`Response Variability, Drug Resistance, and the Effect of Pretreatment
`Platelet Reactivity
`
`Paul A. Gurbel, MD; Kevin P. Bliden, BS; Bonnie L. Hiatt, MD; Christopher M. O’Connor, MD
`
`Background—Clopidogrel is administered to prevent stent thrombosis; however, the uniformity of platelet inhibition after
`treatment and the influence of pretreatment reactivity on drug response have not been described.
`Methods and Results—Platelet aggregation (5 and 20 mol/L ADP), the activation of glycoprotein IIb/IIIa (PAC-1
`antibody), and the expression of P-selectin were measured in patients undergoing elective coronary stenting (n⫽96) at
`baseline and at 2 hours, 24 hours, 5 days, and 30 days after stenting. All patients received aspirin (325 mg). Clopidogrel
`(300 mg) was administered in the catheterization laboratory and followed by 75 mg daily. There was marked
`interindividual variability in drug response as measured by all markers that showed a normal distribution. Resistance,
`defined as baseline aggregation (%) minus posttreatment aggregation (%) ⱕ10% by 5 mol/L ADP, was present in 31%
`and 15% of patients at 5 and 30 days, respectively. Patients with the highest pretreatment platelet reactivity remained
`the most reactive at 24 hours after treatment (P⬍0.0001).
`Conclusions—Interindividual variability in the platelet inhibitory response from clopidogrel occurs in patients undergoing
`elective coronary stenting. Patients with high pretreatment reactivity are least protected. Alternative pharmacological
`strategies and the association of adverse ischemic events should be investigated in these patients. (Circulation. 2003;
`107:2908-2913.)
`
`Key Words: drugs 䡲 platelets 䡲 stents
`
`C lopidogrel with aspirin is the regimen of choice to
`
`thrombosis.1 The CURE study (Clopi-
`prevent stent
`dogrel
`in Unstable angina to prevent Recurrent Events)
`showed that combination clopidogrel and aspirin antiplatelet
`therapy reduces ischemic events compared with aspirin ther-
`apy alone.2 These findings are consistent with those of the
`CAPRIE study (Clopidogrel versus Aspirin in Patients at
`Risk of Ischemic Events), which showed superior reduction
`in ischemic events with clopidogrel therapy compared with
`aspirin, and which may be explained in part by aspirin
`resistance.3 However, the uniformity of inhibition after clo-
`pidogrel therapy and the incidence of drug resistance has not
`been investigated extensively. Interindividual variability in
`response to clopidogrel may affect clinical outcomes.4
`We studied the individual responses to clopidogrel therapy
`in patients undergoing elective coronary artery stenting by
`measuring platelet aggregation and other markers of platelet
`activation by flow cytometry for 30 days after the procedure.5
`The frequency of drug resistance is reported. We also studied
`the influence of pretreatment platelet reactivity on drug
`response.
`
`Methods
`This study was approved by the Investigational Review Board.
`Consecutive patients undergoing elective coronary stenting were
`
`enrolled after giving informed consent. All ages were included. The
`exclusion criteria were a history of bleeding diathesis, acute myo-
`cardial infarction within 48 hours, cerebrovascular event within 3
`months, illicit drug or alcohol abuse, prothrombin time ⬎1.5 times
`control, platelet count ⬍100 000/mm3, hematocrit ⬍25%, creatinine
`⬎4.0 mg/dL, and thienopyridine or glycoprotein (GP) IIb/IIIa use
`before the procedure.
`Per protocol, GP IIb/IIIa inhibitors were not given. Clopidogrel (300
`mg) was given to all patients in the catheterization laboratory after
`successful coronary artery stent implantation followed by 75 mg daily
`for 30 days. In addition, all patients had received at least 81 mg of
`aspirin for 7 days before the procedure (⬎90% received 325 mg) and
`were administered 325 mg on the day of the procedure and daily
`thereafter. Heparin to achieve an activated clotting time ⬎300 seconds
`was administered as a bolus to all patients in the catheterization
`laboratory immediately before stenting.
