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Clopidogrel Pharmacokinetics and Pharmacodynamics Vary Widely Despite
Exclusion or Control of Polymorphisms (CYP2C19, ABCB1, PON1), Noncompliance, Diet, Smoking, Co-medications (including Proton Pump Inhibitors),
and Pre-existent Variability in Platelet Function
Short Title: Clopidogrel Response Variability
Andrew L. Frelinger III, PhD,*† Deepak L. Bhatt, MD, MPH,†‡ Ronald D. Lee, PhD,§ Darcy J.
Mulford, PhD,§ Jingtao Wu, PhD,§ Sai Nudurupati, PhD,§ Anu Nigam, MS,* Michael Lampa,
BS,* Julie K. Brooks, MS,* Marc R. Barnard, MS,* and Alan D. Michelson, MD*†
*Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children’s
Hospital, Dana-Farber Cancer Institute, Boston, MA, †Harvard Medical School, Boston, MA, ‡VA
Boston Healthcare System, Brigham and Women's Hospital, Boston, MA, §Takeda Global
Research & Development Center, Inc., Deerfield, IL
Address for correspondence: Andrew L. Frelinger III, Ph.D., Associate Director, Center for
Platelet Research Studies, Division of Hematology/Oncology, Boston Children’s Hospital, Karp
07212, 300 Longwood Avenue, Boston, MA 02115-5737. Telephone: 617-919-2539. Fax: 617730-4632. e-mail: [email protected].
Online-Only Material for Frelinger; Clopidogrel Response Variability
eMethods:
Enrollment
Subjects were screened for enrollment between Days -28 and Day -2 using predefined inclusion
and exclusion criteria to minimize subject risk and reduce interference with clopidogrel
absorption or metabolism. Subjects provided written, informed consent prior to initiation of study
procedures. Full enrollment criteria are described elsewhere.(1)
Diet, Fluid, and Activity Control
Subjects enrolled in this study (cohorts of 40 subjects at a time) were confined in a clinical
research unit (Celerion, Tempe, AZ) for 10 consecutive days and nights during Periods 1 and 2.
During the confinement period, subjects received standardized meals and an evening snack. On
Day 9 of each of the two periods, when PK samples were collected, all subjects were served
identical meals. All subjects could consume water ad libitum except for 1 hour prior to and postdrug administration. Study drug was administered with 240 mL of water. Subjects drank all of
the water provided with the dose. During confinement, products containing Seville oranges
(sour), grapefruit or grapefruit products (including juice) were prohibited and all subjects were
limited to only standardized meals and snacks provided by the site. Breakfast was provided 1
hour post-dose except for Day 9 of both periods. On Day 9 of both periods, breakfast was not
served. Lunch was served ~4 hours post-dose (after the 4-hour blood collection on Day 9 of
both periods), dinner ~ 9 hours post-dose, and a snack ~12 hours post-dose (after the 12-hour
blood collection on Day 9 of both periods). Breakfast, lunch, dinner and snacks were served at a
consistent time on all other days. Subjects remained upright (sitting, standing, ambulatory) for 2
hours after dosing. Subjects refrained from strenuous exercise beginning upon confinement and
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for the duration of their participation in the study.
Genotype Analysis
Blood, 6 mL, collected into plastic K2 EDTA Vacutainer™ tubes during the screening visit and
stored frozen at -20°C, was used to determine CYP2C19 genotype. Genotyping of the *2, *3,
*4, *5, *6, *7, *8, *9, *10 and *17 (C-806T) alleles of CYP2C19 was performed by Beckman
Coulter Genomics (Morrisville, NC) by published procedures,(2-11) as previously described.(1)
Only homozygous CYP2C19 extensive metabolizers (EM/EM, i.e., not carriers of CYP2C19 *2,
*3, *4, *5, *6, *7, *8, *9, *10 and *17) were randomized into the study. Blood from randomized
subjects was also analyzed for the presence of PON1 (rs662, G192R), CYP3A5 (*3 and *6) and
ABCB1 (MDR1) C3435T allelic variants as described below.
