Randomized Comparison of a High Clopidogrel Maintenance Dose in Patients With Diabetes Mellitus and Coronary Artery Disease Results of the Optimizing Antiplatelet Therapy in Diabetes

Randomized Comparison of a High ClopidogrelMaintenance Dose in Patients With Diabetes Mellitus and

Coronary Artery DiseaseResults of the Optimizing Antiplatelet Therapy in Diabetes Mellitus

(OPTIMUS) Study

Dominick J. Angiolillo, MD, PhD; Steven B. Shoemaker, MD; Bhaloo Desai, PhD; Hang Yuan, PhD;Ronald K. Charlton, PhD; Esther Bernardo, BSc; Martin M. Zenni, MD; Luis A. Guzman, MD;

Theodore A. Bass, MD; Marco A. Costa, MD, PhD

Background—After treatment with clopidogrel, patients with type 2 diabetes mellitus (T2DM) have reduced plateletinhibition compared with patients who are not diabetic. Whether platelet inhibition can be enhanced by increasingclopidogrel maintenance dosage in T2DM patients is unknown. The aim of this pilot study was to assess the functionalimpact of a high maintenance dose in T2DM patients with suboptimal clopidogrel-induced antiplatelet effects.

Methods and Results—T2DM patients on chronic dual antiplatelet therapy were screened to identify suboptimalclopidogrel responders. The latter were randomized to 30-day treatment with a standard (75 mg; n�20) or high (150mg; n�20) daily maintenance dose. Platelet function was assessed at 3 time points: baseline, 30 days afterrandomization, and 30 days after resuming standard dosing. Platelet function parameters included adenosinediphosphate–induced (20 and 5 �mol/L) maximal and late platelet aggregation, inhibition of platelet aggregation,platelet disaggregation, and P2Y12 reactivity index. A total of 64 T2DM patients were screened to identify 40 suboptimalresponders. After randomization, maximal adenosine diphosphate–induced (20 �mol/L) platelet aggregation wassignificantly reduced in the 150-mg group compared with the 75-mg group (P�0.002; primary end point). However,suboptimal clopidogrel response was still present in 60% of patients on the 150-mg regimen. All other platelet functionparameters showed enhanced clopidogrel-induced antiplatelet effects with 150 mg, which returned to baseline valuesafter resumption of standard dosing.

Conclusions—A 150-mg maintenance dose of clopidogrel is associated with enhanced antiplatelet effects compared with75 mg in high-risk T2DM patients. However, enhanced ex vivo platelet reactivity continues to persist, the clinicalimplications of which are unknown and need to be evaluated in large-scale clinical trials. (Circulation. 2007;115:708-716.)

Key Words: clopidogrel � diabetes mellitus � platelets � thrombosis

The P2Y12 receptor plays a pivotal role in platelet aggre-gation.1 This role is emphasized by the results of clinical

trials that demonstrate improvement of long-term clinicaloutcomes in patients treated with the P2Y12 receptor antago-nist clopidogrel.2–4 However, a high interindividual variabil-ity in platelet response to clopidogrel has been described.5,6

The fact that subjects with suboptimal platelet inhibitionby clopidogrel are at increased risk of cardiovascularischemic events7–10 represents an alarming clinical problem.

Clinical Perspective p 716Inadequate dosing has been postulated as one of the

mechanisms for suboptimal clopidogrel-induced antiplatelet

effects.11,12 Previous investigations have shown that admin-istration of a higher clopidogrel loading dose enhancesplatelet inhibition and leads to better response profiles thanstandard dosing.11,12 In addition, results from small studiessuggest that, in patients undergoing percutaneous coronaryinterventions, the use of a high loading dose of clopidogrelmay improve clinical outcomes.13,14 Despite the short-termimprovement in platelet response with high loading dosageregimens of clopidogrel, patients may continue to haveelevated platelet reactivity in the maintenance phase ofantiplatelet therapy.15–19 Of note, readministration of a load-ing dose of clopidogrel in patients already on maintenance

Received October 3, 2006; accepted December 19, 2006.From the Division of Cardiology (D.J.A., S.B.S., B.D., H.Y., E.B., M.M.Z., L.A.G., T.A.B., M.A.C.), University of Florida College of Medicine at

Shands Jacksonville, and the Jacksonville Transplant Center (R.K.C.), Shands Jacksonville, Jacksonville, Florida.Correspondence to Dr Dominick J Angiolillo, Division of Cardiology University of Florida, Shands Jacksonville, 655 West 8th Street, Jacksonville,

FL 32209. E-mail [email protected]© 2007 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.667741

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treatment evokes further platelet inhibition.20 This observa-tion has raised the hypothesis that an increase of the mainte-nance dose of clopidogrel may further enhance plateletinhibition, particularly in high-risk patients. However, plate-let function assessments with a high clopidogrel maintenancedose regimen in high-risk patients are still lacking.