`
`Blood Sampling
`Blood was collected in evacuated container tubes containing 3.8%
`trisodium citrate that were filled to capacity and then inverted 3 to 5
`times for gentle mixing. Samples were obtained before clopidogrel
`administration (baseline) and at 2 hours, 24 hours, 5 days, and 30
`days after stenting.
`
`Platelet Aggregation
`The blood-citrate mixture was centrifuged at 1200g for 2.5 minutes.
`The resulting platelet-rich plasma was kept at room temperature for
`use within 1 hour. The platelet count was determined in the
`
`Received January 29, 2003; revision received March 20, 2003; accepted March 21, 2003.
`From the Sinai Center for Thrombosis Research (P.A.G., K.P.B., B.L.H.), Baltimore, Md, and Duke Clinical Research Institute (C.M.O.), Durham, NC.
`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
`© 2003 American Heart Association, Inc.
`
`Circulation is available at http://www.circulationaha.org
`
`DOI: 10.1161/01.CIR.0000072771.11429.83
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`platelet-rich plasma sample and adjusted to 3.5⫻108/mL with ho-
`mologous platelet-poor plasma. Platelets were stimulated with ADP
`(5 and 20 mol/L), and aggregation was assessed as described
`previously with a Chronolog Lumi-Aggregometer (model 560-Ca)
`with the AggroLink software package.6 Platelet aggregation was
`expressed as the maximal percent change in light transmittance from
`baseline, with platelet-poor plasma used as a reference. Curves were
`analyzed according to accepted standards.7
`
`Flow Cytometry
`The surface expression of platelet receptors was determined by flow
`cytometry with monoclonal antibodies. Briefly, the blood-citrate
`mixture (50 L) was diluted with 450 L of Tris buffered saline
`(10 mmol/L Tris, 0.15 mol/L sodium chloride) and mixed by
`inverting an Eppendorf tube gently 2 times. The corresponding
`antibody was then added (5 L) and incubated at room temperature
`for 30 minutes. After incubation, 400 L of 2% buffered parafor-
`maldehyde was added for fixation. The samples were analyzed on a
`Becton Dickinson FACScan flow cytometer set up to measure
`fluorescent light scatter as described previously.8 All parameters
`were collected with four-decade logarithmic amplification. The data
`were collected in list-mode files and then analyzed. The PAC-1
`antibody (Becton Dickinson) binds only to the active ␣IIb3 receptor,
`and therefore the total amount of ␣IIb3 is not determined.9 PAC-1
`was expressed as log mean fluorescence intensity. P-selectin (Pharm-
`ingen) was measured after stimulation with 200 mol/L ADP and is
`expressed as percent positivity (ie,
`the percentage of platelets
`positive for the antibody) as described previously.10 The dose of
`agonist was chosen on the basis of data reporting maximum
`expression of P-selectin induced by 100 mol/L ADP in the absence
`of an ADP blocker.5
`
`Drug Resistance Definition
`Drug resistance was defined as an absolute difference between
`baseline aggregation and posttreatment aggregation (⌬aggregation
`[%]) of 10% or less with 5 mol/L ADP used as the agonist. Because
`⌬ aggregation (%) ⫽ baseline aggregation (%) ⫺ posttreatment
`aggregation (%), a negative ⌬ aggregation would indicate poststent
`platelet reactivity greater than baseline, and a positive ⌬ aggregation
`would indicate platelet inhibition.
`
`Statistical Analysis
`The responders and nonresponders were compared with t tests.
`Standard regression analysis was used to correlate 5 and 20 mol
`ADP/L-induced aggregation and 5- and 30-day aggregation and
`P-selectin expression (Statistica software).