DNA isolation and purification
DNA from blood samples provided was isolated and purified using the Qiagen QIAamp® DNA
Blood Midi kit(2). All collection tubes had the assigned barcode label and were scanned into the
database prior to transfer of the sample into a new collection tube to maintain the chain-ofcustody.
PON1 rs662 allele: The PON1 rs662 allele is due to a guanine to adenine transition at
nucleotide 575. This mutation results in a substitution of an arginine for a glutamine at position
192 of the protein. PCR products containing the rs662 region were amplified from clinical
genomic DNA samples in parallel with negative (no template) controls, using specific primers.
The PCR product was sequenced using M13F and M13R tailed PCR primers to the region of
interest to provide forward and reverse sequencing reactions. The PCR reactions were purified
using the Exo-SAP-IT® Clean Up kit(3). Sequencing reactions were assembled using the
BigDye® Terminator Cycle Sequencing Kit v3.1.(4) Excess BigDye® Terminator was removed
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using CENTRI-SEP™ purification columns.(5) The purified DNA products were run on the
ABI3730xl Sequencing machine.(6) The raw sequence data from each run was imported into
the Applied Biosystems Variant Reporter™ Software.(7) The reads from each region were
aligned and the genotype for rs662 was called by the software.
CYP3A5 Genotyping:
CYP3A5*3 allele: The CYP3A5*3 mutation is due to an adenine to guanine transition at
nucleotide 6986.(12) This mutation results in a splicing defect and also creates an AlwNl
restriction site. A region around this site was PCR amplified and the PCR product was digested
with AlwNl to produce banding patterns specific for the AlA (*3 non-carrier), AlG (*3
heterozygote), and GIG (*3/*3 homozygote) genotype. The PCR products were gel
electrophoresed and photographed under ultraviolet light.
CYP3A5*6 allele: The CYP3A5*6 mutation is due to a guanine to an adenine transition at
nucleotide 14690.(13) This mutation results in a splicing defect and also destroys a Dde 1
restriction site. A region around this site was PCR amplified and the PCR product digested with
Dde I to produce banding patterns specific for the GIG (*6 non-carrier), G/A (*6 heterozygote),
and the AlA (*6/*6 homozygote) genotype. The PCR products were gel electrophoresed and
photographed under ultraviolet light.
ABCB1 Genotyping:
ABCB1 C3435T allele: The ABCB1 C3435T allele is due to a cytosine to thymine transition at
position 3435 in exon 26.(14) This allele does not cause an amino acid change in the protein. A
235 basepair fragment containing this polymorphism was PCR amplified, and the PCR product
digested with Mbo I to produce banding patterns specific for the C/C, C/T, and T/T genotype.
The PCR products were gel electrophoresed and photographed under ultraviolet light.
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Clopidogrel Pharmacokinetics
Blood (6 mL) for quantification of clopidogrel active metabolite (clopidogrelAM) concentration in
plasma was collected into chilled ethylenediamine-tetraacetic acid tubes on dosing Day 9 at
Pre-dose (no earlier than 30 min pre-dose) (0 hour prior to clopidogrel dosing) and 0.25, 0.5, 1,
1.5, 2, 3, 4, 5, 6, 7, 8, 12, and 24 hours post dose. Immediately following blood collection, 3’methoxyphenacyl bromide was added to chemically stabilize the clopidogrelAM. Plasma was
stored frozen at -80°C until analysis. Concentrations of the H3/H4 pair of Z diastereomers of
clopidogrelAM in human plasma was determined by Advion BioServices, Inc. (Ithaca, NY) using
liquid chromatography/tandem mass spectrometry (LC-MS/MS, with a validated concentration
range of 0.05 ng/mL to 50 ng/mL for clopidogrel, 0.1 ng/mL to 100 ng/mL for clopidogrel active
metabolite. Concentrations below the lower limit of quantification were treated as zero in the
summary statistics and calculation of the PK parameters.