Patients with type 2 diabetes mellitus (T2DM) are charac-terized by enhanced platelet reactivity and reduced in vitroresponsiveness to antiplatelet agents.18,19 Recent studies haveshown that T2DM patients have reduced response to P2Y12

receptor antagonists, including clopidogrel, compared withnondiabetic subjects.18,19,21 T2DM patients are characterizedby an upregulated status of the P2Y12 pathway,21,22 which intheory may be overcome by the use of more potent P2Y12

antagonists or higher doses of clopidogrel.The aim of the this pilot study was to assess the impact of

a high maintenance dose of the P2Y12 receptor antagonistclopidogrel on platelet function profiles in T2DM patientswith coronary artery disease (CAD) on long-term dual anti-platelet therapy who demonstrate suboptimal platelet inhibi-tion. Our hypothesis was that a daily maintenance dose of 150mg clopidogrel will lead to enhanced platelet inhibitioncompared with conventional dosage (75 mg) in these high-risk patients.

MethodsPatient Population and Study DesignPatients were eligible for the study if they had T2DM according tocriteria from the World Health Organization Report, were on insulinor oral hypoglycemic medication, and were between the ages of 25and 80 years.23 All patients included had documented CAD, as allpatients had previously undergone percutaneous coronary interven-tions, and were in a steady state phase of clopidogrel treatment (75mg daily). Pharmacokinetic and pharmacodynamic profiles may varyprofoundly in the initial days or weeks after initiation of dualantiplatelet therapy.5,24 Thus, only patients in the maintenance phaseof treatment (�30 days from initiation) were included. All patientswere also treated with low-dose aspirin (81 mg daily). This dose ofaspirin was chosen to reduce bleeding risk in patients on dualantiplatelet therapy.25 Major exclusion criteria included knownallergies to aspirin or clopidogrel; impaired glucose tolerance orT2DM without pharmacological treatment, gestational diabetes, or

transient hyperglycemia; blood dyscrasia; serum creatinine level �2mg/dL; active bleeding or bleeding diathesis; gastrointestinal bleed-ing within prior 6 months; hemodynamic instability; cerebrovascularaccident within 3 months; any malignancy; concomitant use of otherantithrombotic drugs such as oral anticoagulants, dypiridamole,ticlopidine, or cilostazol, or nonsteroid anti-inflammatory drugs;recent treatment (�30 days before enrollment) with a glycoproteinIIb/IIIa antagonist; platelet count �100�106/�L; hematocrit �25%;liver disease (bilirubin level �2 mg/dL).

The Optimizing anti-Platelet Therapy In diabetes MellitUS(OPTIMUS) study was a randomized, single-center, prospective,parallel-group platelet function study of patients with T2DM andCAD. Patients were recruited from the outpatient cardiology clinic ofour hospital. All patients who met inclusion criteria underwentscreening to define their degree of posttreatment platelet reactivityand identify patients with suboptimal clopidogrel-induced antiplate-let effects (study time point 1). Only patients with suboptimalclopidogrel responses were eligible for randomization. With the useof a computer-based randomization system, suboptimal clopidogrelresponders were assigned to receive a daily clopidogrel maintenancedose with either 150 mg (two 75-mg tablets) or 75 mg (one 75-mgtablets). After randomization, the assigned clopidogrel maintenancedose regimen was maintained for 30 days, at which time plateletfunction was reassessed (study time point 2). Thereafter, all patientsresumed the standard 75-mg daily dose and platelet function wasassessed once again after 30 days (study time point 3). Theflow-diagram of the study is represented in Figure 1. Patientcompliance to antiplatelet treatment was assessed by interview andpill counting.

Blood sampling was performed 2 to 4 hours after drug intake.Samples were processed within 1 hour after blood drawing byoperators blind to patient’s treatment assignment. Laboratory per-sonnel were also blinded to treatment assignments.

The study complied with the Declaration of Helsinki, was ap-proved by the Institutional Review Board of the University of FloridaCollege of Medicine Jacksonville, and all patients gave their in-formed written consent. An independent data safety monitoringcommittee was instituted for adjudication of adverse clinical events.The authors had full access to the data and take full responsibility fortheir integrity. All authors have read and agree to the manuscript aswritten.