`To assess the effect of pretreatment reactivity on drug response,
`patients were divided into high, moderate, and low baseline reactiv-
`ity.11 Two separate analyses were performed on the basis of
`aggregation and P-selectin expression. For 5 mol/L ADP-induced
`aggregation, high reactivity was defined as percent aggregation
`⬎70%; moderate, 60% to 70%; and low, ⬍60%. For P-selectin, high
`reactivity was defined as percent positivity ⬎50%; moderate, 40% to
`50%; and low, ⬍40%. Comparisons were made between groups by
`1-way ANOVA (Statistica software). The Wilks-Shapiro test was
`used to assess conformity with a normal distribution. Curves were
`plotted of the best fit to a normal distribution by Statistica software.
`Given the normal distribution of data, the mean⫾SD and mean⫾SE
`were used. P⬍0.05 was considered significant.
`
`Results
`
`Patient Data
`Ninety-six patients had complete platelet studies performed at
`baseline, and of these patients, 92 had adequate poststent
`samples. The patient demographics on these 92 patients are
`shown with respect to the response to 5 mol/L ADP-induced
`aggregation at day 5 in the Table. The patients were elderly,
`and most were males. Multiple cardiovascular risk factors
`
`Demographics Based on Response to 5 mol/L ADP at Day 5
`
`Parameter
`
`Sex, % male
`
`Age, y
`
`Weight, lb
`
`Smoking, %
`
`⬍6 Months ago
`
`⬎6 Months ago
`
`Never
`
`Previous infarction, %
`
`Previous PTCA, %
`
`Previous CABG, %
`
`Hypercholesterolemia, %
`
`Diabetes, %
`
`Family history, %
`
`Concomitant medications, %
`
`-Blocker
`
`ACE inhibitor
`
`Calcium channel antagonist
`
`Statin
`
`Aspirin
`
`Procedural variables
`
`Responders
`
`Nonresponders
`
`63
`
`66⫾12
`
`190⫾40
`
`50
`
`69⫾10
`
`197⫾36
`
`P
`
`NS
`
`NS
`
`NS
`
`28
`
`38
`
`44
`
`26
`
`23
`
`23
`
`66
`
`42
`
`59
`
`68
`
`57
`
`13
`
`64
`
`13
`
`38
`
`50
`
`44
`
`19
`
`6
`
`50
`
`38
`
`56
`
`50
`
`81
`
`31
`
`56
`
`100
`
`100
`
`NS for all
`
`NS
`
`NS
`
`NS
`
`NS
`
`NS
`
`NS
`
`NS
`
`0.08
`
`0.09
`
`NS
`
`NS
`
`Stent length, mm
`
`19.6⫾11.7
`
`13.8⫾7.5
`
`0.007
`
`Minimal stent
`diameter, mm
`
`3.0⫾0.4
`
`3.1⫾0.5
`
`No. stents/patient
`
`1.5⫾0.4
`
`1.4⫾0.3
`
`NS
`
`NS
`
`CABG indicates coronary artery bypass graft; ACE, angiotensin-converting
`enzyme; and PTCA, percutaneous transluminal coronary angioplasty.
`NS⫽P⬎0.1.
`
`were frequent. Concomitant drug use did not differ signifi-
`cantly between groups. A trend of higher calcium antagonist
`and ACE inhibitor use was observed in the nonresponders.
`Most patients were treated with 1 stent. There were no
`significant procedural differences between responders and
`nonresponders except for total stent length. Follow-up at 30
`days revealed no cases of Q-wave myocardial infarction, stent
`thrombosis, target-vessel revascularization, cerebrovascular
`ischemic events, or death.
