Coronary Heart Disease

Evaluation of Dose-Related Effects of Aspirin on

Platelet Function
Results From the Aspirin-Induced Platelet Effect (ASPECT) Study
Paul A. Gurbel, MD; Kevin P. Bliden, BS; Joseph DiChiara, BS; Justin Newcomer, MS;
Willy Weng, MS; Nagaraj K. Neerchal, PhD; Tania Gesheff, RN; Srivasavi K. Chaganti, MD;
Amena Etherington, MD; Udaya S. Tantry, PhD

Background—The antiplatelet effect of aspirin is attributed to platelet cyclooxygenase-1 inhibition. Controversy exists on
the prevalence of platelet resistance to aspirin in patients with coronary artery disease and effects of aspirin dose on
inhibition. Our primary aim was to determine the degree of platelet aspirin responsiveness in patients, as measured by
commonly used methods, and to study the relation of aspirin dose to platelet inhibition.
Methods and Results—We prospectively studied the effect of aspirin dosing on platelet function in 125 stable outpatients
with coronary artery disease randomized in a double-blind, double-crossover investigation (81, 162, and 325 mg/d for
4 weeks each over a 12-week period). At all doses of aspirin, platelet function was low as indicated by arachidonic acid
(AA)-induced light transmittance aggregation, thrombelastography, and VerifyNow. At any 1 dose, resistance to aspirin
was 0% to 6% in the overall group when AA was used as the agonist, whereas it was 1% to 27% by other methods
[collagen and ADP-induced light transmittance aggregation, platelet function analyzer (PFA-100)]. Platelet response to
aspirin as measured by collagen-induced light transmittance aggregation, ADP-induced light transmittance aggregation,
PFA-100 (81 mg versus 162 mg, Pⱕ0.05), and urinary 11-dehydrothromboxane B2 was dose-related (81 mg versus 325
mg, P⫽0.003). No carryover effects were observed.
Conclusions—The assessment of aspirin resistance is highly assay-dependent; aspirin is an effective blocker of
AA-induced platelet function at all doses, whereas higher estimates of resistance were observed with methods that do
not use AA as the stimulus. The observation of dose-dependent effects despite nearly complete inhibition of AA-induced
aggregation suggests that aspirin may exert antiplatelet properties through non– cyclooxygenase-1 pathways and
deserves further investigation. (Circulation. 2007;115:3156-3164.)
Key Words: aggregation 䡲 aspirin 䡲 coronary disease 䡲 platelets 䡲 resistance

M eta-analyses of clinical trials have indicated that aspirin

treatment of patients with vascular disease is associ-
ated with a 25% to 44% reduction in adverse cardiovascular
events.1,2 The antithrombotic effect of aspirin has been
primarily attributed to the irreversible blockade of the cyclo-
oxygenase-1 (COX-1) enzyme in platelets that leads to
attenuation in the production of an important platelet agonist,
thromboxane A2.1,3 In recent years, an increasing number of
reports about aspirin resistance has led to a growing concern
among clinicians and patients about the efficacy of aspirin
treatment. Various studies have evaluated the antiplatelet
effect of aspirin therapy and have reported the prevalence of
aspirin resistance to be between 0.4% to 35%.3 However,
these studies involved various doses of aspirin and methods
to assess aspirin response. Some methods used agonists that
can stimulate aggregation even in the presence of complete or
nearly complete inhibition of arachidonic acid (AA)-induced
aggregation. Currently no standardized or widely accepted
definition of aspirin resistance exists.3– 6 Therefore, specific
treatment recommendations for patients who exhibit high
platelet reactivity during aspirin therapy or who have poor
platelet inhibition by aspirin are not established.7
Clinical Perspective p 3164
Platelet responsiveness to aspirin during treatment with
325 mg/d aspirin was recently studied in patients who
underwent elective coronary stenting and patients with a
history of stent thrombosis by AA-induced light transmit-
tance aggregometry (LTA) and thrombelastography.8 Ap-
proximately 3% of the patients were found to be noncompli-
ant with aspirin therapy, all of whom were sensitive after

Continuing medical education (CME) credit is available for this article. Go to http://cme.ahajournals.org to take the quiz.
Received October 5, 2006; accepted April 23, 2007.
From the Sinai Center for Thrombosis Research, Baltimore, Md (P.A.G., K.P.B., J.D., T.G., S.K.C., A.E., U.S.T.), and the Department of Mathematics
and Statistics (J.N., W.W., N.K.N.), University of Maryland Baltimore County, Baltimore.
Correspondence to Paul A. Gurbel, MD, Sinai Center for Thrombosis Research, Hoffberger Bldg, Suite 56, 2401 W. Belvedere Ave, Baltimore, MD
21215. E-mail pgurbel@lifebridgehealth.org
© 2007 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.675587

