Journal of Clinical Pharmacy and Therapeutics, 2012
Influence of non-steroidal anti-inflammatory drugs on antiplatelet effect of
aspirin
H. Yokoyama*PhD , N. Ito*MS , S. SoedaBS , M. OzakiMS , Y. SuzukiBS , M. WatanabeBS , E. KashiwakuraBS , T. KawadaBS ,
N. IkedaBS , K. TokuokaMD , Y. KitagawaMD, PhD and Y. Yamada*PhD
*School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Department of Pharmacy, Tokai University Hospital, Isehara,
Department of Pharmacy, Tokai University of Hachioji Hospital, Hachioji, Central Clinical Laboratory, Tokai University Hachioji Hospital, Hachioji,
and Department of Neurology, Tokai University Hachioji Hospital, Hachioji, Japan
Received 01 May 2011, Accepted 09 July 2012
Keywords: antiplatelet effect, aspirin, ibuprofen, interaction, mefenamic acid
SUMMARY
What is known and Objective: It has been reported that ibu-
profen interferes with the antiplatelet effect of low-dose aspi-
rin. This interaction is ascribed to steric hindrance at the
active site of cyclooxygenase-1 by ibuprofen, when aspirin is
administered after ibuprofen. However, whether other non-
steroidal anti-inflammatory drugs (NSAIDs) interact with aspi-
rin similarly is not well defined. The aim of this study was to
assess the influence of nine NSAIDs on the antiplatelet effect
of aspirin.
Methods: We investigated the antiplatelet effect of NSAIDs
using steady-state plasma concentration reported after usual
doses. We studied the in vitro antiplatelet effect of NSAID
alone, aspirin alone, aspirin before NSAID addition and aspi-
rin after NSAID addition to platelet-rich plasma. The rates of
platelet aggregation induced by collagen were determined.
The final concentration of aspirin used was the 50% effective
concentration (EC50) previously estimated in vitro.
Results and Discussion: Ibuprofen and mefenamic acid inter-
fere with the antiplatelet effect of aspirin when added before
the latter. The rate of platelet aggregation was reduced by 481%
and 227%, respectively. The other NSAIDs tested did not sig-
nificantly affect the aspirin antiplatelet effect when exposure
was prior to aspirin. None of the nine NSAIDs altered the aspi-
rin effect if administration followed that of aspirin.
What is new and Conclusion: Naproxen and flurbiprofen have
significant antiplatelet effects at plasma concentrations seen
with usual doses. Our in vitro model suggests that the anti-
platelet effect of aspirin is significantly diminished when
taken after, but not before, ibuprofen or mefenamic acid.
None of the other NSAIDs tested had any effect irrespective
of the timing of dosing.
WHAT IS KNOWN AND OBJECTIVE
It has been reported that non-steroidal anti-inflammatory drugs
(NSAIDs) interact with the antiplatelet effect of aspirin. NSAIDs
inhibit the enzyme cyclooxygenase (COX)-2 and exert anti-
Correspondence: Yasuhiko Yamada, Department of clinical
evaluation of drug efficacy, School of Pharmacy, Tokyo University
of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo
192-0392, Japan. Tel.: (+81) (426) 76 3046; fax: (+81) (426) 76 3069;
e-mail: yamada@ps.toyaku.ac.jp
2012 Blackwell Publishing Ltd 1
doi: 10.1111/j.1365-2710.2012.01373.
inflammatory effects. Furthermore, they depress the production
of prostaglandin (PG) by inhibition of COX-2,1 which is induced
by activation of macrophages, inflammatory mediators and
cytokines on synoviocytes. Because there is homology between
COX-2 and COX-1, most NSAIDs inhibit both, except for drugs
such as coxibs that selectively inhibit COX-2.2 Thus, it is known
that NSAIDs are reversible inhibitors of COX-1 on platelets.
However, none of the package inserts of marketed NSAIDs has
a clear description of potential antiplatelet effects. NSAIDs are
often given to patients with chronic inflammatory disease for
long periods. Therefore, co-administration of aspirin and NSA-
IDs may often occur with patients with vascular disease. Some
reports have suggested a reduction in inhibition of platelet
aggregation in patients who had taken ibuprofen before aspirin,
when compared with aspirin alone.3,4 The mechanism of this
interaction has been ascribed to steric hindrance at the active
site of COX-1 in platelets. Both ibuprofen and aspirin occupy
nearby sites in COX, and the presence of ibuprofen interferes
with aspirin binding.3 Furthermore, it has been speculated that
the effect of aspirin is not fully achieved with concomitant use
of ibuprofen, because the half-life of aspirin (240 78 min) is
shorter than that of ibuprofen (1080 60 min).5,6 Ibuprofen
binds in a reversible manner to COX-1, and thus, the antiplat-
elet effect induced by ibuprofen is expected to be reversible too.
In contrast, aspirin binds to COX-1 in an irreversible manner
and the antiplatelet effect continues for the lifetime of the plate-
lets when aspirin is administrated before ibuprofen. Thus, the
order of administration of these drugs is likely to be important
with respect to this interaction. A reduction by NSAIDs of the
antiplatelet effect of aspirin is likely to increase the risk of vas-
cular events. However, there are no reports of clinically impor-
tant interaction.
The aim of this study was to assess the in vitro influence of
commonly used NSAIDs on the antiplatelet effect of aspirin at
steady-state concentrations seen in vivo.
METHODS
Commercially available aspirin and nine NSAIDs were investi-
gated. We added each NSAID alone, aspirin alone, aspirin
before each NSAID and aspirin after each NSAID to platelet-
rich plasma (PRP), and the rates of collagen-induced platelet
aggregation were determined (Fig. 1). Next, we investigated the
antiplatelet effects of each NSAID and whether each had an
influence on the antiplatelet effect of aspirin.
Interaction of aspirin with NSAIDs H. Yokoyama et al.