Pharmacokinetic parameters were derived using non-compartmental analysis methods
determined from the concentration-time data for all evaluable subjects. Actual sampling times
were used in all computations involving sampling times. The following pharmacokinetic
parameters were calculated: the peak plasma concentration (Cmax), area under the plasma
concentration-time curve from time 0 to the time of last quantifiable concentration (AUCt).
Clopidogrel Pharmacodynamics
VASP P2Y12 Assay. The VASP P2Y12 assay was performed according to the manufacturer’s
instructions by the Center for Platelet Research Studies, Children’s Hospital Boston using citrate
anticoagulated whole blood, shipped overnight at ambient temperature. Samples were treated
with PGE1, and PGE1 plus ADP, fixed, permeabilized, and then reacted with fluorescein
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isothiocyanate (FITC)-conjugated monoclonal antibody 16C2, specific for VASP-Ser239P, and
phycoerythrin (PE)-conjugated monoclonal antibody specific for platelet surface CD61. Platelets
were identified by flow cytometry in a FACSCalibur (Becton Dickinson) flow cytometer using
CD61-PE fluorescence, forward- and side-light scatter and the level of VASP-Ser239P was
determined by 16C2-FITC mean fluorescence intensity (MFI). The results were reported as
platelet reactivity index (PRI) calculated as PRI = MFI(PGE1)-MFI(PGE1+ADP)/MFI(PGE1)×100.
Light Transmission Aggregation. LTA was performed in BioData PAP8 instruments using
platelet-rich plasma (PRP) prepared by centrifugation of citrated whole blood at 100 x g for 10
min and platelet-poor plasma (PPP) prepared by centrifugation of blood at 1500 x g for 10 min.
Platelet counts were not adjusted prior to performing platelet aggregation. Results are reported
as % aggregation, where PRP is set as 0% aggregation and PPP is set as 100% aggregation.
Platelet aggregation was initiated by adding 1/10th volume ADP to achieve a final concentration
of 5 or 20 µM ADP. All LTA assays were performed in duplicate and the results averaged.
Results are reported as maximal percent aggregation (MPA).
Accumetrics VerifyNow P2Y12 Assay. The VerifyNow P2Y12 assay was performed using
VerifyNow model 85005-6 instruments and P2Y12 kit 85064 cartridges. The VerifyNow P2Y12
test cartridge measures platelet aggregation in separate channels in response to ADP and, as a
reference measurement, in response to thrombin receptor activating peptide (TRAP). Results
from the ADP channel are reported as platelet reactivity units (PRU). In addition, the VerifyNow
reports % inhibition of P2Y12 function, calculated by comparing the PRU from the ADP channel
to the PRU from the TRAP channel.
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Online-Only Results:
Clopidogrel pharmacodynamics after 9 days treatment with clopidogrel 75 mg varied widely
(CVs 37%, 53%, 37%, and 32% for VASP PRI, VerifyNow PRU, MPA 5 µM ADP and MPA 20
µM ADP, respectively, Figure 1, panels C - F and Table 3 of the primary manuscript). To
determine if the large observed variation in clopidogrel pharmacodynamic endpoints was the
result of day-to-day subject and/or assay variation, assay results for each subject were
compared across treatment days. eFigure 1 shows results for individual subjects pre-treatment,
and on-treatment for 6, 7, 8, and 9 days. The selected subjects shown are representative of
subjects in quartiles 1 – 4 at 6 days on-treatment. Subjects with a strong response (quartile 1) at
6 days on clopidogrel treatment showed a similar strong response at 7, 8, and 9 days ontreatment. Likewise, subjects with a weak response (quartile 4) at 6 days on clopidogrel showed
a similar weak response at 7, 8, and 9 days on-treatment. The consistency of the responses in
each assay across treatment days 6 – 9 was quantified by calculating intraclass correlation
coefficients (ICCs) eTable 1. High ICC values (all greater than 80%) demonstrate very low
within subject day-to-day variation, insufficient to account for the large overall variation in assay
results observed.