Downloaded from http://circ.ahajournals.org/ at3156

CONS CALIFORNIA DIG LIB on March 16, 2015

in-hospital treatment. Finally, among the entire group only 1
patient (0.4%), who also had a history of stent thrombosis,
was resistant to aspirin treatment. In another study of patients
with a history of myocardial infarction, 9% of patients were
found to be noncompliant with aspirin therapy and only 1
patient was resistant to 325 mg aspirin therapy as measured
by AA-induced LTA.9 These studies suggest that platelet
resistance to aspirin is infrequent in patients administered 325
mg/d aspirin when measured by a laboratory method that is
directly dependent on platelet COX-1 activity.8,9 The high
prevalence of aspirin resistance as reported in previous
studies may in part be explained by the implementation of
assays that do not use AA as the agonist.
Although many investigations have focused on quantifying
the percentage of patients who display aspirin resistance, little
information is available on the effect of varying doses of
aspirin within the individual patient. In addition, limited
information is available to compare simultaneous analyses of
various commonly used assays. On the basis of our previous
study, we hypothesized that the prevalence of platelet resis-
tance to aspirin is rare at all doses when measured by methods
that use AA as the stimulus.8 Our primary aim was to
determine the degree of platelet responsiveness to aspirin as
measured by commonly used methods and study the relation
of dose to platelet inhibition.
Methods
Patients
The study was approved by the Western Institutional Review Board
(Olympia, Wash.) and all patients were ⬎18 years of age. A total of
125 outpatients with coronary artery disease (CAD) documented by
coronary angiography or ultrafast computed tomography scan were
enrolled in a single-center, double-blind, double-crossover study.
Overencapsulated, non– enteric-coated aspirin (Bayer HealthCare,
Morristown, NJ) was prepared on-site. In a 3-period 3-treatment
crossover that followed a Williams design, subjects were assigned to
1 of 6 treatment sequences (81, 162, and 325 mg/d for 4 weeks each
over a 12-week period) according to a randomization schedule
provided by Bayer HealthCare. All subjects, investigators, and
study-related staff were blinded to randomization and treatment
schedules.
Patients with a bleeding diathesis or a history of gastrointestinal
bleeding, hemorrhagic stroke, illicit drug or alcohol abuse, coagu-
lopathy, major surgery within 6 weeks before randomization, platelet
count ⬍100 000/mm3, hematocrit ⬍25%, creatinine ⬎4 mg/dL, or
current use of nonsteroidal antiinflammatory drugs, anticoagulants,
or antiplatelet drugs other than aspirin were excluded from the study.
Because stable CAD patients were studied and were already under
treatment with 81 to 325 mg/d aspirin at the time of enrollment,
discontinuation of aspirin treatment was deemed unethical, and patient
controls could not be studied. Therefore, a pilot study was conducted to
analyze 10 healthy volunteers, aged 28 to 47 years, free of pharmaco-
logical agent use for at least 1 month, to assess the validity of each assay;
these results were compared with the patient group.
Upon completion of each dose, the platelet response to aspirin was
determined by 1, 2, and 5 mmol/L AA-, 2 ␮g/mL collagen-, and 5
␮mol/L ADP-induced aggregation by LTA (Chronolog, Havertown,
Pa); 1 mmol/L AA-induced aggregation by thrombelastography (TEG,
Hemoscope Corp., Niles, Ill); VerifyNow (Accumetrics, San Diego,
Calif); PFA-100 with the collagen/epinephrine cartridge (Dade-Behring,
West Sacramento, Calif); 5 ␮mol/L ADP- and 1 mmol/L AA-induced
platelet expression of activated GPIIb/IIIa and P-selectin by flow
cytometry; and urinary 11-dehydro(dh)-thromboxane(Tx)B2 levels by
the AspirinWorks method (Corgenix, Denver, Colo). Patients were
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Gurbel et al Aspirin Effect on Platelets 3157
instructed to return their bottle of aspirin to count remaining capsules to
calculate percent compliance.
Blood and Urine Sampling
Blood was collected from the antecubital vein into Vacutainer tubes
(Becton-Dickinson, Franklin Lakes, NJ) between 8 AM and 12 PM on
the day of the last aspirin dose. Tubes that contained 3.2% trisodium
citrate were used for LTA and PFA-100; 17 USP/mL lithium heparin
tubes were used for TEG. In addition, 1 tube that contained 3.2%
sodium citrate (Greiner Bio-One Vacuette North America, Inc.,
Monroe, NC) was collected for VerifyNow measurements. After the
first 2 to 3 mL of free-flowing blood was discarded, the tubes were
filled to capacity and gently inverted 3 to 5 times to ensure complete
mixture of the anticoagulant. Urine samples were collected for
measurements of 11-dh-TxB2 at the end of each aspirin treatment
period into tubes that contained indomethacin (Corgenix, Denver,
Colo) and stored at ⫺70°C until analysis.
Light Transmittance Aggregometry
The blood-citrate tubes were centrifuged at 120g for 5 minutes to
recover platelet-rich plasma and further centrifuged at 850g for 10
minutes to recover platelet-poor plasma. The platelet-rich plasma
and platelet-poor plasma were stored at room temperature to be used
within 30 minutes. Platelet aggregation was assessed as described
previously.10 Briefly, platelets were stimulated with 5 ␮mol/L ADP,
2 ␮g/mL collagen, and 1, 2, and 5 mmol/L AA. Aggregation was
assessed with a Chronolog Lumi-Aggregometer (Model 490 to 4D)
with the Aggrolink software package. Aggregation was expressed as
the maximum percent change in light transmittance from baseline,
with platelet-poor plasma used as a reference.
Thrombelastograph PlateletMapping Assay
The TEG Hemostasis Analyzer with PlateletMapping assay relies on
the measurement of thrombin-induced clot strength to enable a
quantitative analysis of platelet function.11 In the PlateletMapping
assay, heparin is used to eliminate thrombin activity in the sample.
Reptilase and factor XIIIa are used to generate a cross-linked fibrin
clot to isolate the fibrin contribution to the clot strength.11,12 The
contribution of the cyclooxygenase pathway to clot formation can be
measured by the addition of 1 mmol/L AA. Blood samples were
analyzed according to the manufacturer’s instructions as described
previously.8
VerifyNow Aspirin Assay
VerifyNow is a turbidimetric-based optical detection system that
measures platelet aggregation in whole blood.13 The cartridge
contains a lyophilized preparation of human fibrinogen-coated
beads, AA, preservative, and buffer. The assay is designed to
measure platelet function on the basis of the ability of activated
platelets to bind to fibrinogen after stimulation. The instrument
measures an optical signal, reported as aspirin reaction units.
PFA-100 Assay
The PFA-100 point-of-care assay utilizes a cartridge that contains a
capillary, a sample reservoir, a collagen/epinephrine-coated mem-
brane, and an aperture that exposes platelets to high shear condi-
tions.14 When platelets in whole blood come in contact with
epinephrine and collagen, they become activated and aggregate at the
aperture, and thus gradually reduce and finally arrest blood flow. The
PFA-100 records the time in seconds from the start of the test until
the platelet plug occludes the aperture (closure time).
Flow Cytometry
The expression of platelet GPIIb/IIIa and P-selectin were determined
by whole blood flow cytometry with a multicolor analysis method
that used the following monoclonal antibodies: fluorescein
isothiocyanate-conjugated PAC-1 (recognizes the active GPIIb/IIIa
receptor), R-phycoerythrin-conjugated CD41a (recognizes the total
GPIIb/IIIa receptor population), and phycoerythrin-cyanine5-
3158 Circulation June 26, 2007