NSAID alone NSAID Collagen

Measurement

Aspirin alone Aspirin Collagen

Measurement

Aspirin NSAID Aspirin NSAID Collagen

Measurement

NSAID Aspirin NSAID Aspirin Collagen

Measurement
Fig. 1. Determination of platelet aggre-
85 105 (min) gation following addition of each
0 1 2 3 4
NSAID alone, aspirin alone and various
combinations of aspirin and NSAIDs.

IDs was used. The steady-state concentration (Css) were calcu-

Materials
Blood samples were collected from six healthy volunteers (three
males, three females). PRP and platelet-poor plasma (PPP) were
prepared from blood collecting from the antecubital media vein
into VENOJECT II tubes (Terumo Medical CO., Tokyo, Japan).
Thereafter, the platelet count of the PRP sample was adjusted to
25 104/lL with PPP.
The drugs used were aspirin (Wako Pure Chemical Indus-
tries, Ltd., Osaka, Japan), loxoprofen trans-OH (the active
metabolite of loxoprofen, kindly provided by Daiichi Sankyo
Company, Japan), ibuprofen (Wako Pure Chemical Industries,
Ltd.), indomethacin (Wako Pure Chemical Industries, Ltd.), dic-
lofenac (Wako Pure Chemical Industries, Ltd.), etodolac (Sigma,
Tokyo, Japan), mefenamic acid (Wako Pure Chemical Industries,
Ltd.), meloxicam (Wako Pure Chemical Industries, Ltd.), nap-
roxen (Wako Pure Chemical Industries, Ltd.) and flurbiprofen
(MP Biomedicals LLC, Ohio, CA, USA). The platelet-aggregating
agent was collagen (Collagen Reagent Horm; Nycomed
Pharma GMBH, Ismaning, Germany) at a final concentration of
10 lg/mL. The final concentration of aspirin was 188 lm, which
we previously found the EC50 in vitro. The final concentrations
of NSAIDs were Css (Table 1). Because protein exists in PRP
and PPP, the concentration including the protein-bound NSA-
Table 1. Pharmacokinetic parameters of NSAIDs
Dose AUC0
Drug (mg) (lg/h/mL) MW
lated from the area under the plasma concentrationtime curve
(AUC0) after administration of a single dose of each NSAID6
14
and the dosing interval (s) of each was based on information
in the package insert.
Determination of platelet aggregation
Aggregation was measured simultaneously using light transmis-
sion (LT) and laser-light scattering methods to determine the
aggregate sizes and numbers with a PA-200 (Kowa Co., Ltd.,
Nagoya, Japan). Measurements produced by adding 30 lL of
collagen after 270 lL of PRP were used as blank values. In the
experiment with aspirin alone, measurements were then per-
formed after adding 30 lL of collagen, 2 min after the addition
of 2 lL of aspirin solution to PRP. A similar measurement proto-
col was used for each NSAID alone. When a NSAID was added
after aspirin to PRP, 2 lL of each NSAID solution was added
2 min after the addition of 2 lL of aspirin solution to PRP. Thirty
micro litres of collagen was added to the PRP at 2 min after
addition of the NSAID, and measurements were then made.
When a NSAID was added before aspirin, the protocol was the
same, with the timing of NSAID and aspirin additions switched.
We found in a preliminary study that there was no difference in
the rate of antiplatelet aggregation for reaction times between 2
and 4 min. The platelet aggregation reaction was evaluated as
the rate of antiplatelet aggregation, which was determined by
subtracting the value for the rate of aggregation of each sample
Css from the blank, divided by the value for the blank.
s (h) (lm)