Online-Only References
1.
Frelinger III AL, Lee RD, Mulford DJ, et al. A Randomized, 2-Period, Crossover Design
Study to Assess the Effects of Dexlansoprazole, Lansoprazole, Esomeprazole and
Omeprazole on the Steady-State Pharmacokinetics and Pharmacodynamics of
Clopidogrel in Healthy Volunteers. 2012;59:1304-11.
2.
QIAamp® DNA Blood Midi/Maxi kit handbook. January 2005.
3.
ExoSAP-IT product numbers 78200/01/02/05/50, Publication P-78200B, Rev. 02/08.
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Online-Only Material for Frelinger; Clopidogrel Response Variability
4.
BigDye® Terminator Cycle Sequencing Kit v3.1, Publication 106PB0901, Rev. 10/02.
5.
Princeton Separations CENTRI-SEP 96 plate or 8 strip protocol product number
79100519D/79- 100613C.
6.
Applied Biosystems 3730/3730xl DNA Analyzers User Guide product number 4331468
Rev. D 01/07.
7.
Applied Biosystems Variant Reporter User Guide product number 4376590 Rev. A
05/07.
8.
De Morais SM, Wilkinson GR, Blaisdell J, Meyer UA, Nakamura K, Goldstein JA.
Identification of a new genetic defect responsible for the polymorphism of (S)mephenytoin metabolism in Japanese. Mol Pharmacol 1994;46:594-8.
9.
de Morais SMF, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The
major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in
humans. J Biol Chem 1994;269:15419-22.
10.
Ferguson RJ, De Morais SM, Benhamou S, et al. A new genetic defect in human
CYP2C19: mutation of the initiation codon is responsible for poor metabolism of Smephenytoin. J Pharmacol Exp Ther 1998;284:356-61.
11.
Sim SC, Risinger C, Dahl ML, et al. A common novel CYP2C19 gene variant causes
ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and
antidepressants. Clin Pharmacol Ther 2006;79:103-13.
12.
Kuehl P, Zhang J, Lin Y, et al. Sequence diversity in CYP3A promoters and
characterization of the genetic basis of polymorphic CYP3A5 expression. 2001;27:38391.
13.
Hoffmeyer S, Burk O, von Richter O, et al. Functional polymorphisms of the human
multidrug-resistance gene: multiple sequence variations and correlation of one allele
with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci U S A
2000;97:3473-8.
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14.
Sakaeda T, Nakamura T, Okumura K. MDR1 genotype-related pharmacokinetics and
pharmacodynamics. Biol. Pharm. Bull. 2002;25:1391-400.
Online-Only Figure Legend
eFigure 1. Day-to-day variation in on-treatment clopidogrel pharmacodynamic endpoints.
Results shown are sequential values for individual subjects representative of quartiles at 6 days
on-treatment. MPA: maximal platelet aggregation; Q1: quartile 1, Q2: quartile 2; Q3: quartile 3;
Q4: quartile 4.
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Online-Only Tables:
eTable 1. Key enrollment criteria related to variability in clopidogrel pharmacokinetics
and/or pharmacodynamics.
Inclusion
Exclusion







Male or female age 18 –
55 years
CYP2C19 EM/EM
(extensive metabolizer)
Healthy by history and
physical examination
Hematology, clinical
chemistry and urinalysis
within reference range
Medication-free
Urine drug screen
negative for substances
of abuse











CYP2C19 *2, *3, *4, *5, *6, *7, *8, *9, *10 and
*17 (poor, intermediate, or ultra-rapid
metabolizer)
Prescription medications within 4 weeks
OTC medications within 2 weeks
Smoking within 6 weeks
Positive cotinine test
Caffeine within 72 h
Alcohol within 72 h
Consumption of products containing Seville
oranges (sour), grapefruit or grapefruit juice
within 14 days
Current or recent history of GI disease,
malabsorption, reflux, GI bleeding
Systolic BP >140 mm Hg or diastolic BP >90
mm Hg
Positive test for HBsAg or anti-HCV
Pregnant or nursing
Abbreviations: BP, blood pressure; GI, gastrointestinal; OTC, over the counter.