conjugated CD62p (recognizes P-selectin). All antibodies were TABLE 1. Patient Demographics
obtained from BD Biosciences (San Diego, Calif). ADP- and
AA-induced expression of GPIIb/IIIa receptors and P-selectin were Patients (n⫽120)
measured as described previously.10 Age, y 65⫾10
Male, n (%) 80 (70)
Urinary 11-dh-TxB2
The AspirinWorks ELISA assay has been described elsewhere.15 Weight, lbs 196⫾51
Briefly, 100 ␮L of urine in assay buffer was incubated with a Systolic BP, mm Hg 130⫾18
monoclonal antibody followed by the addition of 11-dh-Tx B2- Diastolic BP, mm Hg 71⫾6
alkaline phosphate tracer. Urinary 11-dh-TxB2 concentrations were
determined by measurement of color development at 405 nm with an Ethnicity, n (%)
ELISA reader and expressed as pg/mg creatinine. White 79 (69)
Black 32 (28)
Aspirin Resistance Definitions
Asian 3 (3)
Aspirin resistance definitions included ⱖ20% AA-, ⱖ70% ADP-,
and ⱖ70% collagen-induced aggregation by LTA;16 ⱖ50% AA- Risk factors/past medical history, n (%)
induced whole blood aggregation by TEG;8 ⱖ550 aspirin reaction Smoking (previous or current) 46 (40)
units by VerifyNow;13 ⱕ193 seconds by PFA-100;14 and upper
quartile pg 11-dh-TxB2/mg creatinine during treatment with 81 mg.17 Family history of CAD 40 (35)
Hypertension 73 (64)
Statistical Analysis Hyperlipidemia 100 (87)
The PFA-100 is the most widely studied method to assess aspirin
Diabetes mellitus 30 (26)
resistance, and ⬇20% of patients with stable CAD were resistant to
100 mg/d aspirin therapy as assessed by this device.18 Gum et al PVD 9 (8)
found that 5% of patients were resistant to aspirin by 1.6 mmol/L Prior MI 25 (22)
AA-induced platelet aggregation.16 Therefore, the primary outcome
Prior CABG 36 (32)
of the present study was to observe a statistically significant
difference in measurements of aspirin resistance with PFA-100 and Prior PCI 39 (34)
2 mmol/L AA-induced LTA. Prior CVA 6 (5)
The present study used a 3-period 3-treatment crossover design.
Each subject was randomized to 1 of 6 treatment sequences and Medications, n (%)
administered doses of aspirin (81, 162, and 325 mg) according to ␤-Blockers 69 (61)
their treatment sequence. For a given subject, measurements of ACE inhibitors 57 (50)
aspirin resistance were recorded over the 3 treatment periods for each
dose level. As a result, the observations within a given subject Calcium channel blockers 20 (18)
became correlated, which caused the assumptions of the usual Lipid-lowering therapy 96 (84)
ANOVA to be violated. Therefore, the analysis performed here used Laboratory data
a repeated measures logistic regression with a random subject effect
to account for the dependencies from the crossover design. Car- WBC, ⫻1000/mm3 6.4⫾2.1
ryover effects were included in the model because washout periods Platelets, ⫻1000/mm3 237⫾62
were not possible. Carryover effects were modeled with dummy
Hemoglobin, g/dL 13.7⫾2.2
variables (0 or 1) to specify the order in which patients received the
different treatments (ie, if a patient received a dosage of 81 mg in Hematocrit, % 40.5⫾5.0
period 1, an effect for carryover from 81 mg would be included for Creatinine, g/dL 1.0⫾0.2
that patient in period 2). Wald t tests were used to analyze
differences between dosage levels (81 mg versus 162 mg, 81 mg Data are expressed as mean⫾SD unless otherwise indicated. BP indicates
versus 325 mg, and 162 mg versus 325 mg) and to correlate blood pressure; CAD, coronary artery disease; PVD, peripheral vascular
resistance measured by 2 mmol/L AA-induced LTA with resistance disease; MI, myocardial infarction; CABG, coronary artery bypass grafting; PCI,
measured by all other methods.19 In addition, Wald ␹2 tests were percutaneous coronary intervention; CVA, cerebrovascular accident; ACE,
used to test the overall difference between dosage levels and overall angiotensin converting enzyme; and WBC, white blood cells.
carryover effect.
An unpaired t test was used to compare measurements of platelet
function between patients and healthy volunteers at all doses;
P⬍0.05 was considered significant. Statistical analyses were per-
formed with SAS version 9.1.3. (SAS Institute Inc, Cary, NC). were terminated from the study after completion of at least 1
The authors had full access to and take full responsibility for the dose for the following reasons: transient ischemic attack,
integrity of the data. All authors have read and agree to the gastritis, self-withdrawal (n⫽2 patients), and inability to
manuscript as written. obtain blood. Overall compliance with daily aspirin therapy
was 98%.
Results
Patients
Patient demographics are shown in Table 1. Briefly, the Primary Outcome