Statistical analysis
Loxoprofen trans-OH 60 202 (8) 248 8 10
Ibuprofen 200 656 (7) 20628 8 398 Significance was determined using paired t-tests to compare
Indomethacin 50 96 (9) 35779 12 11 aspirin alone with aspirin after the addition of the NSAIDs
Diclofenac 25 0998 (10) 296 8 04 using the software package jmp 7 (SAS, Japan). A value of
Etodolac 200 611 (11) 28735 12 177 P < 005 was considered significant.
Mefenamic acid 250 1554 (12) 24129 6 107
Meloxicam 10 2279 (13) 3514 24 27
Naproxen 250 5605 (14) 23026 8 2434 RESULTS AND DISCUSSION
Flurbiprofen 40 208 (15) 24426 8 106
Antiplatelet effects of NSAIDs
Dose, reported dose (equal to standard dose in Japan); AUC0, area under
The rates of platelet aggregation inhibition of each NSAID are
the plasma concentrationtime curve after administration of a single dose;
MW, molecular weight; s, dosing interval; Css, steady-state concentration in shown in Fig. 2. Those of loxoprofen trans-OH, ibuprofen, indo-
plasma after administration of standard dose; NSAIDs, non-steroidal anti- methacin, diclofenac, etodolac, mefenamic acid and meloxicam
inflammatory drugs. were each <30%, whereas those of naproxen and flurbiprofen

2012 Blackwell Publishing Ltd Journal of Clinical Pharmacy and Therapeutics, 2012
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Interaction of aspirin with NSAIDs
100
Inhibition rate of platelet
aggregation (%)
Fig. 2. Rate of platelet aggregation inhi-
bition by NSAIDs. :Addition at Css of
NSAIDs, : addition at 1/10 of Css of
NSAIDs, values are mean SD (n = 6).
were 810 115% and 783 110%, respectively. In addition,
the rates of platelet aggregation inhibition of those latter two
drugs were determined by adding at 1/10 of Css and found to
be 73 45% and 85 85%, respectively.
Influence of addition of NSAIDs before or after aspirin on anti-
platelet effect of aspirin
Except for ibuprofen and mefenamic acid that decreased the
rate of platelet aggregation inhibition significantly by 481% and
227%, respectively, when given before aspirin, (Fig. 3), none of
the others had an effect, irrespective of the order of NSAID
administration.
DISCUSSION
In the present study, except for naproxen and flurbiprofen, the
tested NSAIDs had low antiplatelet effects, with rates of platelet
aggregation inhibition ranging from 45% to 261%. Naproxen
and flurbiprofen exhibited a significant antiplatelet effect follow-
ing oral administration at usual doses. In a previous study, the
COX-2/COX-1 of IC50 for naproxen and flurbiprofen were esti-
mated to be 17 and 12, respectively, whereas those for indo-
methacin and ibuprofen were estimated to be 27 and 24,
respectively.15 Thus, the authors considered that naproxen and
flurbiprofen at usual doses inhibited COX-1.
We found that most NSAIDs did not influence the antiplat-
elet effect of aspirin. However, the rate of platelet aggregation
inhibition by aspirin showed a significant decrease when aspirin
was added after ibuprofen (481%) and mefenamic acid (227%).
This is consistent with a report that the percentage of antiplat-
elet aggregation decreased when ibuprofen was given before
aspirin.3 There are few reports regarding the interaction
100
Inhibition rate of platelet
aggregation (%)
Fig. 3. Rate of platelet aggregation inhi-
bition following administration of ibu-
profen and mefenamic acid before or
after aspirin. Aspirin+NSAID, aspirin
before NSAID; NSAID + aspirin, aspirin
after NSAID. *P < 005, aspirin alone vs.
NASID + aspirin, paired t-test (n = 6).
2012 Blackwell Publishing Ltd
H. Yokoyama et al.
80
60
40
20
0
between mefenamic acid and aspirin. Although it is not clear
why mefenamic acid showed the interaction, this is likely to be
related to the structures of COX-1 and the drug.3
Catella-Lawson et al.3 reported that there were no interactions
noted between subjects who had taken aspirin (81 mg) before
ibuprofen (400 mg) and aspirin alone. So, it was speculated that
the antiplatelet effects were associated with aspirin in an irre-
versible manner. In contrast, 6 h after administration of ibupro-
fen on day 6, the rate of platelet aggregation inhibition was
approximately 35% in subjects who had taken the same drugs
in reverse order. These results demonstrated an interaction
among the drugs and also indicated an antiplatelet effect
induced by ibuprofen alone.
In another study, 6 h after a single administration of ibupro-
fen (200 mg), the plasma concentration of ibuprofen was
1765 lm.6 Because the dosage and AUC after administration of
ibuprofen showed a linear increase,16 the plasma concentration
of ibuprofen at 6 h after administration of ibuprofen (400 mg)
was calculated to be 353 lm. The rates of platelet aggregation
inhibition in vivo and in vitro were essentially the same at 35%
(concentration of ibuprofen, 353 lm) and 356% (concentration
of ibuprofen, 398 lm), respectively. Our in vitro results are
consistent with the in vivo results reported in the study of
Catella-Lawson et al.
A summary of previous studies of the interactions between
aspirin and NSAIDs is presented in Table 2.3,1722 Our results
were consistent with most of the previous studies in regard to
the interaction between ibuprofen and aspirin. However, one
report of diclofenac18 gave discordant results possibly due to
differences in drug concentrations studied. The concentrations
of diclofenac and aspirin in that report were lower than in our
study (diclofenac, 014 lm vs. 04 lm; aspirin 28 lm vs. 188 lm).
Ibuprofen Mefenamic acid
* 100
*
Inhibition rate of platelet
80 80
aggregation (%)
60 60
40 40
20 20
0 0
Aspirin Aspirin NSAID Aspirin Aspirin NSAID
+NSAID +Aspirin +NSAID +Aspirin
Journal of Clinical Pharmacy and Therapeutics, 2012
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Interaction of aspirin with NSAIDs H. Yokoyama et al.