Platelet Function AnalysisBlood samples for platelet function assays were collected from anantecubital vein using a 21-gauge needle 2 to 4 hours after antiplate-let therapy intake. The first 2 to 4 mL of blood were discarded toavoid spontaneous platelet activation. Platelet function measures

Figure 1. Flow diagram of the study.

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included assessments of platelet aggregation and the P2Y12 reactivityratio.

Platelet aggregation was performed with light transmittance ag-gregometry in all patients according to standard protocols.15–19 Inbrief, blood was collected in sodium-citrated tubes. Platelet aggre-gation was assessed with platelet-rich plasma (PRP) by the turbidi-metric method in a 2-channel aggregometer (Chrono-Log 490Model, Chrono-Log Corp, Havertown, Pa) after stimulus with 5 or20 �mol/L adenosine diphosphate (ADP). PRP obtained as asupernatant after centrifugation of citrated blood at 800 rpm for 10minutes. The isolated PRP was kept at 37°C before use. Platelet-poorplasma was obtained by a second centrifugation of the blood fractionat 2500 rpm for 10 minutes. The platelet count in PRP was adjustedto the range of 250 000/�L by dilution with autologous plasma whenplatelet count was out of range. Light transmission was adjusted to0% with PRP and to 100% platelet-poor plasma for each measure-ment. Curves were recorded for 6 minutes. Aggregation was mea-sured at maximal aggregation (Aggmax) and at 5 minutes (Agglate).Aggmax is reflective of the activity of both the P2Y1 (involved ininitiation of changes in platelet shape and platelet aggregation) andP2Y12 (involved in stabilization of the platelet aggregates) purinergicreceptors.26 However, clopidogrel only blocks the P2Y12 receptor.Therefore, Agglate is more reflective of the activity of the P2Y12

receptor and stabilization of aggregation. Inhibition of plateletaggregation (IPA) was defined as the percent decrease in aggregationvalues (Aggmax and Agglate) obtained at baseline and after treatment:IPA (%) � (intensity of aggregation at baseline) � (intensity ofaggregation at 30 days) � (intensity of aggregation at baseline).Percentage of platelet disaggregation between Aggmax and Agglate

values was defined as: disaggregation (%) � 100 � (1 � Agglate �Aggmax).26 Arachidonic acid-induced platelet aggregation was alsoperformed in order to assess compliance and responsiveness toaspirin,27 defined as Aggmax �20% after stimulus with 0.5 mg/mLarachidonic acid.

The P2Y12 reactivity ratio was determined through assessment ofthe phosphorylation status of the vasodilator-stimulated phosphopro-tein (VASP) by flow cytometry according to standard protocols.28

The phosphorylation status of VASP (VASP-P) was measured byquantitative flow cytometry (Beckman Coulter FC500, Miami, Fla).In brief, VASP-P levels were quantified with labeled monoclonalantibodies with a commercially available kit (Biocytex Inc., Mar-seille, France). The P2Y12 reactivity ratio was calculated aftermeasurement of VASP-P levels after stimulation with PGE1 by meanfluorescence intensity (MFI PGE1) and also PGE1 � ADP (MFIPGE1 � ADP). The P2Y12 reactivity ratio is ([MFI PGE1] � [MFIPGE1 � ADP] � [MFI PGE1]) � 100%. A reduced P2Y12 reactivityratio is indicative of more enhanced clopidogrel-inducedinhibition.28

Definition of Suboptimal Clopidogrel RespondersClopidogrel responsiveness was defined according to the percentageof Aggmax with light transmittance aggregometry. Patients with a 20�mol/L ADP-induced Aggmax �50% were considered as suboptimalresponders. This Aggmax threshold value was selected on the basis ofprevious findings demonstrating that patients with such degrees ofposttreatment platelet reactivity are at higher risk of ischemicevents.10

End Points and Sample Size CalculationThe primary end point of the study was Aggmax after stimulus with 20�mol/L ADP 30 days after randomization (at study time point 2). Wehypothesized that Aggmax at this time point would be 45�15% inpatients randomized to a maintenance dose of 150 mg versus65�15% in patients maintained on a clopidogrel dose of 75 mg.Estimation of platelet function values were based on our previousreports in patients with T2DM.18–19 Thus, 17 patients per groupwould be required to provide a power of 80% to detect statisticaldifference between groups with a 2-sided �-level of 0.05.