majority of patients were elderly white men with a history of A statistically significant difference was observed between
hyperlipidemia who received standard medical treatment for the prevalence of aspirin resistance measured by the PFA-100
CAD, such as statins, ␤-blockers, and angiotensin-converting and 2 mmol/L AA-induced LTA at all doses of aspirin (at 81
enzyme inhibitors. Ninety-six percent (n⫽120 patients) of the mg, n⫽32 versus n⫽2, P⬍0.001; at 162 mg, n⫽14 versus
study population completed platelet testing at all 3 aspirin n⫽0, P⬍0.001; and at 325 mg, n⫽21 versus n⫽0, P⬍0.001,
doses, and data on these patients are reported. Five patients respectively) (Table 2).
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TABLE 2. Effects of Assay and Dose on Measurement of versus 81 mg” are also negative, which indicates a decrease in
Aspirin Resistance the odds of observing resistance for the higher dose. The
Resistance (n)
columns of probability values indicate that the difference is
not statistically significant for some of the methods, which
ⱖ1 2 3 points to possible differences among the methods to detect
81 mg 162 mg 325 mg Dose Doses Doses aspirin resistance. In addition, Table 3 presents the Wald t
LTA statistics (t), standard errors (SE), and the probability values
1 mmol/L AA 1 0 0 1 0 0 for each test that correspond to these estimates. Table 4
2 mmol/L AA 2 0 0 2 0 0
presents the results of the tests for the carryover effects from
each of the 3 dosage levels. For completeness, Tables 3 and
5 mmol/L AA 2 1 0 2 1 0
4 also include ␹2 tests and probability values that test the
2 ␮g/mL Collagen 12 2 1 14 1 0 overall treatment and carryover effects. Results are further
5 ␮mol/L ADP 19 11 10 27 7 3 described under each method.
TEG-1 mmol/L AA 5 3 5 11 2 0
VerifyNow 7 4 4 13 2 0 AA-Induced Platelet Aggregation Measured
Urinary 11-dehydro- 31 22 14 42 16 5 by LTA
Thromboxane B2 Aspirin resistance was rare at all doses (Figure 1, Table 2).
The treatment and carryover effects of aspirin dosing on
PFA-100 32 14 21 42 15 5
AA-induced platelet aggregation are analyzed in Tables 3 and
4. AA-induced aggregation was low in patients, and no
Analysis of Aspirin Treatment Effects and additional reduction in aggregation occurred at doses ⬎81 mg
Carryover Effects (Table 5). One patient was resistant at 1 mmol/L AA-LTA
Table 3 presents the estimated difference in resistance be- while on 81 mg aspirin. At 2 mmol/L AA-LTA, the latter
tween aspirin doses. For the logistic regression model, this patient and another patient were resistant while on 81 mg
estimated difference corresponds to an estimate of the log of aspirin. At 5 mmol/L AA-LTA, these 2 patients were resistant
the odds ratio between 2 doses. The odds ratio is the ratio of at 81 mg and 1 of these 2 patients was resistant at 162 mg. No
the probability that resistance is present to the probability that patients were found to be aspirin resistant during treatment
resistance is not present. For consistency, each odds ratio with 325 mg daily aspirin (Figure 1).
refers to the odds for the higher dose divided by the odds for
the lower dose. For example, the first column of Table 3 AA-Induced Platelet Aggregation Measured
corresponds to the log of the odds ratio for the patients who by TEG
In total, 11 patients were resistant at ⱖ1 dose of aspirin; 5,
received 162 mg versus 81 mg. Similarly, the columns titled
3, and 5 patients were aspirin-resistant during treatment
“325 mg versus 81 mg” and “325 mg versus 162 mg” provide
with 81 mg, 162 mg, and 325 mg, respectively (Table 2).
the comparisons between 325 mg versus 81 mg and 325 mg
One patient was resistant at both 81 mg and 162 mg, and
versus 162 mg. A log of the odds ratio of 0 would charac-
another patient was resistant at both 162 mg and 325 mg;
terize that the odds of aspirin resistance for 2 different doses none were resistant at all 3 doses (Table 2). Similar to
are equal, whereas a negative value would indicate that a LTA, aggregation was low; treatment and carryover effects
decrease in resistance occurs as the dose increases. Positive of aspirin doses on AA-induced platelet aggregation were
values are similarly interpreted. For example, the estimate for not observed (Tables 3 to 5), and aspirin resistance was
5 ␮mol/L ADP in the 162 mg versus 81 mg column of Table infrequent at all doses (Table 2).
3, ⫺1.2104, indicates that the odds of resistance measured by
5 ␮mol/L ADP LTA when given 162 mg of aspirin is less VerifyNow Aspirin Assay
than the odds of resistance measured by the 5 ␮mol/L ADP In total, 13 patients were resistant at ⱖ1 dose of aspirin; 7, 4,
when given 81 mg. The log of the odds ratios for “325 mg and 4 patients were aspirin-resistant during treatment with 81