`
`Platelet Aggregation
`Histograms of the response to clopidogrel are shown in
`Figures 1 and 2. Baseline aggregation to 5 and 20 mol/L
`ADP was 62⫾18% and 83⫾21%, respectively. The response
`to 5 and 20 mol/L ADP showed a shift to the right between
`2 and 24 hours after treatment, which indicates increased
`platelet
`inhibition. Aggregation by 5 mol/L ADP was
`maximally inhibited by 24 hours (P⬍0.05 compared with
`baseline). Platelet aggregation was 58⫾22% at 2 hours,
`37⫾22% at 24 hours, 32⫾18% at 5 days, and 31⫾15% at 30
`days. At 2 hours after stenting, 63% of patients met the
`definition of resistance and platelet reactivity was greatest,
`with 42% of patients having greater aggregation than at
`baseline. At 24 hours, resistance fell to 31%, and 24% of
`patients still had greater aggregation than at baseline. No
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`Figure 1. Relationship between fre-
`quency of patients and absolute change
`in aggregation (⌬Aggregation [%]) in
`response to 5 mol/L ADP at 2 hours
`(A), 24 hours (B), 5 days (C), and 30 days
`(D) after stenting. ⌬Aggregation (%) is
`defined as baseline aggregation (%)
`minus posttreatment aggregation (%).
`Resistance, as defined herein, is ⌬
`Aggregation (%) ⱕ10%. Resistance is
`present in those patients subtended by
`double-headed arrow. Curves represent
`normal distribution of data and were cre-
`ated by Statistica software.
`
`further changes were seen at 5 days, when resistance was
`observed in 31%. However, at 30 days after stenting, the
`incidence of resistance fell
`to 15%, but 11% still had
`aggregation greater than baseline.
`The response to 20 mol/L ADP showed a similar pattern.
`Aggregation was 80⫾24% at 2 hours and fell to 60⫾25% at
`24 hours (P⬍0.05 compared with baseline). At 5 days,
`aggregation remained stable (57⫾23%), with a nonsignificant
`decrease at 30 days (52⫾14%). A ⌬ aggregation of 10% or
`less was present in 53% of patients at 2 hours, 35% at 24
`
`hours, 32% at 5 days, and 21% at 30 days. The correlation
`between the 5- and 20-mol/L ADP aggregation response
`was strong (r⫽0.6).
`
`Correlation of Responses at 5 and 30 Days
`A strong correlation was observed between the 5- and 30-day
`responses to 5 mol/L ADP (r⫽0.8). Moreover, strong
`correlations were also observed between 5- and 30-day
`responses for 20 mol/L ADP (r⫽0.8) and P-selectin
`(r⫽0.7).
`
`Figure 2. Relationship between fre-
`quency of patients and absolute change
`in aggregation (⌬Aggregation [%]) in
`response to 20 mol/L ADP at 2 hours
`(A), 24 hours (B), 5 days (C), and 30 days
`(D) after stenting. ⌬Aggregation (%) is
`defined as baseline aggregation (%)
`minus posttreatment aggregation (%).
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`Figure 3. Relationship between fre-
`quency of patients and absolute change
`in % positivity of stimulated P-selectin
`expression (⌬% Positivity) at 2 hours (A),
`24 hours (B), 5 days (C), and 30 days (D)
`after stenting. ⌬% Positivity is defined as
`baseline positivity (%) minus posttreat-
`ment positivity (%).
`
`Platelet Receptor Expression
`P-Selectin
`Baseline stimulated P-selectin expression was 45⫾16% and fell
`over 24 hours, as indicated by a shift in the curve to the right (Figure
`3). Maximum inhibition of P-selectin expression occurred within 24
`hours (24⫾13%; P⬍0.05 compared with baseline) and was un-
`changed at 5 days (22⫾13%) and 30 days (23⫾10%), as observed
`in the aggregation studies. An absolute change in percent positivity
`of 10% or less was observed in 44% of patients at 2 hours, 25% at
`24 hours, 12% at 5 days, and 29% at 30 days, which again suggests
`resistance to the standard clopidogrel regimen.
`
`PAC-1
`PAC-1 binding showed similar response variability (Figure
`4). Baseline expression was 14.9⫾13.1, and inhibition of the
`
`expression of active GP IIb/IIIa was maximal within 24 hours
`(8.5⫾5.1; P⬍0.05 compared with baseline). No significant
`changes as compared with 24 hours were observed at 5 days
`(8.1⫾3.7) or 30 days (8.6⫾5.3).
`
`Effect of Pretreatment Platelet Reactivity on
`Drug Response
`High pretreatment reactivity, defined by the response to 5
`mol/L ADP, was present in 31 patients, moderate reactivity
`in 25 patients, and low reactivity in 40 patients. High-
`reactivity patients had a greater incidence of diabetes (71%;
`P⬍0.05) than those with moderate (24%) and low (40%)
`reactivity. Patient weight, gender, age, statin use, smoking
`history, incidence of hyperlipidemia, history of prior infarc-
`
`Figure 4. Relationship between fre-
`quency of patients and absolute change
`in mean fluorescence intensity (MFI) of
`PAC-1 binding (⌬MFI) at 2 hours (A), 24
`hours (B), 5 days (C), and 30 days (D)
`after stenting. ⌬MFI is defined as base-
`line MFI minus posttreatment MFI.
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`Figure 5. A, ADP-induced platelet aggre-
`gation (5 mol/L ADP) in high-,
`moderate-, and low-reactivity groups at
`baseline and at 1 and 5 days after clopi-
`dogrel therapy. High-reactivity patients
`were defined as pretreatment percent
`aggregation ⬎70%; moderate, 60% to
`70%; and low, ⬍60%. B, Stimulated
`P-selectin expression in high-,
`moderate-, and low-reactivity groups at
`baseline and at 1 and 5 days after clopi-
`dogrel therapy. High-reactivity patients
`were defined as pretreatment percent
`positivity ⬎50%; moderate, 40% to
`50%; and low, ⬍40%.
`
`tion, total contrast load, procedure duration, and number of
`vessels treated were not significantly different between
`groups.
`
`Platelet Aggregation
`the high-reactivity group had
`At baseline, by definition,
`markedly greater reactivity (78⫾6%) than the moderate
`(65⫾3%; P⬍0.00001) and low (48⫾9%; P⬍0.00001) groups
`(Figure 5A). At day 1 after stenting, the high-reactivity group
`continued to have the most reactive platelets (67⫾11%;
`P⫽0.004 versus moderate [56⫾15%] and P⬍0.0001 versus
`low [52⫾15%]). By day 5, platelet reactivity by this marker
`was similar among groups.
`
`P-Selectin Expression
`At baseline, the high-reactivity group had greater expression
`(67⫾7%) than the moderate (44⫾3%; P⬍0.00001) and low
`(29⫾6%; P⬍0.00001) groups (Figure 5B). The effect that
`pretreatment reactivity had on the inhibitory response to
`clopidogrel at day 1 was remarkably similar to findings with
`ADP-induced light-transmittance aggregometry. At day 1,
`patients with high pretreatment P-selectin expression re-
`mained the most reactive (P⬍0.001 versus low reactivity). At
`day 5 of therapy, patients in the high-reactivity group had a
`trend to greater P-selectin expression than the moderate
`(31⫾12% versus 19⫾13%; P⫽0.06) and low (20⫾7%;
`P⫽0.04) groups.
`
`Discussion
`The present study illustrates the variable platelet inhibitory
`response to the standard administered dose of clopidogrel.
`The platelet aggregation studies used 2 agonist concentrations
`that showed a strong correlation. In addition, platelet receptor
`expression showed similar findings. These uniform observa-
`tions,
`irrespective of the methodology chosen to detect
`inhibition, strengthen our conclusions that the response to
`clopidogrel therapy is indeed heterogeneous and that drug
`resistance occurs. Our observations are in agreement with one
`other report of 18 patients with stable angina treated with the
`same clopidogrel
`regimen after coronary intervention.12
`Those investigators demonstrated variable inhibition of ADP-
`induced fibrinogen binding on day 2 after stenting.
`Our definition of drug resistance was empirical because
`there have been no extensive reports on this subject among
`patients treated with clopidogrel. Clopidogrel inhibits aggre-
`gation in response to ADP, and therefore, we studied the
`response to 2 different concentrations of this agonist with
`light-transmittance aggregometry. Moreover, we assessed the
`expression of an established marker of platelet activation
`(P-selectin) in response to a maximal agonist concentration
`and studied the response of a sensitive platelet activation–
`dependent marker (PAC-1 binding) in nonstimulated blood.5,9
`The present study suggests that the maximum inhibitory
`response to a 300-mg loading dose followed by 75 mg/d
`occurs within 24 hours. These findings are consistent with
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`reports in healthy volunteers and in patients undergoing
`coronary stenting.13,14
`The response to clopidogrel appears to be patient specific.
`The robust correlations demonstrated that in most patients,
`the 30-day inhibitory response from clopidogrel was pre-
`dicted by the 5-day response. Of equal
`importance,
`the
`present investigation also suggests that resistance to clopi-
`dogrel does not accrue over time.
`The present study is the first to demonstrate that the level
`of platelet reactivity after the standard clopidogrel regimen
`for coronary stenting is critically dependent on the pretreat-
`ment reactivity. The present in vitro tests suggest that patients
`with the greatest pretreatment platelet activity have the least
`antithrombotic protection, particularly within the first 24
`hours of therapy. The level of platelet reactivity has been
`correlated with adverse events by others.4 Moreover, an
`examination of P-selectin expression suggests that this rela-
`tionship is true even after 5 days of therapy, when those
`patients with the greatest baseline expression of
`this
`activation-dependent receptor tended to be more reactive than
`those with baseline low or moderate expression. The present
`findings may help to explain why ticlopidine without a
`loading dose did not prevent stent thrombosis in the first 3
`days after the procedure.15 The similar findings at 24 hours
`using 2 different established markers of platelet activity
`strengthen our conclusion that the response to clopidogrel
`therapy is indeed dependent on pretreatment reactivity. Pre-
`vious investigations using aggregometry and P-selectin ex-
`pression as markers of reactivity have shown that loading
`doses higher than 300 mg may enhance and accelerate
`platelet inhibition in patients undergoing coronary interven-
`tions.16 –19 Similar strategies may particularly benefit patients
`with high pretreatment reactivity.
`
`Limitations
`The present study included patients undergoing elective
`coronary stenting, which is known to increase platelet reac-
`tivity.8 Because pretreatment reactivity affected the reactivity
`measured after antiplatelet therapy, postdrug platelet reactiv-
`ity may be less in studies of healthy volunteers and in patients
`with stable coronary artery disease. Our definition of resis-
`tance involves the amplitude of maximal platelet aggregation
`and can be influenced by various factors, including intrapa-
`tient variability. The current rates of stent thrombosis ob-
`served in elective stenting are much lower than the incidence
`of clopidogrel resistance in the present study, which suggests
`that our definition may be an overestimate or that resistance
`to clopidogrel
`is not a primary factor influencing stent
`thrombosis in these patients. However, the present data imply
`that nonresponders with high pretreatment reactivity may be
`at greatest risk.
`inhibitory response to the
`the platelet
`In conclusion,
`standard dosing regimen of clopidogrel for coronary stenting
`is variable, follows a normal distribution, and appears stable
`over 30 days. Patients with high pretreatment reactivity are
`the least protected within the first 5 days of treatment. Further
`study is necessary to investigate the mechanisms of these
`findings and how they correlate with the occurrence of
`
`ischemic events. The present work would also support further
`investigations to determine whether higher clopidogrel doses
`may overcome interindividual differences in drug response.
`
`Acknowledgments
`Supported by the Sinai Center for Thrombosis Research and Platelet
`and Thrombosis Research, LLC.
`
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`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1036, p. 6 of 6
`
`
`
`
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