Other platelet function measures included Aggmax after stimuluswith 5 �mol/L ADP; Agglate after stimulus with 5 and 20 �mol/LADP; IPA of Aggmax and Agglate after stimulus with 5 and 20 �mol/L

ADP; disaggregation after stimulus of 5 and 20 �mol/L ADP; andP2Y12 reactivity ratio. Inter- and intragroup group comparisons ofthese platelet function measures were performed at various timepoints.

Statistical AnalysisNormally distributed continuous variables are presented asmeans�SD. Variables were analyzed for a normal distribution withthe Kolmogorov-Smirnov test. Categorical variables are expressed asfrequencies and percentages. Differences between groups wereassessed with the Fisher exact test for categorical variables. Paired ttests were used for comparison of normally distributed continuousvariables in the same group. Unpaired t tests were used forcomparison of normally distributed continuous variables between the2 groups. P�0.05 was considered statistically significant. Statisticalanalysis was performed with SPSSv14.0 software (SPSS Inc. Chi-cago, Ill).

ResultsPlatelet function screening was performed in a total of 64patients with an Aggmax of 58.05�14.5% induced by 20�mol/L ADP. Of these, 40 patients (62.5%) showed subop-timal clopidogrel response and were randomized. At studytime point 1, Aggmax induced by 20 �mol/L ADP was66.2�8% in the 40 patients with suboptimal clopidogrelresponse, versus 39.9�8% in the remaining 24 patients noteligible for randomization. No differences in baseline demo-graphics existed between patients eligible and ineligible forrandomization (data not shown). Baseline demographics ofpatients randomized to daily clopidogrel maintenance dosesof 75 mg (n�20) versus 150 mg (n�20) are shown in Table1. No differences in baseline characteristics existed except forslightly higher age in patients randomized to the maintenancedose of 150 mg clopidogrel. Use of CYP3A4- and non–CYP3A4-metabolizing statins was similar between the 2groups. CYP3A4-metabolizing statins were not associatedwith clopidogrel-induced antiplatelet effects in the presentstudy. Platelet counts (109/mL) were similar between the 2groups: 307�86 (75-mg group) versus 279�54 (150-mggroup) (P�0.21). All patients were compliant and responsiveto aspirin. No bleeding complications occurred during thestudy. No changes in medical therapy, which included T2DMmedications or antianginal therapy, occurred during thestudy. Finally, no patients interrupted clopidogrel or aspirintherapy as a result of side effects.

Maximal ADP-Induced Platelet Aggregation ProfilesNo differences existed in baseline (study time point 1) Aggmax

after stimulus with 20 �mol/L ADP between the 2 groups(Table 2). One month after randomization (study time point2), patients assigned to a clopidogrel maintenance dose of 150mg experienced a significant reduction in Aggmax comparedwith their baseline values and compared with patients ran-domized to a dose of 75 mg (P�0.002; Figure 2). No changesin Aggmax were observed among patients randomized tostandard dosing. Eight patients in the 150-mg maintenancedose group (40%) had an Aggmax after stimulus with 20�mol/L ADP �50% at study time point 2 versus none in the75-mg dose group. Intergroup comparisons also showedhigher IPA in the 150-mg maintenance dose group (Figure3A). Cumulative distribution curves of changes in Aggmax andIPA after stimulus with 20 �mol/L ADP illustrate the

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individual changes in clopidogrel-induced antiplatelet effectsbetween baseline (study time point 1) and 30 days afterrandomization (study time point 2) (Figure 4). In patients onstandard dosing, individual response profiles varied over timebut overall did not yield any significant changes, and allpatients persisted at both time points with Aggmax �50%. Inpatients randomized to a high-dose regimen, all patients had

a decrease in posttreatment platelet reactivity values and anincrease in IPA. The change in posttreatment platelet reac-tivity and IPA was �10% in 75% and 85% of these patients,respectively.

No differences existed in baseline Aggmax values afterstimulus with 5 �mol/L ADP between the 2 groups (Table 2).One month after randomization, patients assigned to a150-mg clopidogrel maintenance dose experienced a signifi-cant reduction in Aggmax, whereas no significant changes wereobserved in patients kept on standard dosing (Table 2).Intergroup comparison showed lower posttreatment plateletreactivity (Table 2) and higher IPA (Figure 3A) in the highmaintenance group.

At study time point 3, patients assigned to 150-mg doseexperienced a return to Aggmax values comparable to thoseobserved at baseline after a switch back to standard dosing(Table 2). In patients assigned to standard dosing, no signif-icant differences in platelet aggregation existed at all 3 studytime points (Table 2), a finding that demonstrated a persistentsuboptimal response to standard clopidogrel maintenancedose.

Late ADP-Induced Platelet Aggregation ProfilesNo differences existed in baseline (study time point 1) Agglate

parameters between the 2 groups (Table 2). One month afterrandomization (study time point 2), patients assigned to a150-mg clopidogrel maintenance dose experienced a 20%and 15% reduction in Agglate after stimulus with 20 and 5�mol/L ADP, respectively (Table 2). No significant changeswere observed in patients on the 75-mg dose (Table 2).Intergroup comparison also showed lower platelet reactivity(Table 2) and higher IPA (Figure 3B) in patients assigned toa 150-mg dose at study time point 2. At study time point 3,results paralleled those of Aggmax. (Table 2).

Platelet DisaggregationDisaggregation of ADP-aggregated platelets by clopidogrelwas similar in both groups at baseline after stimulus with 20and 5 �mol/L (Figure 5). One month after randomization,patients assigned to a high clopidogrel maintenance doseexperienced an increase in platelet disaggregation, whereasno significant changes were observed in patients kept onstandard dosing (Figure 5). Enhanced platelet disaggregation

TABLE 1. Demographics of the Study Population

75 mg(n�20)

150 mg(n�20) P

Age, y 59�10 64�8 0.05

Male, n (%) 14 (70) 12 (60) 0.75

Race, n (%)

White 11 (55) 13 (65) 0.75

Black 8 (40) 6 (30) 0.74

Hispanic 1 (5) 1 (5) 1.00

Risk factors/past medical history, n (%)

Insulin-dependent diabetes mellitus 6 (30) 8 (40) 0.74

Non–insulin-dependent diabetes mellitus 14 (70) 12 (60) 0.74

HbA1C 7.0�1.1 7.1�1.5 0.93

Smoking 4 (20) 3 (15) 1.00

Hyperlipidemia 19 (95) 18 (90) 1.00

Hypertension 19 (95) 18 (90) 1.00

Body mass index, kg/m2 32.4�7 33.5�6 0.61

Prior myocardial infarction 8 (40) 7 (35) 1.00

Prior CABG 4 (20) 5 (25) 1.00

Multivessel CAD 15 (75) 14 (70) 1.00

Treatment, n (%)

�-Blockers 17 (85) 16 (80) 0.74

Nitrates 6 (30) 8 (40) 1.00

ACE inhibitors/ARB 13 (65) 14 (70) 1.00

Lipid-lowering agents

CYP3A4 metabolizing statin 17 (85) 15 (75) 0.69

Non-CYP3A4 metabolizing statin 1 (5) 4 (20) 0.34

HbA1C indicates hemoglobin A1C; CAD, coronary artery disease; CABG,coronary artery bypass grafting; ACE, angiotensin-converting enzyme; ARB,angiotensin II receptor blockers; and CYP3A4, cytochrome P450 3A4 isoen-zyme.

Values are expressed as mean�SD unless otherwise indicated

TABLE 2. Aggmax and Agglate Values After Stimulus With 20 �mol/L and 5 �mol/L ADP in Patients Randomized to a Standard (75mg) or High (150 mg) Daily Maintenance Dose

Study Time Point 1 Study Time Point 2 Study Time Point 3

20 �mol/L ADP 5 �mol/L ADP 20 �mol/L ADP 5 �mol/L ADP 20 �mol/L ADP 5 �mol/L ADP

Aggmax Agglate Aggmax Agglate Aggmax Agglate Aggmax Agglate Aggmax Agglate Aggmax Agglate

75 mg 64.9�9 59.7�12 50.7�11 41.3�15 63.1�7 58.0�11 51.0�9 41.1�13 62.5�11 58.3�14 48.4�12 38.7�18

150 mg 67.4�6 63.2�7 51.2�8 41.5�11 52.3�13* 43.3�13* 39.1�12* 26.1�17* 64.3�6 58.5�10 48.5�8 39.1�11

P 0.32 0.28 0.84 0.96 0.002 �0.0001 0.001 0.003 0.55 0.97 0.98 0.93

Values are expressed as mean�SD of percentage (%) platelet aggregation. P values reported in the table refer to intergroup comparisons. At study time point 2,patients in the 150-mg dose group had Aggmax and Agglate significantly lower versus study time points 1 and 3 (*P�0.001 for intra- and intergroup comparisons).Intra- and intergroup comparisons showed no differences in Aggmax and Agglate between study time points 1 and 3. Intergroup comparisons of Aggmax and Agglate atstudy time point 2 in the 75-mg dose group were not different from values at study time points 1 and 3 in the 150-mg dose group. No differences in Aggmax andAgglate were observed in the 75-mg group at all study time points.

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was observed in patients assigned to a 150-mg versus thosereceiving a 75-mg maintenance dose. Thirty days afterdiscontinuation of high clopidogrel dose, disaggregation val-ues returned to values comparable with baseline (Figure 5).

P2Y12 Reactivity RatioAt baseline (study time point 1), the P2Y12 reactivity ratio wassimilar in patients randomized to standard and high clopi-dogrel maintenance dosing (Figure 6). One month afterrandomization (study time point 2), the P2Y12 reactivity ratiosignificantly reduced in patients assigned to the high clopi-dogrel maintenance dose regimen; the P2Y12 reactivity ratiowas also lower compared with patients kept on standarddosing. The P2Y12 reactivity ratio returned to values compa-rable with baseline in patients randomized to a high clopi-dogrel maintenance dose regimen after resumption of astandard clopidogrel maintenance dose, whereas no signifi-cant changes were observed in patients on standard dosing(Figure 6).

DiscussionThis randomized prospective study is the first to confirm thehypothesis that a high maintenance dose (150 mg daily) ofclopidogrel in a high-risk group of patients with T2DM andCAD enhances platelet inhibition, as assessed by multiplebiological determinants of platelet function, compared with astandard dose (75 mg daily). The dose-dependent effect ofclopidogrel was confirmed by the return to baseline values ofall platelet function measures after discontinuation of thehigh-dose regimen and recommencement of standard dosing.However, despite an overall improvement in platelet functionprofiles among patients randomized to a high maintenancedose of clopidogrel, response to antiplatelet therapy remainedvariable and a considerable proportion of patients persistedwith enhanced platelet reactivity. In addition, our studyprovides further evidence that most patients with T2DMcontinue to exhibit increased platelet reactivity despite dual

antiplatelet therapy, which makes this high-risk group a targetpopulation for evaluation of more aggressive antiplatelettreatment regimens.

In patients diagnosed with acute coronary syndromesand/or treated with percutaneous coronary interventions,adjunctive treatment with clopidogrel in combination withaspirin has been shown to be superior to aspirin alone forsecondary prevention of ischemic events, including in pa-tients with diabetes mellitus.3,4 In addition, clopidogrel hasbeen shown to be superior to aspirin, especially in patientswith diabetes mellitus.2,29 However, despite the clinical ben-efit associated with clopidogrel treatment, diabetic patientscontinue to have a higher risk of ischemic events comparedwith nondiabetic patients, a difference that may be attributed,at least in part, to suboptimal responsiveness to antiplatelettherapy.18,19

Suboptimal clopidogrel-induced antiplatelet effects observedwith currently recommended dosing regimens have been re-ported by various investigators with the suggestion that theycontribute to poor clinical outcomes.5–14 Dose-finding studiesthat support the currently recommended doses of clopidogrelwere designed to achieve a level of platelet inhibition similar to500 mg of ticlopidine per day.30 These dose-finding studies didnot take into consideration the prothrombotic milieu of high-risksubjects, such as T2DM. Previous reports have shown thatsuboptimal clopidogrel responsiveness poses considerable riskof ischemic events.7–10,28,31 High loading doses (�600 mg) havebeen proposed as a strategy to accelerate and enhance plateletinhibition compared with a standard loading dose of 300 mg andto improve short-term clinical outcomes compared with standardclopidogrel therapy.11–14 However, the antiplatelet effects ofclopidogrel front-loading are confined to the initial phase oftherapy, and patients rely on their daily maintenance dose forlong-term prevention of ischemic events. Our study confirmsthat a long-term maintenance strategy with standard recom-mended doses is suboptimal in most patients with T2DM andCAD. Furthermore, our study shows that improvements in

Figure 2. Maximal platelet aggregationafter stimulus with 20 �mol/L ADPassessed at study time points 1 (T1), 2(T2), and 3 (T3). Values are expressed aspercentage (%) of maximum plateletaggregation. Boxes represent individualmeasurements; bars denote means�SD.

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platelet inhibition are transient and confined to the period of highdoses of clopidogrel therapy, and that high platelet reactivity isa persistent state in these patients.

