirin and oral anticoagulants. On the one hand it is not
surprising that this drug combination has been reported
to be more effective than oral anticoagulants alone in
preventing thrombotic complications in patients with
aortic ball valves.49 On the other hand it could explain
the high frequency of bleeding complications encountered in other trials.50 Two recent reports in rabbits5"
and in rats52 have suggested that high doses of aspirin
may prolong bleeding time independently of an effect
on platelet cyclooxygenase and also of formation of
salicylate.
Effects of aspirin and salicylate on leukocytes. The observation that aspirin and salicylate equipotently inhibit
the generation of chemotactic 12-HETE from 12HPETE in the lipoxygenase pathway of the arachidonic acid metabolism,18 although controversial, has
been considered as diminishing the importance of the
cyclooxygenase pathway in the anti-inflammatory activity of salicylate.7
A key event in any inflammatory reaction is the
migration of leukocytes from the blood into the inflammatory lesion. Leukocytes metabolize arachidonic
acid by lipoxygenase53 and the products of this enzyme
are potent chemotactic agents54 that induce leukocyte
aggregation.55 56 It has recently been proposed57 that
migrating leukocytes contribute to the tissue injury
accompanying myocardial ischemia, possibly by release of proinflammatory mediators and free radicals
0
DY ~~~~~F
Rh
FIGURE 5. Chemical structures of salicylate and
warfarin.
OH
SALICYLIC ACID
Vol. 72, No. 6, December 1985
OH
CH2COCH3
WARFARIN
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DE GAETANO et al.
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(oxygen metabolites). Drugs that reduce the migration
and/or activation of leukocytes might be useful in reducing infarct size.
Aspirin and salicylate inhibit the production of superoxide anion by activated leukocytes7 but do not
reduce leukocyte invasion into an inflammatory exudate.58 Thus, although an interaction with superoxide
anion could be considered as a possible additional
mechanism of the anti-inflammatory action of salicylates, the biological consequences of this pharmacologic effect are not obvious. A recent observation in
our laboratory (A. Lorico and S. Villa, unpublished)
may also be of interest in this context: gentisic acid,59 a
hydroxylated metabolite of salicylate, not only prevented superoxide anion production by human leukocytes stimulated with either arachidonic acid or phorbol myristate acetate, but also prevented leukocyte
aggregation. The concentrations of gentisic acid active
in vitro (0.4 to 1.2 mM) are probably not reached in the
human circulation after current therapeutic doses of
aspirin.60 However, this finding merits further investigation.
Conclusions. Aspirin inhibits cyclooxygenase. This
effect distinguishes it from salicylate, its major metabolite, with regard to inhibition of the platelet function
induced by certain stimuli. Although inactive on cyclooxygenase, salicylate may interfere with the effect
of aspirin on this enzyme both in platelets and (even
more effectively) in the vasculature. Knowledge of the
pharmacokinetics of aspirin and salicylate is essential
for optimal use of aspirin as a selective inhibitor of
platelet but not of vascular cyclooxygenase activity.
The mechanism involving cyclooxygenase does not,
however, appear to fully explain the antithrombotic
action of aspirin. Therefore, it is best to assume that in
preventing thrombosis aspirin acts by at least two
mechanisms, one of which is the inhibition of platelet
cyclooxygenase.
The best candidate mechanisms, most likely to be
linked to the salicylate moiety of aspirin, are the inhibition of vitamin K-dependent clotting factor synthesis, cell-mediated activation of blood fibrinolysis, and
interference with the platelet and/or leukocyte lipoxygenase pathway of arachidonic acid metabolism.
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therapeutic action. Adv Pharmacol Chemother 17: 233, 1980
8. de Gaetano G, Cerletti C, Berteld V: Pharmacology of antiplatelet
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Lancet 2: 974, 1982
9. Cattaneo M, Chahil A, Somers D, Kinlough-Rathbone RL, Packham MA, Mustard JF: Effect of aspirin and sodium salicylate on
thrombosis, fibrinolysis, prothrombin time, and platelet survival in
rabbits with indwelling aortic catheters. Blood 61: 353, 1983
10. Marcus AJ: Aspirin as an antithrombotic medication. N Engl J Med
309: 1515, 1983
11. Ezer E, Palosi E, Hajos Gy, Szpomy L: Antagonism of the gastrointestinal ulcerogenic effect of some non-steroidal anti-inflammatory agents by sodium salicylate. J Pharm Pharnmacol 28: 655, 1976
12. Merino J, Livio M, Rajtar G, de Gaetano G: Salicylate reverses in
vitro aspirin inhibition of rat platelet and vascular prostaglandin
generation. Biochem Pharmacol 29: 1093, 1980
13. Vargaftig BB: The inhibition of cyclo-oxygenase of rabbit platelets
by aspirin is prevented by salicylic acid and by phenanthrolines.
Eur J Pharmacol 50: 231, 1978
14. Vargaftig BB: Salicylic acid fails to inhibit generation of thromboxane A2 activity in platelets after in vivo administration to the rat. J
Pharm Pharmacol 30: 101, 1978
15. Cerletti C, Livio M, de Gaetano G: Non-steroidal anti-inflammatory drugs react with two sites on platelet cyclo-oxygenase: evidence from in vivo drug interaction studies in rats. Biochim
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Judith Baggott, Antonella Bottazzi, Vincenzo de Ceglie, and
Felice de Ceglie helped prepare the manuscript.
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1193
Pharmacology of platelet inhibition in humans: implications of the salicylate-aspirin
interaction.
G de Gaetano, C Cerletti, E Dejana and R Latini
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Circulation. 1985;72:1185-1193
doi: 10.1161/01.CIR.72.6.1185
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