TABLE 3. Analysis of Aspirin Treatment Effects

Overall
162 mg vs 81 mg 325 mg vs 81 mg 325 mg vs 162 mg Treatment

Method Estimated SE t P Estimated SE t P Estimated SE t P ␹2 P

LTA
5 ␮mol/L ADP ⫺1.2104 0.6107 ⫺1.98 0.049 ⫺1.1307 0.6078 ⫺1.86 0.065 0.0797 0.6064 0.13 0.896 4.92078 0.085
2 ␮g/mL collagen ⫺2.9794 1.1674 ⫺2.55 0.012 ⫺3.7225 1.4375 ⫺2.59 0.011 ⫺0.743 1.2643 ⫺0.59 0.558 8.55392 0.014
2 mmol/L AA ⫺2.9012 4.3951 ⫺0.66 0.510 ⫺6.5075 7.0303 ⫺0.93 0.357 ⫺3.6063 5.8005 ⫺0.63 0.535 0.88346 0.643
TEG-1 mmol/L AA ⫺1.3392 1.0162 ⫺1.32 0.190 ⫺0.5896 1.073 ⫺0.55 0.584 0.7496 0.9598 0.78 0.437 1.8054 0.405
PFA-100 ⫺1.6059 0.4912 ⫺3.27 0.001 ⫺0.9091 0.4472 ⫺2.03 0.044 0.6968 0.4684 1.49 0.140 10.8833 0.004
VerifyNow ⫺0.8034 0.7557 ⫺1.06 0.290 ⫺0.6159 0.8032 ⫺0.77 0.445 0.1875 0.8253 0.23 0.821 1.24041 0.538
Urinary thromboxane ⫺0.7756 0.4438 ⫺1.75 0.083 ⫺1.5271 0.4951 ⫺3.08 0.003 ⫺0.7515 0.4749 ⫺1.58 0.116 9.58268 0.008

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3160 Circulation June 26, 2007

TABLE 4. Analysis of Carryover Effects

Carryover From 81 mg Carryover From 162 mg Carryover From 325 mg Overall Carryover

Method Estimated SE t P Estimated SE t P Estimated SE t P ␹2 P

LTA
5 ␮mol/L ADP ⫺0.9391 0.6908 ⫺1.36 0.177 ⫺0.9806 0.6504 ⫺1.51 0.134 ⫺0.6823 0.6233 ⫺1.09 0.276 3.65743 0.301
2 ␮g/mL collagen 0.9348 1.2637 0.74 0.461 ⫺1.6206 0.9107 ⫺1.78 0.078 ⫺1.11 0.7831 ⫺1.42 0.159 4.89409 0.180
2 mmol/L AA 2.9798 4.3875 0.68 0.498 0.06461 1.4716 0.04 0.965 ⫺3.5324 6.0881 ⫺0.58 0.563 0.84129 0.840
TEG-1 mmol/L AA 2.2977 1.1178 2.06 0.060 ⫺0.4254 1.2213 ⫺0.35 0.728 0.871 1.0994 0.79 0.430 5.48605 0.139
PFA-100 0.1525 0.496 0.31 0.759 ⫺0.0318 0.4615 ⫺0.07 0.945 ⫺0.6994 0.515 ⫺1.36 0.177 2.23757 0.525
VerifyNow ⫺0.7626 0.9175 ⫺0.83 0.408 ⫺1.1865 0.9233 ⫺1.29 0.201 ⫺0.263 0.7559 ⫺0.35 0.729 2.07524 0.557
Urinary thromboxane 0.2431 0.4922 0.49 0.622 ⫺0.2994 0.5086 ⫺0.59 0.557 ⫺0.2982 0.4752 ⫺0.63 0.532 1.15715 0.763

mg, 162 mg, and 325 mg, respectively (Table 2). Two 162 mg and 325 mg (Tables 3 and 5). No carryover effects
patients were resistant at both 81 mg and 162 mg doses; none between aspirin dosing were observed (Table 4).
were resistant at all 3 doses (Table 2). Significant treatment or
carryover effects were not observed (Tables 3 and 4). ADP-Induced Platelet Aggregation Measured
by LTA
Urinary 11-dh-TxB2 Overall, 27 patients were resistant at ⱖ1 dose of aspirin;
In total, 42 patients were resistant at ⱖ1 dose of aspirin; 31, 19, 11, and 10 patients were aspirin-resistant during
22, and 14 patients were aspirin-resistant during treatment treatment with 81 mg, 162 mg, and 325 mg, respectively
with 81 mg, 162 mg, and 325 mg, respectively (Table 2). (Table 2). In total, 7 patients were resistant at 2 aspirin
Sixteen patients were resistant at 2 doses, and 5 patients were doses, and 3 patients were resistant at all 3 doses of aspirin
resistant at all 3 doses (Table 2). An incremental treatment (Table 2). A significant effect of aspirin treatment on
effect of aspirin dosing on urinary 11-dh-TxB2 levels was ADP-induced platelet aggregation was observed between
observed in patients, with significant differences in levels 81 mg and 162 mg (P⫽0.049), with no further inhibition
(P⫽0.003) demonstrated between 81 mg and 325 mg (Tables demonstrated between 162 mg and 325 mg (Tables 3 and
3 and 5), uninfluenced by carryover effects (Table 4). 5). No carryover effects between aspirin dosing were
observed (Table 4).
Collagen-Induced Platelet Aggregation Measured
by LTA PFA-100 Assay
In total, 14 patients were resistant at ⱖ1 dose of aspirin; 12, With the PFA-100 method, 42 patients were found to be
2, and 1 patients were aspirin-resistant during treatment with aspirin-resistant at ⱖ1 dose of aspirin; 32, 14, and 21
81 mg, 162 mg, and 325 mg, respectively (Table 2). Only 1 patients were aspirin-resistant during treatment with 81
patient was resistant at both 81 mg and 325 mg aspirin doses; mg, 162 mg, and 325 mg, respectively (Table 2). In total,
none were resistant at all 3 doses (Table 2). A significant 15 patients were resistant at any 2 aspirin doses, and 5
effect of aspirin treatment on collagen-induced platelet ag- patients were resistant at all 3 doses of aspirin (Table 2).
gregation was observed between 81 mg and 162 mg Interestingly, treatment with 325 mg aspirin was associ-
(P⫽0.012) with no further inhibition demonstrated between ated with shorter closure times and increased rates of

