High on clopidogrel treatment platelet reactivity is frequent in acute and rare in elective stenting and can be functionally overcome by switch of therapy

Thrombosis Research 133 (2014) 257–264

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Regular Article

High on clopidogrel treatment platelet reactivity is frequent in acute andrare in elective stenting and can be functionally overcome by switchof therapy

Sarolta Leé ⁎, Katarina Vargová, István Hizoh, Zsófia Horváth, Petra Gulácsi-Bárdos, Zsófia Sztupinszki,Anna Apró, Andrea Kovács, István Préda, Emese Tóth-Zsámboki, Róbert G. KissMedical Center, Hungarian Defence Forces, Department of Cardiology, Budapest, Hungary

Abbreviations: AA, arachidonic-acid; ACEI, angiotensinACS, acute coronary syndrome; ADP, adenosine-diphosphablocker; BMI, body mass index; CABG, coronary artery bydisease; COLL, collagen; COX-1, cyclooxygenase-1; CV, coeluting stent; EPI, epinephrine; HPR, high on-clopidogreLM, left main coronary artery branch; LTA, light transmmajor adverse coronary events;MI, myocardial infarction;NSTEMI, non ST segment elevation myocardial infarction;ous coronary intervention; PsNR, clopidogrel pseudo noreal non-responder; rpm, rate per minute; SA, stable anginmyocardial infarction; TIMI, thrombolysis in myocardial ipectoris.⁎ Corresponding author at: Medical Center, Hungarian

Cardiology, Róbert Károly krt. 44., Budapest, Hungary, Hfax: +36 14651857.

E-mail address: [email protected] (S. Leé).

0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.thromres.2013.11.029

a b s t r a c t

Article history:Received 10 August 2013Received in revised form 10 November 2013Accepted 29 November 2013Available online 6 December 2013

Keywords:Platelet reactivityLight transmission aggregometryClopidogrelTherapy adjustmentPercutaneous coronary interventionMyocardial infarction

The benefit of adjusted antiplatelet therapy in patients with myocardial infarction after primary percutaneouscoronary intervention is not well elucidated. We aimed to identify patients with high on treatment platelet reac-tivity and to gradually adjust antiplatelet therapy.Materials and Methods: We enrolled 133 acute myocardial infarction and 67 stable angina patients undergoingintracoronary stenting into our study. Maximal aggregation was determined with light transmissionaggregometry. Aggregation N50% induced by 5 μMADPwas indexedwith high on-clopidogrel treatment plateletreactivity. In these cases 75 mg clopidogrel was doubled and control test was performed. Patients effectivelyinhibitedwith 150 mg clopidogrelwere defined as clopidogrel pseudo non-responders. Patientswith high plate-let reactivity even on 150 mg clopidogrelwere considered as clopidogrel real non-responders andwere switchedto ticlopidine.Results: Aggregations (5ADP; p = 0.046) and the ratio of real non-responders (p = 0.013) were significantlyhigher in themyocardial infarction group.Most real non-responderswere effectively treatedwith switch of ther-apy. The ratio of pseudo non-responders also tended to be higher in myocardial infarction. Platelet reactivityremained constant during follow-up; however, a new appearance of high platelet reactivity was observed at 6and at 12 months.Conclusions: Patients with acutemyocardial infarction undergoing percutaneous coronary interventionmay ben-efit from prospective platelet function testing, because of higher platelet reactivity and much higher ratio ofclopidogrel real non-response. Switch of therapy may effectively overcome clopidogrel non-response. A newappearance of high platelet reactivity with unknown clinical significance is observed in both groups among thepatients on clopidogrel.

© 2013 Elsevier Ltd. All rights reserved.

-converting-enzyme inhibitor;te; ARB, angiotensin-II receptorpass graft; CAD, coronary arteryefficient of variation; DES, drugl treatment platelet reactivity;ission aggregometry; MACE,

NPR, normal platelet reactivity;OR, odds ratio; PCI, percutane-n-responder; RNR, clopidogrela; STEMI, ST segment elevationnfarction; UAP, unstable angina

Defence Forces, Department of-1134. Tel.: +36 702464686;

ghts reserved.

Introduction

The success of percutaneous coronary intervention therapy requiresinduction of dual antiplatelet treatment. In the last decade the COX-1inhibitor aspirin and the P2Y12 ADP-receptor blocker clopidogrelbecame the primary choice of treatment to prevent recurrent ischemicevents after intracoronary stenting [1–3]. However high variability ofindividual clopidogrel response raised doubts in „one-size-fits-all”dosing method, therefore clinical benefit of platelet function testingand tailored antiplatelet therapy became an excessively researchedfield in the past few years.

Evidences accumulated that high on-clopidogrel treatment plateletreactivity after intracoronary stenting is an independent risk factor ofrecurrent ischemic events such as cardiovascular death, myocardialinfarction or stent thrombosis [4–11]. Therefore clinical need emergedfor measuring the efficacy of antiplatelet therapy and for developingmore effective ADP-receptor blocking agents.

258 S. Leé et al. / Thrombosis Research 133 (2014) 257–264

In the last few years several point-of-care platelet function testsunderwent clinical validation beside the reference light transmissionaggregometrymethod [9,12,13]. These platelet function tests establisheda link between high on-treatment platelet reactivity and ischemicevents, but most of the tests failed to predict bleeding risk in patientson dual antiplatelet therapy [12].

Novel antiplatelet agents (prasugrel and ticagrelor) were alreadyintroduced in everyday clinical practice. Undoubtedly, they improvedplatelet inhibition compared to clopidogrel [14–16] and took theirplace in current clinical guidelines [17,18], still their net clinical benefitis not obvious in all cases as theymay excessively increase overall bleed-ing risk. Therefore a subsegment of acute and all elective CAD patientsare still depending on clopidogrel. The prognostic role of bleedingconsequences is as important as that of ischemic events; an optimalbalance in platelet inhibition would be desirable to improve long termclinical outcomes.

Nevertheless, platelet function is only one of multiple influencingfactors. Therapeutical decisions at individual level are further aggravat-ed by widened supply of antiplatelet agents, different biological drugprofiles, considerable cost/benefit ratios and the pronounced heteroge-neity of cardiovascular patients. Recently closed large clinical trials[19–21] failed to confirm the beneficial effect of tailored antiplatelettherapy on clinical outcomes in low and intermediate risk cardiacpatients or in medically treated ACS patients [22]. However, there isstill a lack of knowledge regarding the benefit of tailored antiplatelettherapy in high risk STEMI patients after primary PCI. Hencemonitoringplatelet reactivity beside obvious clinical parameters still seems to beuseful to find a „therapeutic window” in platelet inhibition and throughthat to improve clinical outcomes of certain cardiology patients.

Therefore, the primary objectives of our prospective study were toidentify patients with high on-clopidogrel treatment platelet reactivity(HPR)with platelet function testing among acute and elective cardiac pa-tients, to optimize antiplatelet therapy in “clopidogrel non-responders”until laboratory certified platelet inhibition is achieved and to completea long-term follow up of individually tailored antiplatelet therapy.

Materials and Methods

Patient Population and Study Design

We enrolled 200 patients into our study: 133 patients with myocar-dial infarction (MI) (105 patients with STEMI, 28 patientswithNSTEMI)and 67 patients with stable angina (SA). All patients underwent PCI andintracoronary stenting (bare metal stent or drug eluting stentimplantation).

A loading dose of 600 mg clopidogrel was given to all patients in theMI group and to clopidogrel naive patients in the SA group. MI patientswere given 75 or 150 mg clopidogrel daily based on the physician’s deci-sion. Determining factors were age, body weight, comorbidities, stent lo-calization, length and number of stents implanted. According to ourcurrent institutional practice, higher doses of clopidogrel were appliedin the first 30 days after PCI, then standard 75 mg clopidogrel for thewhole study period. In the SA group, clopidogrel maintenance dose was75 mg daily. All patients were given ≥100 mg aspirin daily (in 5 cases300 mg daily dose was applied according to the physician’s decision.)

The exclusion criteria were as follows: history of severe renal orhepatic disease, haematological or haemostatic disorders, acute orchronic inflammatory disease, active malignancy and active bleeding.The study protocolwas approved by the institutional and regional ethicsreview committee, and a written informed consent was obtained fromall patients before enrolment.

Platelet Function Testing

Platelet function was measured with LTA (Carat TX4, Hungary)72 hours after PCI in the MI group and 24 hours after PCI in the SA

group. Measurement details are described previously [23]. Briefly,blood was drawn from the cubital vein into citrate-anticoagulatedtubes (final concentrationwas 3.2%). Platelet rich plasmawas separatedby centrifugation at 980 rpm for 10 minutes. Platelet poor plasma wasprepared by further centrifugation at 3000 rpm for 10 minutes. Plateletagonists were 10 μM epinephrine (EPI), 1 μg/ml collagen (COLL) and0,5 μg/ml arachidonic-acid (AA) to assess overall platelet reactivityand 1,25 μM; 5 μM; 10 μM adenosine-diphosphate (ADP) to assessclopidogrel’s effectiveness specifically. Measuring time was 7 minutesand maximal aggregation was determined in percentage. Control LTAwas performed 5 days after therapeutic adjustments. The intra- andinter-assay CVs of 5 μM ADP induced aggregation were 7.98% and3.34%, respectively.

Patients were re-interviewed and repeated LTA was performed at 6and 12 month. The investigators were in close contact with the patientsduring the study; patients were fully informed about the importance ofthe applied antiplatelet therapy andwere asked to report modificationsof their medication. Number of participants decreased gradually at theconsecutive control points: at 6 month 102 MI and 48 SA patientswere available for control LTA testing and interviewing. At 12 month91 MI and 44 SA patients completed the study.

Statistical Analysis

Statistical analyses were performed using MedCalc for Windows,version 12.7 (MedCalc Software, Ostend, Belgium). Between-groupdifferences were evaluated with the Mann-Whitney test. ANCOVAanalysis was used to eliminate the distortion effect of the differentdemographic data. Adjustment was performed for the followingcovariates: age, gender, BMI, smoking, hypertension, diabetesmellitus, hypercholesterinaemia, elapsed time between the PCI andplatelet function testing, history of myocardial infarction, previousPCI or CABG operation, aspirin and clopidogrel medication on admis-sion. The Wilcoxon signed-rank test or repeated measures ANOVAtest was used for repeated measures. Categorical variables in 2 × 2contingency tables were analyzed using Fisher’s exact test.

Results

Patients Demography

Baseline characteristics of the different patient groups are listedon Table 1. The mean age, the prevalence of hypertension, hyper-cholesterinaemia, previous myocardial infarction, percutaneouscoronary intervention and coronary artery bypass graft operationwas significantly higher in the SA group, while ratio of smokerswas higher in the MI group. Previous ACEI, beta-blocker, statin, aspi-rin and clopidogrel medication was significantly higher in the SAgroup.

Initial Clopidogrel Regimens in MI and SA Patients

In the MI group, 41 patients (30.8%) were on standard 75 mgclopidogrel maintenance dose and 92 individuals (69.2%) were on150 mg, high dose clopidogrel initially (Table 1).

Baseline Platelet Aggregations in the MI and SA Patients

Baseline platelet reactivity in respect to most agonists was signifi-cantly higher in patients receiving 75 mg clopidogrel in the MI groupthan in the SA group (detailed demographic description and aggrega-tion values of these subgroups are shown in Table 2). After adjustmentto demographic parameters, the difference diminished in case of epi-nephrine and arachidonic-acid induced aggregations (p = 0.359 andp = 0.861, respectively), however, the difference remained significant

Table 1Demographic data and clinical endpoints of the MI and SA patients.

Demographic data MIn = 133

SAn = 67

p value

Age,mean ± SD, years 59.2 ± 10.3 66.2 ± 9.2 p b 0.01Males/females, n (%) 91(67.4)/

44(32.6)44(65.7)/23(34.3)

NS

Body mass index (kg/m2, mean ± SD) 28.4 ± 4.9 27.5 ± 4.3 NSRisk factors, n (%)History of tobacco use, n (%) 77 (57.9) 19 (28.4) p b 0.01Hypertension, n (%) 88 (65.2) 57 (85) p b 0.01Diabetes mellitus, n (%) 36 (26.7) 24 (35.8) NSHypercholesterinaemia, n (%) 31 (22.9) 32 (47.8) p b 0.01Previous MI, n (%) 15 (11.1) 22 (32.8) p b 0.01Previous PCI, n (%) 22 (16.3) 30 (44.8) p b 0.01Previous CABG, n (%) 0 9 (13.43) p b 0.01Medication on admissionAspirin, n (%) 31 (22.9) 55 (82.1) p b 0.01Clopidogrel, n (%) 10 (7.4) 32 (47.8) p b 0.01Beta blockers, n (%) 49 (36.3) 51 (76.1) p b 0.01ACE- inhibitor or ARB, n (%) 60 (44.4) 61 (91) p b 0.01Lipid-lowering agents, n (%) 36 (26.7) 60 (89.6) p b 0.01Nitroglycerine or nitrates, n (%) 22 (16.3) 34 (50.7) p b 0.01Coronarography resultsNumber of diseased vessels n (%)1 66 (48.9) 34 (50.7) NS2 35 (25.9) 20 (29.9) NS3 or LM 34 (25.2) 13 (19.4) NS

Number of implanted stents,(mean ± SD)

1.7 ± 0.9 1.6 ± 0.8 NS

Total stented length (mm),(mean ± SD)

39.7 ± 23.9 36.7 ± 22.3 NS

Clopidogrel maintenance dose atbaseline, n (%)75 mg 41 (30.8) 67 (100) p b 0.001150 mg 92 (69.2) 0 (0) p b 0.001

PPI medication at baselineOn PPI, n (%) 121 (91) 36 (53.7) p b 0.001Pantoprazole, n (%) 121 (91) 34 (50.7) p b 0.001Lansoprazole, n (%) 0 1 (1.5) NSRabeprazole, n (%) 0 1 (1.5) NS

Clinical end points at 12 month, nDeath from any cause 4 1Stroke 1 0Reinfarction 0 0Urgent revascularization 2 0Non-urgent target vesselrevascularization

4 0

Stent thrombosis 0 0TIMI minor bleeding 0 0TIMI major bleeding 0 0

Table 2Demographic data and baseline platelet reactivity of MI patients treated with 75 mgclopidogrel and the SA group.

Demographic data MI on 75 mgclon = 41

SAn = 67

p value

Age, mean ± SD, years 63.9 ± 9.49 66.2 ± 9.2 NSMales/females, n (%) 21(51.2)/

20(48.8)44(65.7)/23(34.3)

NS

Body mass index, kg/m2,mean ± SD

26.7 ± 5.6 27.4 ± 4.3 NS

Risk factors, n (%)History of tobacco use, n (%) 20 (48.8) 19 (28.4) p b 0.05Hypertension, n (%) 28 (68.3) 57 (85) p b 0.05Diabetes mellitus, n (%) 9 (21.9) 24 (35.8) NSHypercholesterinaemia, n (%) 7 (17.1) 32 (47.8) p b 0.01Previous MI, n (%) 5 (12.2) 22 (32.8) p b 0.05Previous PCI, n (%) 6 (14.6) 30 (44.8) p b 0.01Previous CABG, n (%) 0 9 (13.43) p b 0.05Medication on admissionAspirin, n (%) 11 (26.8) 55 (82.1) p b 0.001Clopidogrel, n (%) 4 (9.8) 32 (47.8) p b 0.001Beta blockers, n (%) 17 (41.5) 51 (76.1) p b 0.001ACE- inhibitor or ARB, n (%) 21 (51.2) 61 (91) p b 0.001Lipid-lowering agents, n (%) 12 (29.3) 60 (89.6) p b 0.001Nitroglycerine or nitrates, n (%) 4 (9.8) 34 (50.7) p b 0.001Coronarography resultsNumber of diseased vessels n (%)1 17 (41.5) 34 (50.7) NS2 14 (34.1) 20 (29.9) NS3 or LM 10 (24.4) 13 (19.4) NS

Number of implanted stents,mean ± SD

1.7 ± 0.7 1.6 ± 0.8 NS

Total stented length (mm),mean ± SD

38.5 ± 17.4 36.7 ± 22.3 NS

Baseline platelet reactivity, aggr.max. %, median (IQR)

1 μg/ml collagen 20 (12-31.5) 18.5 (10-35.5) p = 0.6991,25 μM ADP 16 (9.75-25) 8 (2.25-15) p = 0.0015 μM ADP 43 (36-53) 39 (28-48) p = 0.01810 μM ADP 54 (45.75-

62.25)46 (35.25-56.75)

p = 0.008

10 μM epinephrine 43 (30-59) 24 (17-46.75) p = 0.0040,5 μg/ml arachidonic acid 4 (1.75-6.25) 2 (1-4.75) p = 0.011

259S. Leé et al. / Thrombosis Research 133 (2014) 257–264

with all concentrations of the ADP agonist (1.25ADP: p = 0.005; 5ADP:p = 0.046; 10ADP: p = 0.023; Fig. 1).

Interestingly, patients receiving 150 mg clopidogrel compared tothose taking the standard dose within the MI group, showed only atendency of higher platelet reactivity,without reaching statistical signif-icance. Moreover, after adjusting to demographic parameters, this ten-dency completely diminished regarding all agonists (Fig. 2). Detaileddescription of patient subgroups and aggregation values are shown inTable 3.

Definition of High on Clopidogrel Treatment Platelet Reactivity Based onLTA Results in MI and SA Patients

5 μM ADP induced maximal aggregation (AGGRmax 5ADP) wasconsidered as the indicator of the efficacy of clopidogrel therapy withthe cut-off value of AGGRmax 5ADP = 50 %. Below this value normalplatelet reactivity (NPR), and above high on-clopidogrel treatmentplatelet reactivity (HPR) was defined, independently of the appliedclopidogrel maintenance dose.

The number of patients with high platelet reactivity tended to bemore in the MI than in the SA group (MI: 19.5% vs SA: 11.9%;p = 0,123). Similarly to the baseline platelet aggregation data, propor-tion of HPR patientswithin theMI group tended to be higher among pa-tients on 150 mg treatment dose (26.8% vs. 16.3%, p = 0.121).

Management, Definition and Ratio of Clopidogrel Pseudo and RealNon-responders

In HPR patients, antiplatelet therapywas adjusted as follows: in caseof 75 mg clopidogrel, the drug dose was doubled. In patients with HPRalready on 150 mg clopidogrel, 2 × 250 mg ticlopidine was induced(the study was conducted before the prasugrel/ticagrelor era in ourcountry; patient management and therapeutic modifications are indi-cated in Fig. 6 in the Supplement).

Patients with high initial platelet reactivity on 75 mg but reachingnormal platelet reactivity on 150 mg clopidogrel were defined asclopidogrel pseudo non-responders (PsNR). In contrast, patients withpersisting high platelet reactivity even on 150 mg clopidogrel were de-fined as clopidogrel real non-responders (RNR, Fig. 6).

The ratio of real non-responders was significantly higher in theMI group compared to SA group (MI: 18/133 = 13.5% vs SA: 2/67 =2.9%; p = 0.013). The ratio of pseudo non-responders also tended tobe higher in the MI group but did not reach statistical significance (MI:8/41 = 19.5% vs SA: 6/67 = 8.9%, p = 0.099, Fig. 6).

1.25 µM ADP 5 µM ADP 10 µM ADP

MI 75 mg clopidogrel SA 75 mg clopidogrel

Max

imal

agg

rega

tion

(%)

100

90

80

70

60

50

40

30

20

10

0

p=0.005

p=0.047

p=0.022

Fig. 1. Baseline platelet reactivity of MI and SA patients being on identical 75 mgclopidogrel therapy. In the MI group platelet reactivity was significantly higher with allconcentrations of ADP. Results are expressed as mean (represented by columns) ± SEM(represented by bars). p b 0.05 was considered significant after adjustment for demo-graphic differences.

Table 3Demographic data and baseline platelet reactivity of the 75 mg and 150 mg clopidogrelsubgroups of the MI patients.

Demographic data MI on 75 mg clon = 41

MI on 150 mgclon = 92

p value

Age, mean ± SD, years 63.9 ± 9.5 56.9 ± 9.8 p b 0.001Males/females, n (%) 21(51.2)/

20(48.8)69(75)/23(25) p b 0.01

Bodymass index, kg/m2,mean ± SD 26.7 ± 5.6 29.2 ± 4.4 p b 0.01Risk factors, n (%)History of tobacco use, n (%) 20 (48.8) 57 (61.9) NSHypertension, n (%) 28 (68.3) 59 (64.1) NSDiabetes mellitus, n (%) 9 (21.9) 27 (29.3) NSHypercholesterinaemia, n (%) 7 (17.1) 24 (26.1) NSPrevious MI, n (%) 5 (12.2) 10 (10.9) NSPrevious PCI, n (%) 6 (14.6) 15 (16.3) NSPrevious CABG, n (%) 0 0Medication on admissionAspirin, n (%) 11 (26.8) 19 (20.7) NSClopidogrel, n (%) 4 (9.8) 6 (6.5) NSBeta blockers, n (%) 17 (41.5) 31 (33.7) NSACE- inhibitor or ARB, n (%) 21 (51.2) 52 (56.5) NSLipid-lowering agents, n (%) 12 (29.3) 23 (25) NSNitroglycerine or nitrates, n (%) 4 (9.8) 10 (10.9) NSCoronarography resultsNumber of diseased vessels n (%)1 17 (41.5) 48 (52.2) NS2 14 (34.1) 21 (22.8) NS3 or LM 10 (24.4) 23 (25) NS

Number of implanted stents,mean ± SD

1.7 ± 0.7 1.7 ± 1 NS

Total stented length (mm),mean ± SD

38.5 ± 17.4 40.2 ± 26.4 NS

Baseline platelet reactivity, aggr.max. %, median (IQR)

1 μg/ml collagen 20 (13-30) 25 (12.5-44.5) p = 0.3921,25 μM ADP 16 (10-25) 12 (6-21) p = 0.135

260 S. Leé et al. / Thrombosis Research 133 (2014) 257–264

Platelet Hyperreactivity is Efficiently TreatedwithModification of AntiplateletTherapy

In pseudo non-responders clopidogrel dose doubling resulted ineffective platelet inhibition. On the other hand, switch of therapy toticlopidine also resulted in normal platelet reactivity in all patients inthe SA group and in the majority of real non-responders in the MIgroup (16 out of 18 individuals, Fig. 6). Platelet reactivity in MI and SApatients before and after therapy adjustment or switch is shown inFig. 3A, B, C andD. Patients undergoing therapy intensification remainedon modified therapy for the whole study period.

5 μM ADP 43 (36-53) 39 (29.5-50) p = 0.06510 μM ADP 54 (46-62) 48 (37-59) p = 0.08810 μM epinephrine 43 (30-58) 34.5 (20-55) p = 0.0810,5 μg/ml arachidonic acid 4 (2-6) 3 (1-7) p = 0.663

Kinetics of Platelet Function During Long Term Follow-up in MI and SAPatients

In the MI group most of the patients (n = 84) were already onstandard 75 mg clopidogrel therapy by 6 month, however 8 patientsreceived 150 mg clopidogrel and 10 patients received ticlopidine as aresult of therapy intensification (Fig. 6). At 12 month, 60 patientswere on standard, 5 patients were on high dose clopidogrel and 10patients were on ticlopidine. Platelet reactivity remained on the samelevel throughout 12 months follow-up in the MI group (Fig. 4A).

1 µg/ml coll 1.25 µM ADP 5 µM ADP

Max

imal

agg

rega

tion

(%)

100

90

80

70

60

50

40

30

20

10

0

p=0.102

p=0.812

p=0.581

MI 75 mg clopidog

Fig. 2. Platelet reactivity at baseline in the 75 mg and 150 mg clopidogrel subgroups of Msubgroups. Results are expressed as mean (represented by columns) ± SEM (represented by b

In the SA group 44 patients were on standard, 2 patients were onhigh dose clopidogrel and 2 patients were on ticlopidine at 6 month.At 12 month, 38 patients on 75 mg, 1 patient on 150 mg clopidogreland 1 patient on ticlopidine were available for retesting (Fig. 6). In SApatients, platelet reactivity showed slight oscillation: at 6 months plate-let aggregation was significantly higher compared to the 12 monthvalues (5ADP6month vs. 5ADP12month p = 0.005; Fig. 4B).

10 µM ADP 10 µM EPI 0.5 µg/ml AA

p=0.638

p=0.399

p=0.812

rel MI 150 mg clopidogrel

I patients. After adjustment for covariates no difference was observed between the twoars). p b 0.05 was considered significant.

0

10

20

30

40

50

60

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100

p= 0,093

p= 0,012

p= 0,012

p= 0,012

p= 0,069

p= 0,529Max

imal

agg

rega

tion

%

1 µg/ml coll

1,25 µM ADP

5 µM ADP

10 µM ADP

10 µM EPI

0,5 µg/ml AA

1 µg/ml coll

1,25 µM ADP

5 µM ADP

10 µM ADP

10 µM EPI

0,5 µg/ml AA

p= 0,075

p= 0,028

p= 0,028

p= 0,028

p= 0,075

p= 0,500

1 µg/ml coll

1,25 µM ADP

5 µM ADP

10 µM ADP

10 µM EPI

0,5 µg/ml AA

0

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0

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Max

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%

p= 0,007

p= 0,000

p= 0,005p= 0,018

p= 0,011

p= 0,001

1 µg/ml coll

1,25 µM ADP

5 µM ADP

10 µM ADP

10 µM EPI

0,5 µg/ml AA

TIC150 mg CLON=18 TIC150 mg CLON=2

150 mg CLO75 mg CLON=6150 mg CLO75 mg CLON=8

A B

C D

Fig. 3. Platelet reactivity aftermodification of therapy in clopidogrel pseudo non-responders (PsNR) in theMI (A) and in the SA (B) group and in clopidogrel real non-responders(RNR) in the MI (C) and SA group (D). Clopidogrel dose doubling in PsNR patients resulted in significantly lower platelet aggregation induced by ADP in both patient groups (A and B).Platelet reactivity induced by collagen, epinephrine and arachidonic-acid also tended to be lower in PsNR patients. Platelet reactivity was significantly lower after ticlopidine inductioninduced by all agonists in RNR patients in the MI group (C) and was also substantially lower induced by ADP in the SA group (D, statistical analysis was not performed because of lowcase number). Results are expressed as median (represented by squares), IQR (represented by boxes) and non-outlier range (represented by bars). p b 0.05 was considered significant.

261S. Leé et al. / Thrombosis Research 133 (2014) 257–264

Platelet aggregation did not differ in the MI and SA group at 6 and12 month and platelet reactivity was also irrespective of the 75 mg or150 mg clopidogrel maintenance therapy at 6 and at 12 months.

Long Term Follow-up of Platelet Function in MI and SA Patients – Incidenceof New HPR

Despite the fact, that out of our 200patients, 198 individualswere oneffective antiplatelet therapy aftermodifications at baseline, therewas aremarkable incidence of new HPR at 6 month in both patient groups(Fig. 5). In the MI group new HPR occurred in 12 patients receivingstandard and in 2 patients receiving high dose clopidogrel therapy(13.7%, Fig. 6). Interestingly, in the SA group incidence of new HPRwas unexpectedly high; 14 patients were on 75 mg and 1 patient wason 150 mg clopidogrel (Fig. 6).

In these patients, further therapeutic interventions were not per-formed. Interestingly, the majority of patients with HPR at 6 month,returned with normal platelet reactivity at 12 month (MI group: 8patients on 75 mg and 1 patient on 150 mg clopidogrel; SA group: 11patients on 75 mg and 1 patient on 150 mg clopidogrel). