`© 2005 by the American College of Cardiology Foundation
`Published by Elsevier Inc.
`
`Vol. 45, No. 2, 2005
`ISSN 0735-1097/05/$30.00
`doi:10.1016/j.jacc.2004.09.067
`
`Antiplatelet Therapy
`
`Variability in Platelet Responsiveness
`to Clopidogrel Among 544 Individuals
`Victor L. Serebruany, MD, PHD,* Steven R. Steinhubl, MD, FACC,† Peter B. Berger, MD, FACC,‡
`Alex I. Malinin, MD,* Deepak L. Bhatt, MD, FACC,§ Eric J. Topol, MD, FACC§
`Towson, Maryland; Lexington, Kentucky; Durham, North Carolina; and Cleveland, Ohio
`
`RESULTS
`
`METHODS
`
`OBJECTIVES We sought to describe the responses of patients to clopidogrel using ex vivo measures of
`platelet aggregation and activation in a large, heterogeneous population.
`BACKGROUND Recently, a number of reports, using various definitions, have dichotomized patients who are
`treated with clopidogrel into a minority of “non-responders” and a majority of “responders.”
`Such classifications imply that treatment leads to an all-or-none response, with potentially
`important clinical implications.
`We conducted secondary post-hoc analyses of a dataset consisting of volunteers (n ⫽ 94) and
`patients after coronary stenting (n ⫽ 405), with heart failure (n ⫽ 25), and after stroke (n ⫽
`20).
`The response of subjects to clopidogrel followed a normal, bell-shaped distribution, with a
`mean and standard deviation of 41.9 ⫾ 20.8% when aggregation was induced by 5 mol/l of
`adenosine diphosphate. When hyporesponsiveness and hyper-responsiveness to clopidogrel
`were considered to be two standard deviations less than and greater than the mean,
`respectively, the prevalence of hyporesponsiveness and hyper-responsiveness in these patients
`was 4.2% and 4.8%, respectively. Pretreatment platelet activity and clinical characteristics
`were not associated with responsiveness to clopidogrel.
`CONCLUSIONS Individuals receiving clopidogrel exhibit a wide variability in response that follows a normal
`distribution. The clinical implications of this variability are unknown but potentially are
`important. Clinical trials are needed to define whether hyporesponders to clopidogrel are at
`increased risk for thrombotic events and whether hyper-responders are at increased risk for
`bleeding. If so, the individualization of antiplatelet therapy, including clopidogrel dosing, may
`be possible in the future but will require the ability to easily and reproducibly measure
`responsiveness by a method that has been proven to be predictive of clinical events.
`(J Am
`Coll Cardiol 2005;45:246 –51) © 2005 by the American College of Cardiology Foundation
`
`Variability among patients in the measured response to
`treatment with an antiplatelet agent has been recognized
`since 1966 (1). Since that time, numerous small studies have
`suggested a correlation between clinical outcomes and ex
`vivo aspirin “non-responsiveness” or “resistance” (2,3), but
`such a relationship has yet to be proven in adequately
`powered, large-scale, prospective clinical trials. More re-
`cently, a number of reports have been published identifying
`variability in the level of platelet inhibition achieved with
`the administration of clopidogrel, with a minority of indi-
`viduals again being classified as “non-responders” or “resis-
`tant” (4 – 6). Like the studies of aspirin that preceded them,
`these studies are relatively small and use different platelet
`assessments and definitions for determining responsiveness.
`
`From the *HeartDrug Research Laboratories, Towson, Maryland; †University of
`Kentucky, Lexington, Kentucky; ‡Duke Clinical Research Institute, Durham, North
`Carolina; and §Cleveland Clinic, Cleveland, Ohio. Drs. Serebruany, Steinhubl,
`Berger, and Topol have received research support from the Sanofi-BMS Partnership.
`Drs. Serebruany, Steinhubl, and Topol are consultants for Sanofi-BMS Partnership.
`Drs. Serebruany, Steinhubl, Berger, and Bhatt received honoraria for educational
`presentations for Sanofi-Synthelabo/Bristol-Myers Squibb Partnership. Dr. Sereb-
`ruany is an owner of HeartDrug Research LLC. The sponsors had no role in study
`design, data collection, data analyses, interpretation of results, or manuscript writing.
`The first two authors contributed equally to this study.
`Manuscript received July 7, 2004; revised manuscript received September 22, 2004,
`accepted September 27, 2004.
`
`Defining patients as either “responsive” or “unresponsive”
`to an antiplatelet therapy suggests a dichotomous response
`that is quite different from what is expected and clinically
`observed in nearly all other therapeutic interventions. Mul-
`tiple genetic and environmental influences have been shown
`to affect ex vivo platelet responsiveness in humans, in vivo
`thrombosis in animal models, and responsiveness to anti-
`platelet therapies (7–9). Therefore, like other biologic sys-
`tems under polygenetic and environmental influence, plate-
`let function and response to antiplatelet therapy would be
`expected to demonstrate a wide range in responses among
`subjects.
`To better understand the interindividual variation in
`response to clopidogrel therapy, we analyzed a combined
`database (n ⫽ 544) of studies conducted in the Baltimore
`metropolitan area (1998 to 2004), which we believe makes
`up the largest clinical data set of clopidogrel-treated subjects
`in which platelet function has been assessed serially.
`
`METHODS
`
`Population (general inclusion and exclusion criteria). Five
`hundred forty-four subjects who had been treated with
`clopidogrel or with aspirin and clopidogrel were eligible for
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`
`247
`
`Abbreviations and Acronyms
`ADP ⫽ adenosine diphosphate
`PRP ⫽ platelet-rich plasma
`
`this analysis. To be included in the present analyses, all
`patients had a baseline sample (before treatment with
`clopidogrel) and at least one additional sample with evalu-
`able platelet data after treatment with clopidogrel. Volun-
`teers and patients were excluded if they had any clinical
`issues that could influence measured platelet response. We
`assessed patient compliance to the study by interview and by
`pill counting. To reflect an individual’s full response to
`clopidogrel, only those patients whose platelet function tests
`were performed at least 3 to 4 h or longer after a 300-mg
`loading dose or, in those not receiving a loading dose, 5 days
`or longer were included in this analysis. The four cohorts of
`individuals included in the current analysis are subsequently
`listed.
`
`HUMAN VOLUNTEERS WITH MULTIPLE RISK FACTORS OR
`DOCUMENTED VASCULAR DISEASE (n ⴝ 94). Subjects were
`eligible for this study if they met all of the following
`inclusion criteria: a documented history of vascular disease
`or multiple risk factors for vascular disease. All subjects were
`free of aspirin upon beginning the study and received 75 mg
`of clopidogrel immediately after the baseline sample fol-
`lowed by 75 mg once daily thereafter for seven days, at
`which time platelets were assessed.
`
`PATIENTS UNDERGOING CORONARY STENTING (n ⴝ 405). All
`patients had received 325 mg of aspirin daily for at least one
`week. Most patients (94%) also received a 300-mg
`clopidogrel-loading dose immediately before intervention,
`followed by 75 mg of clopidogrel once daily for at least 30
`days. Platelet function was assessed at multiple time points
`in different patients: at baseline, 2 h, 3 h, 4 h, 24 h, 5 days,
`and 30 days after a loading dose. Because 2 h is not sufficient
`
`for clopidogrel to exhibit its full antiplatelet efficacy, we used
`the next evaluable sample (4 to 24 h) in the present analyses.
`
`PATIENTS WITH HEART FAILURE (n ⴝ 25). Eighty-eight out-
`patients with a left ventricular ejection fraction ⬍40% or
`New York Heart Association functional class II to IV
`congestive heart failure symptoms in the setting of preserved
`systolic function (10) were included. Only patients who
`were found to have increased platelet activation at baseline
`and who were treated with both clopidogrel 75 mg and
`aspirin 325 mg (n ⫽ 25) were included in the current
`analysis. Platelet function was assessed at baseline and at 30
`days after randomization.
`
`POST-STROKE PATIENTS OR PATIENTS WITH TRANSIENT
`ISCHEMIC ATTACK (n ⴝ 20). Patients age ⱖ40 years were
`eligible if they had suffered from ischemic stroke between
`two and six months earlier and were receiving aspirin (81
`mg/day). Twenty patients who were assigned to clopidogrel
`75 mg and aspirin 81 mg daily for 30 days were included in
`the current analysis. Platelet activity was measured at
`baseline and at 30 days after randomization.
`Samples. Blood samples were obtained with a 19-gauge
`needle by direct venipuncture and drawn into 7-ml Vacu-
`tainer tubes at room temperature containing 3.8% trisodium
`citrate. All samples were labeled with a coded number and
`analyzed by blinded technicians. Research coordinators were
`not aware of the platelet data, and laboratory personnel did
`not know the treatment allocation. Platelet studies were
`performed at baseline and at prespecified time points as
`noted previously in this work.
`Platelet assessment. CONVENTIONAL OPTICAL PLATELET
`AGGREGOMETRY. The blood-citrate mixture was centri-
`fuged at 1,200 g for 2.5 min. The resulting platelet-rich
`plasma (PRP) was kept at room temperature for use within
`1 h. The platelet count was determined in the PRP sample
`and adjusted to 3.5 ⫻ 108/ml with homologous platelet-
`poor plasma. Platelets were stimulated with 5 mol of
`adenosine diphosphate (ADP), and aggregation was as-
`sessed using a Chronolog Lumi-Aggregometer (model 560-
`
`Figure 1. Distribution of changes in 5 mol of adenosine diphosphate
`(ADP)-induced platelet aggregation in 544 patients after receiving clopi-
`dogrel therapy. Negative changes in aggregation values represent aggrega-
`tion values after the administration of clopidogrel that were higher than the
`baseline readings.
`
`Figure 2. Distribution of 5 mol of adenosine diphosphate (ADP)-
`induced residual platelet aggregation in 544 patients after receiving
`clopidogrel therapy.
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`Characteristics of Clopidogrel Response
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`JACC Vol. 45, No. 2, 2005
`January 18, 2005:246 –51
`
`Figure 3. Correlation between inhibition of platelet activation as deter-
`mined by the change in platelet/endothelial cell adhesion molecule-1
`(PECAM-1) expression and the change in 5 mol of adenosine diphos-
`phate (ADP)-induced platelet aggregation after treatment with clopi-
`dogrel. Negative values resulted from the higher readings after treatment
`when compared with the baseline measures.
`
`Ca; Chronolog Corp., Haverton, Pennsylvania) with the
`AggroLink software package (Chronolog Corp.).
`
`FLOW CYTOMETRY. The following monoclonal antibodies
`were used in at least one of the four patient cohorts included
`in the current analysis: CD41 antigen (glycoprotein IIb) and
`CD62P (P-selectin; DAKO Corp., Carpenteria, California)
`and PAC1 (activated glycoprotein IIb/IIIa), CD31 (plate-
`let/endothelial cell adhesion molecule-1), and CD51/CD61
`(integrin ␣v3, vitronectin receptor; PharMingen, San
`Diego, California). The formation of platelet-leukocyte
`aggregates was assessed by dual labeling with pan-platelet
`marker (CD151) and then with CD14, the macrophage
`receptor for endotoxin lipopolysaccharides. The samples
`were analyzed on a Becton Dickinson FACScan flow
`cytometer (Becton Dickinson, San Diego, California) set up
`to measure fluorescent light scatter as previously described.
`P selectin was expressed as percent positive cells. Other
`antigens were expressed as log mean fluorescence intensity.
`Definition of clopidogrel response. Platelet response to
`clopidogrel was defined as hyporesponders (two standard
`deviations below the mean), hyper-responders (two stan-
`dard deviations above the mean), and the rest individuals
`were defined as standard responders.
`Statistical analysis. Categorical data are displayed as fre-
`quencies and percentages. The chi-square test was used for
`dichotomous analyses of categorical data. Continuous data
`are presented as mean values ⫾ SD and were compared
`using one-way repeated measures analysis of variance. Nor-
`mal distribution of the data was tested with Anderson-
`Darling and D’Agostino omnibus tests. Skewness between
`0.0 and 0.5 was considered as a minimum (fairly symmet-
`ric). The Pearson linear correlation coefficient (r) was
`computed and applied for analysis. Differences between
`individual flow cytometric histograms were assessed using
`the Smirnov-Kolmogorov test
`incorporated in the
`CELLQuest (Becton Dickinson) software. Statistical anal-
`yses were performed using SPSS/11.5 (SPSS, Inc., Chicago,
`Illinois).
`
`Figure 4. Platelet inhibition over the course of time for multiple time point
`assessments (⬎2) after loading with 300 mg and maintenance dose of 75
`mg of clopidogrel.
`
`RESULTS
`
`Platelet function before and after clopidogrel therapy was
`analyzed in all 544 individuals by conventional aggregom-
`etry. In most patients (88%), the expression of platelet
`receptors using flow cytometry also was measured. Using
`light-transmittance aggregometry and analyzing the change
`in maximal platelet aggregation with 5 mol of ADP as the
`agonist, we discovered that the mean change in aggregation
`from baseline after the initiation of clopidogrel therapy was
`41.9%, with a SD of 20.8% (Fig. 1). The histogram of the
`study population is consistent with a normal, bell-shaped
`distribution. A negative skewness of ⫺0.1 confirmed
`almost-ideal symmetric normal distribution, with a very
`slight trend towards hyporesponsiveness. There were 23
`subjects (4.2%) with a change in ADP-induced platelet
`aggregation greater than two standard deviations above the
`mean (⬎83.5%);
`they were considered to be hyper-
`responders. Similarly, 26 subjects (4.8%) experienced almost
`no measurable change in aggregation (⬍2 standard devia-
`tion reductions in aggregation from the mean); they were
`considered to be hyporesponders. The change in ADP-
`induced platelet aggregation after the administration of
`clopidogrel ranged from ⫺32% (i.e., greater aggregation
`than before the administration of clopidogrel) to 94%
`(almost complete inhibition of aggregation).
`When clopidogrel responsiveness was described by the
`maximal platelet aggregation induced by 5 mol of ADP
`after clopidogrel (Fig. 2) rather than the change in aggre-
`gation, a normal distribution again was observed, with a
`skewness of 0.35. The mean residual aggregation after the
`administration of clopidogrel was 37.9%, with a standard
`deviation of 16.8%. The range of residual ADP-induced
`aggregation after clopidogrel was 3% to 84%. Platelet
`aggregation after the administration of clopidogrel was two
`standard deviations less than the mean in 19 subjects (3.5%)
`and two standard deviations greater than the mean in 27
`subjects (5.0%).
`Measures of inhibition of platelet aggregation by light-
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`Characteristics of Clopidogrel Response
`
`249
`
`Table 1. Baseline Characteristics of Standard Responders, Hyper-Responders (⬎2 SD), and
`Hyporesponders (⬍2 SD)
`
`Characteristics
`
`Demographics
`Age, yrs (⫾ SD)
`Male gender, n (%)
`Caucasian, n (%)
`Risk factors, n (%)
`Tobacco use
`Hypertension
`Diabetes
`Hypercholesterolemia
`Family history
`Medical history, n (%)
`Previous MI
`Previous stroke
`Heart failure
`Heart surgery
`Medications, n (%)
`Beta-blockers
`ACE inhibitors
`Calcium-channel blockers
`AT-receptor antagonists
`Diuretics
`Statins
`Atorvastatin
`Antidepressants
`Aggregometry (5M ADP)
`Platelet aggregation (%)
`
`Hyporesponders
`(n ⴝ 18)
`
`Hyper-
`Responders
`(n ⴝ 18)
`
`Standard
`Responders
`(n ⴝ 404)
`
`p Value
`(ANOVA)
`
`62.7 ⫾ 11.1
`12 (67)
`11 (61)
`
`60.8 ⫾ 11.9
`10 (56)
`12 (67)
`
`64.0 ⫾ 10.7
`287 (71)
`264 (65)
`
`9 (50)
`15 (83)
`7 (39)
`11 (61)
`12 (67)
`
`4 (22)
`2 (11)
`5 (27)
`2 (11)
`
`6 (33)
`4 (22)
`3 (17)
`3 (17)
`4 (22)
`9 (50)
`5/9 (56)
`3 (17)
`
`67 ⫾ 13
`
`10 (56)
`13 (72)
`7 (39)
`10 (54)
`11 (61)
`
`4 (22)
`1 (6)
`4 (22)
`2 (11)
`
`8 (44)
`4 (22)
`5 (28)
`4 (22)
`3 (17)
`10 (54)
`6/10 (60)
`2 (11)
`
`214 (53)
`275 (68)
`177 (44)
`233 (58)
`281 (69)
`
`96 (24)
`30 (7)
`33 (8)
`36 (9)
`
`140 (35)
`137 (35)
`87 (20)
`86 (20)
`71 (18)
`221 (55)
`134/221 (61)
`45 (11)
`
`68 ⫾ 10
`
`65 ⫾ 10
`
`0.36
`0.28
`0.89
`
`0.77
`0.08
`0.61
`0.8
`0.67
`
`0.74
`0.5
`0.36
`0.75
`
`0.45
`0.28
`0.22
`0.6
`0.42
`0.51
`0.47
`0.2
`
`0.6
`
`Data are presented as the mean value ⫾ SD or percentage of patients.
`ACE ⫽ angiotensin-converting enzyme; ADP ⫽ adenosine diphasphate; ANOVA ⫽ analysis of variance; AT ⫽
`angiotensin; MI ⫽ myocardial infarction.
`
`transmittance aggregometry and platelet activation by plate-
`let/endothelial cell adhesion molecule-1 expression deter-
`mined by flow cytometry were available from 374 subjects at
`identical time points from the same blood samples (Fig. 3).
`Regression analysis revealed a moderate positive correlation
`(r ⫽ 0.51, p ⫽ 0.023) between these measures of platelet
`aggregation and activation-dependent receptor expression
`after the administration of clopidogrel.
`Of the 544 subjects studied, 380 had more than one
`measurement of platelet function after the maximal anti-
`platelet effects of clopidogrel were achieved (Fig. 4). Most of
`these serial measurements were within 48 h of the initiation
`of therapy with a loading dose. Only a few patients (n ⫽ 30)
`were evaluated after 30 days of receiving daily clopidogrel.
`No significant change in the mean level of inhibition of
`ADP-induced platelet aggregation was observed over the
`course of time, although the small numbers studied at the
`longest durations of therapy limit the ability to make any
`definitive conclusions.
`Hyporesponders and hyper-responders to clopidogrel, as
`determined by change in ADP-induced platelet aggrega-
`tion, did not differ significantly in clinical characteristics
`from those whose responses were in the standard range
`(Table 1). Hyporesponders to clopidogrel had a trend
`toward a greater prevalence of hypertension. Platelet activity
`before the administration of clopidogrel, which was defined
`
`by baseline platelet aggregation response to ADP, did not
`appear to be associated with the response to clopidogrel
`(Table 1).
`In some of the subjects included in this analysis, up to six
`separate measures of platelet inhibition were conducted.
`Whether subjects who were hyporesponders or hyper-
`responders to ADP-induced aggregation also were hypore-
`sponsive or hyper-responsive to other tests of platelet
`inhibition was analyzed (Table 2). Of the 26 patients
`identified as hyporesponders, 50% to 89% met the criteria
`for being hyporesponsive when other measures of platelet
`inhibition were used. Similarly, of the 23 patients identified
`as hyper-responsive by their aggregation response, 51% to
`74% also remained hyper-responsive when additional tests
`of platelet function were used.
`
`DISCUSSION
`
`This study, which comprises the largest population base of
`its kind to date, demonstrates a marked variability in
`response after standard dosing of the antiplatelet agent
`clopidogrel. The normal distribution of response to clopi-
`dogrel is consistent with the large number of recognized and
`unrecognized genetic and environmental factors that influ-
`ence platelet function and responsiveness to other antiplate-
`let therapies. The clinical
`implications of these ex vivo
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`Table 2. Number of Patients Who Were Hyporesponsive or Hyper-Responsive to Clopidogrel Based on the Change in ADP-Induced
`Aggregation Who Were Determined to be Hyporesponsive and Hyper-Responsive by Other Methods of Measuring Platelet Inhibition
`
`Group*
`
`Hyporesponders
`Hyper-responders
`
`n
`
`26/544
`23/544
`
`GP IIb/IIIa
`
`P-Selectin
`
`PECAM-1
`
`15 (58%)
`13 (57%)
`
`18 (69%)
`15 (65%)
`
`23 (89%)
`16 (70%)
`
`VTR
`
`15 (58%)
`17 (74%)
`
`CD151-CD14
`
`13 (50%)
`12 (51%)
`
`*Based on (2SD) ADP-induced aggregation hyporesponders, hyper-responders definition, and after quality control and quality assurance sample validation.
`CD151-CD14 ⫽ formation of platelet-monocyte aggregates; GP ⫽ glycoprotein; PECAM-1 ⫽ platelet/endothelial cell adhesion molecule-1; VTR ⫽ vitronectin receptor.
`
`findings are unknown but likely are to be important, based
`on the high prevalence of atherosclerotic disease and the
`central role of antiplatelet therapies in the prevention and
`treatment of its complications.
`Clopidogrel, administered with or without aspirin, has
`been evaluated in prospective, placebo-controlled trials in-
`volving more than 30,000 patients to date (11–13). Treat-
`ment with the combination of clopidogrel and aspirin for as
`short a period as one year can decrease the occurrence of
`death, myocardial infarction, and stroke by 20% to 27%
`compared with the use of aspirin alone (12,13). However,
`despite the recognition for several decades of wide interpa-
`tient variability in the measured response to antiplatelet
`therapy, a true relationship between any test of platelet
`inhibition and clinical outcomes has yet to be proven.
`Clopidogrel “non-responsiveness” has been reported to be
`present in as little as 5% to as many as 56% of patients who
`are undergoing coronary stenting. Previous studies (4 – 6)
`labeled patients as non-responders based on the arbitrary
`definitions of the change in ADP-induced platelet aggre-
`gation before and after the start of clopidogrel therapy. In
`this study, we chose to identify responsiveness to clopidogrel
`in a manner more consistent with standard laboratory
`practice when describing normally distributed values, with
`abnormal values being those greater and less than two
`standard deviations from the mean. By doing so, we found
`4.8% of subjects to be hyporesponsive and 4.3% to be
`hyper-responsive to clopidogrel.
`The definition of clopidogrel response in our analyses is
`also arbitrary but seems more physiologic for assessment in
`the large cohorts compared with the single-patient measures
`based only on the differences in the platelet activity. On the
`basis of the present data, the concept of triaging patients
`into “responder” and “non-responder” must be performed
`with great caution. The present dataset reveals a normal,
`bell-shaped distribution of clopidogrel response, thereby
`suggesting too wide of a range of response to be simply
`dichotomized. We were not able to identify any clinical
`characteristics associated with hyper-responsiveness or hy-
`poresponsiveness, nor were we able to confirm the results of
`previous investigators who reported a relationship between
`baseline platelet activity and response to clopidogrel (6).
`Although, like other investigators, we focused on the use of
`standard light-transmittance aggregometry in PRP as a
`means of evaluating individual responsiveness to clopi-
`dogrel, we also used a wide selection of other studies of
`platelet function to assess a patient’s responsiveness to
`clopidogrel. We found a relatively strong correlation be-
`
`tween the measured inhibition of platelet aggregation and
`the inhibition of platelet activation using flow cytometry but
`also showed that the classification of individuals as hypore-
`sponders or hyper-responders does vary depending on the
`test used. All of these measures of platelet function are
`limited in their applicability to clinical practice because they
`require specialized equipment, complicated sample prepa-
`ration, and technical expertise. Although several point-of-
`care tests are available, their clinical value has yet to be
`proven. The term “clopidogrel resistance” (as opposed to
`clopidogrel response variability) can only be accurately used
`when and if there is documentation that administration of
`clopidogrel not only results in a lack of platelet inhibition
`but also yields less clinical benefit than in patients achieving
`greater levels of platelet inhibition.
`Study limitations. Several
`limitations merit mention.
`First, we present post-hoc second analyses; therefore, the
`data were not collected in the prospective fashion. Second,
`different protocols were used for the primary studies. High
`frequency of the use of concomitant medications may have
`affected the platelet characteristics. Despite the fact that in
`most patients platelets were assessed with more than 10
`characteristics, some established biomarkers of platelet ac-
`tivity, such as beta-thromboglobulin, platelet
`factor-4,
`thromboxane, and nitric oxide, were not measured. Im-
`portantly, different doses of aspirin (if any) were used,
`although this should not influence ADP-induced effects.
`Finally, clinical outcome data were available for the
`present analyses.
`Conclusions. The results of this study demonstrate that in
`patients treated with clopidogrel, there is a very large range
`of responsiveness to ex vivo testing that represents a nor-
`mally distributed bell-shaped curve. If these ex vivo results
`correspond to clinical outcomes, which remains to be
`proven, it is likely that a small but significant portion of
`patients are receiving inadequate protection from throm-
`botic events despite currently standard antiplatelet therapy,
`whereas a similar proportion may be at higher risk for
`bleeding complications. There is a great need for clinical
`trials to prospectively identify a measure of platelet function
`that can consistently and reproducibly measure the response
`of a patient to an antiplatelet therapy and then be able to
`correlate that result to the risk of adverse clinical outcomes.
`Once this is established, individualized treatment regimens
`should then be studied in an attempt to maximize the
`benefit and minimize the risk to the tens of millions of
`patients treated with daily, life-long antiplatelet therapy.
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`JACC Vol. 45, No. 2, 2005
`January 18, 2005:246 –51
`
`Serebruany et al.
`Characteristics of Clopidogrel Response
`
`251
`
`Reprint requests and correspondence: Dr. Victor L. Serebruany,
`HeartDrug Research Laboratories, Osler Medical Building, 7600
`Osler Drive, Suite 307, Towson, Maryland 21204. E-mail:
`heartdrug@aol.com.
`
`REFERENCES
`
`1. Quick AJ. Salicylates and bleeding: the aspirin tolerance test. Am J
`Med Sci 1966;252:265–9.
`2. Grotemeyer KH, Scharafinski HW, Husstedt IW. Two-year
`follow-up of aspirin responder and aspirin non-responder. A pilot
`study including 180 post-stroke patients. Thrombosis Res 1993;71:
`397– 403.
`3. Gum P, Kottke-Marchant K, Welsh PA, White J, Topol EJ. A
`prospective, blinded determination of the natural history of aspirin
`resistance among stable patients with cardiovascular disease. J Am Coll
`Cardiol 2003;41:961–5.
`4. Jaremo P, Lindahl TL, Fransson SG, Richter A. Individual variations
`of platelet inhibition after loading doses of clopidogrel. J Intern Med
`2002;252:233– 8.
`5. Muller I, Besta F, Schulz C, Massberg S, Schonig A, Gawaz M.
`Prevalence of clopidogrel non-responders among patients with stable
`angina pectoris scheduled for elective coronary stent placement.
`Thromb Haemost 2003;89:783–7.
`
`6. Gurbel PA, Bliden KP, Hiatt BL, O’Connor CM. Clopidogrel for
`coronary stenting. Response variability, drug resistance, and the effect of
`pretreatment platelet reactivity. Circulation 2003;107:2908–13.
`7. O’Donnell CJ, Larson MG, Feng D, et al. Genetic and environmental
`contributions to platelet aggregation: the Framingham Heart Study.
`Circulation 2001;103:3051– 6.
`8. Fontana P, Dupont A, Gandrille S, et al. Adenosine diphosphate-
`induced platelet aggregation is associated with P2Y12 gene sequence
`variations in healthy subjects. Circulation 2003;108:989 –95.
`9. Michelson AD, Furman MI, Goldschmidt-Clermont P, et al. Platelet
`GP IIIa PlA polymorphisms display different sensitivities to agonists.
`Circulation 2000;101:1013– 8.
`10. Serebruany VL, Malinin AI, Jerome SD, et al. Effect of clopidogrel
`and aspirin combination versus aspirin alone on platelet aggregation
`and major receptor expression in patients with heart failure: the Plavix
`Use for Treatment Of Congestive Heart Failure (PLUTO-CHF) trial.
`Am Heart J 2003;146:713–20.
`11. CAPRIE Steering Committee. A randomised, blinded trial of clopi-
`dogrel versus aspirin in patients at risk of ischemic events (CAPRIE).
`Lancet 1996;348:1329 –39.
`12. The Clopidogrel in Unstable Angina to Prevent Recurrent Events
`Trial Investigators. Effects of clopidogrel in addition to aspirin in
`patients with acute coronary syndromes without ST-segment eleva-
`tion. N Engl J Med 2001;345:494 –502.
`13. Steinhubl SR, Berger PB, Mann JT 3rd, et al. Early and sustained dual
`oral antiplatelet therapy following percutaneous coronary intervention.
`A randomized controlled trial. JAMA 2002;288:2411–20.
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`
`Ex. 1039, p. 6 of 6
`
`
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