100

90

80
Figure 1. Individual platelet aggregation data mea-
70
Aggregation (%)

sured after stimulation by 3 concentrations of AA

60 by LTA at 3 different doses of aspirin. At 1 mmol/L
AA-LTA, 1 patient (␱) while on 81 mg aspirin was
50 resistant. At 2 mmol/L AA-LTA, this patient and
another patient (▫) were resistant while on 81 mg
40
aspirin. At 5 mm AA-LTA, these 2 patients at 81
Resistance
30 mg were resistant and 1 of these 2 patients was
resistant at 162 mg. No patients were found to be
20 aspirin resistant during treatment with 325 mg
10
aspirin daily. Dashed line indicates cut point for
resistance. AA indicates arachidonic acid; LTA,
0 light transmittance aggregometry.
81 mg

162 mg

162 mg

162 mg
325 mg

81 mg

325 mg

81 mg

325 mg

1 mmol AA 2 mmol AA 5 mmol AA

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 Gurbel et al Aspirin Effect on Platelets 3161

TABLE 5. Platelet Function in Healthy Volunteers and Patients

Healthy
Volunteers CAD Patients

No ASA 81 mg ASA 162 mg ASA 325 mg ASA

(n⫽10) (n⫽120) (n⫽120) (n⫽120)
LTA, % aggregation
5 ␮mol/L ADP 70⫾10 58⫾13* 58⫾10* 58⫾10*
2 ␮g/mL collagen 80⫾13 38⫾26* 29⫾2* 27⫾17*
1 mmol/L AA 77⫾10 3⫾9* 3⫾2* 3⫾2*
2 mmol/L AA 80⫾11 6⫾13* 4⫾5* 4⫾2*
5 mmol/L AA 78⫾5 7⫾15* 5⫾7* 4⫾2*
TEG, % aggregation
1 mmol/L AA 95⫾9 12⫾18* 11⫾16* 9⫾8*
VerifyNow, ARU 627⫾39 454⫾58* 434⫾44* 428⫾45*
PFA-100 closure time, secs 142⫾39 222⫾73* 257⫾63* 240⫾68*
Urinary thromboxane, pg 614⫾108 378⫾182* 321⫾129* 291⫾13*
11-dh-TxB2/mg Cr
Data are expressed as mean⫾SD. ASA indicates aspirin.
*Pⱕ0.005 for healthy volunteers compared to CAD patients.