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eTable 2. ClopidogrelAM levels and platelet function pre-treatment and following 9 days
treatment with clopidogrel 75 mg in a homogeneous population of normal subjects.
Pre-Treatment
N=156 (a)
Mean ± SD
CV
On-Treatment (9 days)
Mean ± SD
CV
p-value (b)
ClopidogrelAM AUCt, ng-hr/mL
---
41.3 ± 13.96
34%
-
ClopidogrelAM Cmax, ng/mL
---
39.6 ± 15.93
40%
-
VASP PRI, %
87.0 ± 6.49
7%
44.0 ± 16.41
37%
<0.0001
VerifyNow P2Y12, PRU
303.5 ± 55.0
18%
121.7 ± 64.14
53%
<0.0001
MPA 5 µM ADP, %
77.9 ± 9.84
13%
31.7 ± 11.89
37%
<0.0001
MPA 20 µM ADP, %
78.7 ± 8.31
11%
40.6 ± 12.88
32%
<0.0001
a) n = 155 for clopidogrelAM AUCt and Cmax (unreliable pharmacokinetic results were obtained
from 1 subject); n = 156 for all other assays.
b) Paired t-test, pre-treatment vs. on-treatment.
Abbreviations: ADP = adenosine diphosphate; CV = coefficient of variation; MPA = maximal
platelet aggregation; PRI = platelet reactivity index; PRU = platelet reaction units; VASP =
vasodilator-stimulated phosphoprotein.
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eTable 3. Intraclass Correlation Coefficient (ICC) for assessment of the steady state of
clopidogrel PD end points after multiple clopidogrel doses. Least squares means estimated
from a linear mixed effect model with repeated measures. Day is a fixed factor in the model. LS
= least squares; MPA = maximal platelet aggregation; PD = pharmacodynamic; PRI = platelet
reactivity index; PRU = P2Y12 reaction units.
On-treatment
On-treatment
On-treatment
On-treatment
6 Days
7 Days
8 Days
9 Days
LS Mean (SE)
LS Mean (SE)
LS Mean (SE)
LS Mean (SE)
ICC (%)
VASP PRI (%)
46.0 (1.30)
44.5 (1.31)
42.9 (1.30)
44.0 (1.30)
80.2
MPA 5 µM ADP (%)
32.0 (1.06)
32.5 (1.06)
32.4 (1.06)
31.7 (1.05)
82.5
MPA 20 µM ADP (%)
42.2 (1.08)
41.2 (1.07)
40.2 (1.07)
40.6 (1.07)
87.3
VerifyNow PRU
116.1 (5.02)
108.9 (5.02)
110.7 (5.02)
121.7 (5.02)
93.0
PD End Point
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eTable 4. ClopidogrelAM AUCt and Cmax in subjects grouped by polymorphisms in PON1,
CYP3A5 or ABCB1.