Patients with T2DM have increased platelet reactivity andreduced in vitro responsiveness to antiplatelet agents, whichinclude P2Y12 receptor antagonists, compared with nondia-betic subjects.18,19,21 Numerous mechanisms can account forplatelet dysfunction in T2DM.31 In vitro studies have shownthat insulin reduces platelet aggregation by inhibition of theP2Y12 pathway.21,22,32 In fact, human platelets are targets ofthe effects of insulin, which interacts with its own receptor onthe platelet surface and leads to loss of Gi activity. This lossreduces suppression of cyclic adenosine monophosphate,which thus inhibits P2Y12 signaling and reduces plateletreactivity.21,22 However, platelets of T2DM patients are alsotargets of the insulin resistance phenomenon that typicallyaffects T2DM patients and results in decreased sensitivity toinsulin.32 This decreased sensitivity leads in turn to upregu-lation of the P2Y12 pathway and increased platelet reactivityin diabetic platelets. Other mechanisms responsible for sub-optimal clopidogrel-induced antiplatelet effects in T2DMinclude increased exposure to ADP, increased cytosolic levelsof calcium, and increased platelet turnover.32

Suboptimal responsiveness to clopidogrel represents anemerging clinical entity. This phenomenon has been associ-

ated with recurrence of ischemic events, which include stentthrombosis.7–10,28,31 Stent thrombosis may lead to severeconsequences and has become a major concern in the era ofdrug-eluting stents. Importantly, diabetes mellitus has beenidentified as an independent predictor of stent thrombosis.33

Current guidelines (class IIb indication, level of evidence C)state that in patients in whom stent thrombosis may becatastrophic or lethal, platelet aggregation studies may beconsidered and the dose of clopidogrel increased to 150 mgper day if �50% inhibition of platelet aggregation is demon-strated.34 A similar approach may be utilized in clinicalpractice, but essentially no data exist on the efficacy, eitherbiological or clinical, of this treatment regimen.

The present study provides the first biological evidencethat 150 mg of clopidogrel may indeed improve plateletinhibition in high-risk patients who are suboptimal respond-ers under current standard antiplatelet therapy. Nevertheless,our study also shown that more than half of the patientscontinued to exhibit platelet aggregation �50%, which hasbeen considered a therapeutic threshold for P2Y12 inhibi-tion.10 These findings highlight the potential need for moreeffective antithrombotic strategies to tackle the diabeticplatelet and that individualized therapy may be required.35

However, individualized therapy with higher maintenancedoses of current medications or with additional or morepotent antiplatelet agents is not yet supported by clinicalstudies. Furthermore, large-scale clinical studies have clearlyshown the benefit of standard antiplatelet treatment regimens

Figure 3. Inhibition of maximal (A) and late (B) platelet aggrega-tion between baseline (study time point 1) and 30 days (studytime point 2) assessed after stimulus with 20 �mol/L and 5�mol/L ADP in patients randomized to clopidogrel maintenancedoses of 75 mg and 150 mg. Values are expressed as percent-age (%) of platelet aggregation. Error bars indicate standarddeviations of the mean.

Figure 4. Cumulative distribution curves of change of maximalplatelet aggregation (A) and inhibition of maximal platelet aggre-gation (B) between baseline (study time point 1) and 30 days(study time point 2) after stimulus with 20 �mol/L ADP inpatients randomized to clopidogrel maintenance doses of 75 mgand 150 mg. Values are expressed as percentage (%) plateletaggregation. Boxes represent individual changes.

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in diabetic patients, as recommended in current guidelines,and the results of this pilot ex vivo platelet functionanalysis should not lead physicians to change their medicalpractice, as they have not been shown to have clinicalimplications.2–4,29,34 Novel and more potent oral P2Y12 recep-tor antagonists and drugs that inhibit platelet thrombin recep-tors are currently under clinical investigation and have beenshown to have less response variability. These agents mayrepresent potential future alternatives for treating these high-risk patients.36,37

ConclusionIn conclusion, this study demonstrates that the currentlyrecommended maintenance dose of clopidogrel is associated

with a high incidence of suboptimal response among patientswith T2DM and CAD. Based on the results of various plateletfunction assessments, our study demonstrates the overallbiological effectiveness of a 150-mg maintenance dose regi-men compared with standard doses of 75 mg in high-riskpatients. At the same time, our study found that some patientscontinue to show high platelet aggregation levels despiteincreased doses of clopidogrel, a finding that suggests theneed for further improvement in our antiplatelet strategies,the clinical implications of which need to be evaluated inlarge-scale clinical trials.

Study LimitationsThe present study did not account for performance of multi-ple significance tests or for potential correlation amongvarious measures of platelet function. Also, our study was notpowered to evaluate the risk of bleeding with the use ofhigh-dose clopidogrel in association with aspirin. Thus, thelack of adverse effects in the present study should beinterpreted with caution. In addition, the study was notdesigned to measure clinical outcomes. Therefore, the resultsof this pilot study need to be considered as exploratory andour laboratory findings should not be applied to clinicalpractice. Before development of large randomized clinicaleffectiveness trials that test aggressive antiplatelet therapy,the safety and biological effectiveness, as comprehensivelyinvestigated in the present study, of such strategies must bedetermined. Ultimately, numerous definitions have been re-ported to describe clopidogrel-induced antiplatelet effects.However, recent studies suggest that posttreatment plateletreactivity rather than responsiveness is a better determinant ofischemic risk.10,38 Given that our patient population wascomposed of patients already treated with clopidogrel, post-treatment platelet reactivity was the most applicable defini-tion of poor response to clopidogrel-induced antiplatelet

Figure 5. Platelet disaggregation at T1, T2, and T3 after stimulus with 20 �mol/L (left) and 5 �mol/L (right) ADP in patients randomizedto clopidogrel maintenance doses of 75 mg and 150 mg. Values are expressed as percentage (%). Error bars indicate standard devia-tions of the mean.

Figure 6. P2Y12 reactivity index at T1, T2, and T3 in patientsrandomized to clopidogrel maintenance doses of 75 mg and150 mg. Values are expressed as percentage (%). Error barsindicate standard deviations of the mean.

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effects in our study. In addition, the methods and cut-offvalues used in our study have been shown to identify patientsat highest risk of development of future ischemic events, andthis work has been suggested as a therapeutic guide inclopidogrel-treated patients.10

Sources of FundingThis study was funded by a University of Florida College ofMedicine at Jacksonville Research Grant (2005/70992). Dr Angio-lillo received the 2006 Stop Heart Disease Researcher of the YearAward sponsored by the Florida Heart Research Institute and FloridaChapter of the American College of Cardiology.

DisclosuresDr Angiolillo and Dr Zenni are on the speakers’ bureau and theconsultant/advisory board for Bristol Myers Squibb and Sanofi-Aventis. The other authors report no conflicts.

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CLINICAL PERSPECTIVESuboptimal response to antiplatelet agents is an emerging clinical entity. Recent investigations have focused onidentification of patients with suboptimal antiplatelet drug response. In particular, numerous investigations have identifiedpatients with diabetes mellitus (DM) to exhibit reduced platelet inhibition compared with non-DM patients treated withrecommended doses of antiplatelet agents. These findings may explain why DM patients continue to have an increasedatherothrombotic risk even with the use of antiplatelet agents. Results from recent investigations demonstrate that selectiveuse of specific antiplatelet agents or modification of their dosage may improve functional response profiles in DM patients.In particular, the Optimizing anti-Platelet Therapy In diabetes MellitUS (OPTIMUS) study demonstrates that specifictargeting of the upregulated P2Y12 pathway of platelets from high-risk DM patients, with a higher clopidogrel maintenancedosage, significantly increases platelet inhibition. However, a considerable number of patients continue to exhibit enhancedplatelet reactivity. Whether the laboratory findings from this study translate into better clinical outcomes without anincrease in bleeding hazards warrants further investigation. Future investigations will need to evaluate how specificblockade of dysfunctional targets of the diabetic platelet as well as tackling plasmatic components extrinsic to the platelet(ie, thrombin), but which contribute to platelet activation and thrombosis, will hamper the prothrombotic status of thesehigh-risk patients. Hopefully, this will lead to an era of individualized antithrombotic treatment regimens to overcome the“one size fits all” concept, performed through routine measurements of platelet activity in the same way that blood sugar,blood pressure, and cholesterol are followed to help guide therapy.

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Esther Bernardo, Martin M. Zenni, Luis A. Guzman, Theodore A. Bass and Marco A. CostaDominick J. Angiolillo, Steven B. Shoemaker, Bhaloo Desai, Hang Yuan, Ronald K. Charlton,

Therapy in Diabetes Mellitus (OPTIMUS) StudyDiabetes Mellitus and Coronary Artery Disease: Results of the Optimizing Antiplatelet

Randomized Comparison of a High Clopidogrel Maintenance Dose in Patients With

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2007 American Heart Association, Inc. All rights reserved.

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