resistance compared with 162 mg aspirin (Tables 2 and 5). significant correlations existed between 2 mmol/L AA-
Like collagen- and ADP-induced platelet aggregation, induced LTA and any other methods of aspirin resistance
there was a statistically significant treatment effect, unin- assessment.
fluenced by carryover effects, between 81 mg and 162 mg
as determined by PFA-100 (P⫽0.001), and no further Flow Cytometry
treatment effect was observed between 162 mg and 325 mg A comparison of platelet GPIIb/IIIa and P-selectin expression
(Tables 3 to 5). in patients and healthy volunteers is shown in Figure 2.
Nonstimulated total GPIIb/IIIa expression was nonsignifi-
Comparison of Platelet Function Measurements in cantly higher in patients versus healthy volunteers
Healthy Volunteers and Patients (P⫽0.134), whereas activated GPIIb/IIIa reached signifi-
A significant effect of aspirin treatment in patients compared cance (P⫽0.033). AA-stimulated expression of total and
with healthy volunteers who were not given aspirin at all activated GPIIb/IIIa was significantly lower in patients
doses was observed by use of ADP-, collagen-, and AA- treated with aspirin as compared with healthy volunteers
induced LTA, AA-induced TEG, VerifyNow, PFA-100, and (Pⱕ0.0001) (Figure 2A). Despite potent inhibition of AA–
urinary thromboxane measurements (Table 5; Pⱕ0.05 for all induced aggregation by aspirin therapy, we observed promi-
measurements). nent activation of GPIIb/IIIa and P-selectin expression after
AA stimulation.
Correlation Between Methods ADP-stimulated expression of total GPIIb/IIIa was nonsig-
Table 6 compares the log of the odds ratios of resistance for
nificantly higher (P⫽0.108) in patients treated with aspirin as
the various assays with 2 mmol/L AA LTA. With the
compared with healthy volunteers, whereas expression of
exception of 1 mmol/L and 5 mmol/L AA-induced LTA, no
activated GPIIb/IIIa reached significance (P⫽0.02) (Figure
2B). Nonstimulated P-selectin expression was nonsignifi-
TABLE 6. Analysis of Method Effects cantly higher in patients versus healthy volunteers (P⫽0.06).
Difference from LTA (2 mmol/L AA) AA-stimulated P-selectin expression was lower in patients
than in healthy volunteers (P⬍0.0001). In contrast, ADP-
Method Estimated SE t P
stimulated P-selectin expression remained higher in patients
LTA than in healthy volunteers (P⫽0.032) (Figure 2C) despite
1 mmol/L AA ⫺1.1185 1.1633 ⫺0.96 0.3383 lower aggregation (Table 5). A dose-related effect of aspirin
5 mmol/L AA 0 0.8283 0 1 therapy was not observed by any flow cytometric measure-
2 ␮g/mL collagen 1.7194 0.6472 2.66 0.009 ment (Figure 2).
5 ␮mol/L ADP 2.9707 0.6138 4.84 ⬍0.0001
TEG-1 mmol/L AA 1.7194 0.6472 2.66 0.009 Discussion
VerifyNow 1.7194 0.6472 2.66 0.009
The present study assessed platelet responsiveness to 3
different, frequently used aspirin doses by various commonly
Urinary 11-dehydro-thromboxane B2 3.609 0.6079 5.94 ⬍0.0001
used assays in patients with CAD. Our study was in part
PFA-100 3.7205 0.6074 6.13 ⬍0.0001
stimulated by the controversy that surrounds the relationship
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3162 Circulation June 26, 2007
of aspirin dose to treatment effect.20,21 As a double-crossover
study, each patient served as his or her own control. The
results of the study have several significant implications in
the interpretation of platelet responsiveness to aspirin and
resistance. First, the estimation of aspirin resistance is highly
assay-dependent. Assays that use AA to stimulate platelet
aggregation (LTA, TEG, VerifyNow) result in resistance
estimates that are lower than methods that use stimuli other
than AA. Second, aspirin is a highly effective inhibitor of
AA-induced aggregation. However, despite complete or near-
complete inhibition of AA-induced aggregation at all agonist
concentrations and at all doses of aspirin, platelet aggregation
stimulated by other agonists can be robust. Third, the platelet
response to aspirin as measured by collagen- and ADP-
induced LTA, PFA-100, and urinary thromboxane was dose-
related. Last, the overall variability in the measurement of
aspirin resistance was most notable for assays that did not use
AA as the stimulus.
High Aspirin Efficacy in the Inhibition of
AA-Induced Aggregation
Earlier studies demonstrated that repeated 50 to 100 mg
daily doses of aspirin were associated with highly effective
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Figure 2. A, Total GPIIb/IIIa expression in
patients and healthy volunteers measured by
flow cytometry before and after stimulation
with 1 mmol/L AA and 1 ␮mol/L ADP at 3
different doses of aspirin. Data are reported
as mean fluorescence intensity (MFI).
*Pⱕ0.05 for difference in MFI values between
healthy volunteers and patients. B, Activated
GPIIb/IIIa expression in patients and healthy
volunteers measured by flow cytometry
before and after stimulation with 1 mmol/L
AA and 1 ␮mol/L ADP at 3 different doses of
aspirin. Data are reported as MFI. *Pⱕ0.05
for difference in MFI values between healthy
volunteers and patients. C, P-selectin expres-
sion in patients and healthy volunteers mea-
sured by flow cytometry before and after
stimulation with 1 mmol/L AA and 1 ␮mol/L
ADP at 3 different doses of aspirin. Data are
reported as % positive cells. *Pⱕ0.05 for dif-
ference in % positive cells between healthy
volunteers and patients.
inhibition of COX-1 as demonstrated by ⬎95% inhibition
of serum TxB2.1 Maximum inhibition of serum TxB2 can
also be attained within 4 hours by a single 325-mg aspirin
dose.22 Our results agree with these observations. Maxi-
mum inhibition of COX-1 activity was present at the
lowest aspirin dose as measured by AA-induced aggrega-
tion in platelet-rich plasma at 3 different agonist concen-
trations. When the cut points for resistance defined by the
manufacturers are used, the prevalence of resistance was
low by all 3 methods that measured platelet aggregation
after stimulation by AA (LTA, TEG, VerifyNow). The
results also agree with a previous study by our group that
demonstrated the high efficacy of a 325-mg aspirin dose in
the suppression of AA-induced aggregation in patients
who underwent coronary stenting.8
Previous investigations with VerifyNow have reported a
higher prevalence of aspirin resistance than the present
investigation.13,23 It is interesting that these earlier inves-
tigations used cationic propyl gallate as the agonist instead
of AA. The use of AA as the agonist in the present
investigation resulted in low estimates of resistance by
VerifyNow measurements. The presence of uninhibited
COX-2 in whole blood that contributed to thromboxane A2