PON1 rs662
ClopidogrelAM AUCt, ng*hr/mL
ClopidogrelAM Cmax, ng/mL
Mean (SD, n)
Mean (SD, n)
A/A (Q/Q)
Low activity
40.45 (12.787, 33)
40.32 (14.113, 33)
A/G (Q/R)
Intermediate activity
40.38 (11.123, 89)
38.08 (12.494, 89)
G/G (R/R)
High activity
44.54 (20.506, 33)
43.19 (23.942, 33)
P-valuea
0.4217
0.5723
ClopidogrelAM AUCt, ng*hr/mL
ClopidogrelAM Cmax, ng/mL
Mean (SD, n)
Mean (SD, n)
CYP3A5 *3
*1/*1
High expression
44.01 (8.916, 9)
41.44 (8.439, 9)
*1/*3
Intermediate expression
39.28 (11.013, 43)
38.12 (13.187, 43)
*3/*3
Low expression
41.87 (15.344, 103)
40.13 (17.454, 103)
P-valuea
0.1613
0.3197
ClopidogrelAM AUCt, ng*hr/mL
ClopidogrelAM Cmax, ng/mL
Mean (SD, n)
Mean (SD, n)
ABCB1 C3435T
aP-values
C/C
High activity
42.28 (9.656, 43)
40.37 (12.4, 43)
C/T
Intermediate activity
40.99 (15.27, 77)
39.64 (17.431, 77)
T/T
Low activity
40.67 (15.644, 35)
38.77 (16.718, 35)
P-valuea
0.4533
0.5484
shown are from ANOVA models using log-transformed values of clopidogrelAM AUCt and Cmax and which
included factors for genotype, cohort, period, and treatment group.
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eTable 5. Factors associated with clopidogrelAM pharmacokinetics in multivariable model.
Independent predictor
ClopidogrelAM AUCt
ClopidogrelAM Cmax
General Linear Model
General Linear Model
adjusted r2 = 0.1789
adjusted r2 = 0.1552
Squared
Squared
semipartial
p-value
semipartial
ˆ 2)
correlation ( 
(a)
ˆ 2)
correlation ( 
p-value
Age (years)
0.0384
0.0082
0.0315
0.0179
Weight (kg)
0.0372
0.0092
0.0328
0.0157
(adjusted for cohort, period,
and treatment group)
a) P-values are from ANOVA models using log-transformed values of clopidogrelAM AUCt and
Cmax.
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eTable 6. VASP PRI in subjects grouped by polymorphisms in PON1, CYP3A5 or ABCB1.
VASP PRI, %
PON1 rs662
Mean (SD, n)
A/A (Q/Q)
Low activity
43.51 (17.703, 33)
A/G (Q/R)
Intermediate activity
44.88 (15.546, 90)
G/G (R/R)
High activity
42.10 (17.666, 33)
P-valuea
0.8190
VASP PRI, %
CYP3A5 *3
Mean (SD, n)
*1/*1
High expression
34.32 (16.604, 9)
*1/*3
Intermediate expression
44.96 (14.052, 43)
*3/*3
Low expression
44.44 (17.157, 104)
P-valuea
0.1402
VASP PRI, %
ABCB1 C3435T
aP-values
Mean (SD, n)
C/C
High activity
42.14(16.445, 44)
C/T
Intermediate activity
44.18 (16.174, 77)
T/T
Low activity
45.95 (17.084, 35)
P-valuea
0.5075
shown are from ANOVA models which included factors for genotype, cohort, period and treatment group.
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eTable 7. Platelet aggregation in subjects grouped by polymorphisms in PON1, CYP3A5 or ABCB1.
PON1 rs662
MPA 5 µM ADP, %
MPA 20 µM ADP, %
VerifyNow P2Y12, PRU
VerifyNow P2Y12, % Inh.
Mean (SD, n)
Mean (SD, n)
Mean (SD, n)
Mean (SD, n)
A/A (Q/Q)
Low activity
32.05 (14.135, 33)
39.59 (13.988, 33)
118.12 (75.564, 33)
59.70 (23.174, 33)
A/G (Q/R)
Intermediate activity
31.52 (11.696, 90)
40.20 (12.369, 90)
120.24 (60.569, 90)
58.43 (18.188, 90)
G/G (R/R)
High activity
32.03 (10.206, 33)
42.64 (13.310, 33)
129.27 (62.748, 33)
56.94 (19.224, 33)
p-valuea
0.9954
0.6400
0.8417
0.9620
MPA 5 µM ADP, %
MPA 20 µM ADP, %
VerifyNow P2Y12, PRU
VerifyNow P2Y12, % Inh.