Table 2. Summary of previous studies of interactions between aspirin and NSAIDs

NSAID Study schedule Method Result Subject population Ref

Diclofenac, 50 mg On day 1, NSAID only, then NSAID at 15:00, 23:00 TXB2 ND Healthy volunteers 17
and 7:00, and aspirin (80 mg) at 15:00 for 7 days
a 
Diclofenac, 014 lm NSAID followed by aspirin (28 lm) Closure time (PFA-100 ) SD (decrease) Healthy volunteers 18
Flurbiprofen, 100 mg Concurrent NSAID and aspirin (325 mg) Platelet aggregation ND Patients 19
Ibuprofen, 400 mg NSAID at 8:00, aspirin (81 mg) at 10:00 for 6 days Platelet aggregation SD (decrease) Patients 3
Ibuprofen, 800 mg On day 1, NSAID only, then NSAID at 15:00, 23:00 TXB2 SD (decrease) Healthy volunteers 17
and 7:00, an aspirin (80 mg) at 15:00 for 7 days
Ibuprofen, 400 mg NSAID followed by aspirin (325 mg) 2 h later Impedance SD (decrease) Healthy volunteers 20
Meloxicam, 15 mg From 1 to 4 days, NSAID alone, from 5 to 10 days, Platelet aggregation ND Healthy volunteers 21
NSAID followed by aspirin (100 mg) 2 h later
Naproxen, 500 mg NSAID at 8:00 and 20:00, aspirin (300 mg) at 10:00 Platelet aggregation ND Healthy volunteers 22
for 6 days
Rofecoxib 25 mg NSAID at 8:00, aspirin (81 mg) at 10:00 for 6 days Platelet aggregation ND Patients 3

SD, significant difference as compared with aspirin followed by NSAID; ND, no difference as compared with aspirin followed by NSAID; Ref, reference;
NSAIDs, non-steroidal anti-inflammatory drugs.
a
in vitro.

We consider that there is a risk of a decreased effect of aspi-
rin when administered after ibuprofen or mefenamic acid. Nota-
bly, patients with thrombotic infarction who are treated with
NSAIDs for pain relief immediately after a cardiac catheteriza-
tion post-test or post-operative stenting may not be receiving
appropriate antiplatelet therapy if NSAIDs such as ibuprofen or
mefenamic acid are administrated before aspirin. When a
patient begins therapy with aspirin after those NSAIDs, a wash-
out period is recommended. The washout period should be at
least 79 h with ibuprofen (t1/2: 108 min) and at least 1114 h
with mefenamic acid (t1/2: 162 min). Patients who are receiving
non-prescription NSAIDs when they begin to take aspirin
should be advised accordingly.
WHAT IS NEW AND CONCLUSION
Naproxen and flurbiprofen have significant antiplatelet effects
at plasma concentrations seen with usual doses. In addition, the
antiplatelet effect of aspirin is decreased significantly when aspi-
rin is taken after ibuprofen or mefenamic acid administration.

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