production may explain a higher prevalence of resistance
as compared with LTA.24 The measurement of urinary
thromboxane also suggested a dose-related effect with
superior reduction in levels observed with doses ⬎81 mg.
Because urinary thromboxane levels indicate total in vivo
thromboxane production, an unblocked COX-2 source may
partially explain our findings.6,25
Platelet Aggregation Despite COX-1 Inhibition
Our results agree with prior studies that demonstrated the
presence of residual platelet reactivity to agonists other than
AA in patients treated with aspirin.26 –29 We have referred to
the latter methods as COX-1–nonspecific because they may
not directly indicate residual COX-1 activity. The presence of
high platelet aggregation in the latter methods has been
interpreted as evidence for aspirin resistance.30 Our study,
which used various concentrations of AA, suggests that
platelet COX-1 activity is inhibited in the majority of pa-
tients. Our results therefore argue against residual COX-1
activity as the primary explanation for aspirin resistance as
indicated by the presence of high platelet reactivity to either
collagen or ADP, or rapid shear-induced aggregation as
measured by PFA-100.
Because the currently accepted explanation for the antithrom-
botic effect of aspirin is the inhibition of COX-1, aspirin
resistance has been defined as the presence of persistent COX-1
activity during aspirin therapy.30 However, our data suggest that
aspirin may influence targets in addition to COX-1 in platelets.
Consideration should be given to the observed dose-dependent
effects of aspirin in the present study when platelet responsive-
ness to aspirin is evaluated in future studies.
COX-1–Independent Effects of Aspirin
The occurrence of lower collagen-, ADP-, and shear-induced
aggregation in patients treated with aspirin as compared with
healthy volunteers clearly demonstrates that aspirin plays an
important role in the modification of the response to these
stimuli. Our findings are consistent with numerous prior
reports that confirm the same.26 –29 However, the dose-related
effect of aspirin observed after stimulation by collagen in the
presence of complete or near-complete inhibition of AA-
induced aggregation suggests that aspirin may exert antiplate-
let effects beyond acetylation of COX-1. Persistent
thromboxane production after collagen stimulation cannot be
ruled out on the basis of our study; however non–COX-1–
mediated effects deserve further investigation. Interestingly,
maximum inhibition of aggregation stimulated by collagen
and shear occurred at a 162-mg dose of aspirin with no further
reduction observed at the 325-mg dose.
Flow Cytometry as a Method to Assess
Aspirin Responsiveness
Our study demonstrates that, despite aspirin therapy, patients
exhibit higher platelet reactivity than healthy volunteers not
on aspirin therapy. The latter conclusion is supported by
higher levels of circulating active platelets as measured by
both P-selectin and activated GPIIb/IIIa expression on non-
stimulated platelets as well as greater expression of these
receptors after ADP stimulation. Interestingly, we observed
an uncoupling of the relationship between platelet function
and receptor expression in patients on aspirin therapy. De-
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Gurbel et al Aspirin Effect on Platelets 3163
spite greater expression of P-selectin and activated GPIIb/IIIa
after ADP stimulation in patients, lower ADP-induced aggre-
gation was observed than in healthy volunteers. Moreover,
only partial inhibition of AA-induced expression of activated
GPIIb/IIIa and P-selectin occurred in patients treated with
aspirin, although platelet function was profoundly inhibited.
The former findings suggest that aspirin therapy may inhibit
the function of the activated GPIIb/IIIa receptor. The latter
data also suggest that AA stimulation may directly increase
P-selectin and activated GPIIb/IIIa expression through a
mechanism independent of COX-1.
Limitations
Pretreatment studies could not be conducted because all
patients had CAD and were therefore on aspirin therapy at the
time of enrollment. The patients in the present investigation
will be followed to track the development of clinical events
that will be correlated with their laboratory findings. Finally,
although in vitro thromboxane generation was not measured,
the observation of very low platelet aggregation induced by
various concentrations of AA lends support to the conclusion
that aspirin is highly effective in the blockade of COX-1. The
primary goal of our study was to assess commonly used
platelet functional measurements. The present study repre-
sents an exhaustive analysis that addresses platelet response
to multiple doses of aspirin with the use of common func-
tional assays. In the present study we determined 11-dh-TxB2
levels, a method that has been linked to clinical events in the
Heart Outcomes Prevention Evaluation (HOPE) Study.17
In conclusion, aspirin is a highly effective blocker of AA-
induced platelet function in stable outpatients with CAD. The
prevalence of aspirin resistance was found to be highly assay-
dependent, with significantly higher measurements of resistance
found with methods that use agonists other than AA. The
observation of dose-related effects despite near-complete inhi-
bition of AA-induced aggregation suggests that aspirin may also
exert antiplatelet effects through non–COX-1 pathways. In
addition, despite greater ADP-induced expression of activated
GPIIb/IIIa in patients treated with aspirin than in healthy
volunteers, platelet function in patients was lower, which sug-
gests that aspirin therapy may inhibit the function of the
activated GPIIb/IIIa receptor. Finally, our observations suggest
that AA may also stimulate platelet activation through a COX-
1–independent pathway. Further investigations are required to
better define the underlying mechanisms for the antithrombotic
effects of aspirin. A clearer understanding of the mechanistic
effects of aspirin is necessary to establish a uniform definition of
aspirin “resistance”.
Sources of Funding
The study was supported by Bayer HealthCare LLC, Morristown,
NJ, and Sinai Hospital of Baltimore, Md.
Disclosures
Dr Gurbel has received research grants and honoraria from Hemo-
scope, AstraZeneca, Schering Plough, Medtronic, Lilly/Sankyo,
Sanofi, Boston-Scientific, and Bayer. The remaining authors report
no conflicts.
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CLINICAL PERSPECTIVE
Controversy surrounds the optimal dose of aspirin for the treatment of patients with coronary artery disease and the concept
of resistance to aspirin therapy. Currently, aspirin is administered without assessment of its effect on platelet function.
Low-dose daily aspirin is a uniformly accepted strategy for secondary prevention in patients with cardiovascular disease.
However, prospective information is limited on potential dose-related platelet effects of aspirin and their relation to clinical
outcomes. In the present study we assessed platelet responsiveness to 3 different frequently used aspirin doses by various
assays in patients with stable coronary artery disease. We demonstrated that aspirin at all doses is a highly effective blocker
of platelet function when measured by methods that use arachidonic acid as the agonist. A significantly higher prevalence
of resistance was observed with methods that used agonists other than arachidonic acid to stimulate platelets. The
observation of dose-dependent and non– dose-dependent effects of aspirin on platelet function suggests that aspirin also
exerts antithrombotic effects through pathways other than cyclooxygenase-1. A clearer understanding of the mechanistic
effects of aspirin is necessary to establish a definition for aspirin resistance and its clinical relevance. On the basis of the
dose-dependent antiplatelet effects observed in the present study, we believe that higher aspirin doses may improve clinical
outcomes in selected patients. Large-scale prospective clinical trials are needed to support the link between superior
dose-related effects of aspirin on platelet function and clinical outcomes.

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 Evaluation of Dose-Related Effects of Aspirin on Platelet Function: Results From the
Aspirin-Induced Platelet Effect (ASPECT) Study
Paul A. Gurbel, Kevin P. Bliden, Joseph DiChiara, Justin Newcomer, Willy Weng, Nagaraj K.
Neerchal, Tania Gesheff, Srivasavi K. Chaganti, Amena Etherington and Udaya S. Tantry

Circulation. 2007;115:3156-3164; originally published online June 11, 2007;

doi: 10.1161/CIRCULATIONAHA.106.675587
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Copyright © 2007 American Heart Association, Inc. All rights reserved.
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