Mean (SD, n)
Mean (SD, n)
Mean (SD, n)
Mean (SD, n)
CYP3A5 *3
*1/*1
High expression
29.83 (10.428, 9)
38.39 (9.236, 9)
99.89 (73.833, 9)
67.00 (21.604, 9)
*1/*3
Intermediate expression
31.71 (10.812, 43)
40.57 (11.851, 43)
126.21 (66.704, 43)
57.49 (19.560, 43)
*3/*3
Low expression
31.92 (12.501, 104)
40.78 (13.619, 104)
121.73 (62.479, 104)
58.01 (19.222, 104)
p-valuea
0.5921
0.5656
0.5765
0.4656
MPA 5 µM ADP, %
MPA 20 µM ADP, %
VerifyNow P2Y12, PRU
VerifyNow P2Y12, % Inh.
Mean (SD, n)
Mean (SD, n)
Mean (SD, n)
Mean (SD, n)
ABCB1 C3435T
C/C
High activity
30.52 (12.062, 44)
38.64 (13.480, 44)
118.02 (63.659, 44)
59.05 (20.596, 44)
C/T
Intermediate activity
31.69 (10.620, 77)
41.34 (12.260, 77)
121.68 (64.832, 77)
58.77 (18.843, 77)
T/T
Low activity
33.39 (14.271, 35)
41.37 (13.571, 35)
126.40 (64.775, 35)
56.71 (19.721, 35)
p-valuea
0.7806
0.7745
0.8557
0.8174
aP-values
shown are from ANOVA models which included factors for genotype, cohort, period and treatment group.
Inh. = inhibition.
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eTable 8. Factors associated with on-treatment VerifyNow PRU and % inhibition and MPA
to 5 and 20 µM ADP in multivariable models.
VerifyNow PRU
VerifyNow % Inhibition
General Linear Model
General Linear Model
2
adjusted r = 0.6500
adjusted r2 = 0.5738
Squared
Squared
semipartial
Independent predictor
P-value
semipartial
ˆ )
correlation ( 
(a)
ˆ 2)
correlation ( 
p-value
0.0874
<0.0001
0.1033
<0.0001
2
ClopidogrelAM AUCt
ClopidogrelAM Cmax
VASP PRI
Hematocrit (%)
Pre-treatment VerifyNow PRU
or % Inhibition
Independent predictor
0.0443
<0.0001
0.1373
<0.0001
0.0293
0.0005
0.1015
<0.0001
0.0235
0.0016
MPA 5 µM ADP
MPA 20 µM ADP
General Linear Model
General Linear Model
adjusted r2 = 0.3519
adjusted r2 = 0.4357
Squared
Squared
semipartial
semipartial
ˆ 2)
correlation ( 
p-value
ˆ 2)
correlation ( 
p-value
0.2118
<0.0001
0.1086
<0.0001
0.0181
0.0277
ClopidogrelAM AUCt
VASP PRI
Hematocrit (%)
0.0548
0.0004
Gender
Pre-treatment MPA to 5 or 20
µM ADP
0.0203
0.0297

0.0541
0.0002
0.0218
0.0159
a) P-values are from ANOVA models using log-transformed values of clopidogrelAM AUCt and
Cmax. Abbreviations: see eTable 2.
Page 17 of 18
Online-Only Material for Frelinger; Clopidogrel Response Variability
Online-Only Figure:
eFigure 1. Day-to-day variation in on-treatment clopidogrel pharmacodynamic end
points. Results shown are sequential values for individual subjects representative of quartiles at
6 days on-treatment. MPA: maximal platelet aggregation; Q1: quartile 1, Q2: quartile 2; Q3:
quartile 3; Q4: quartile 4.
Page 18 of 18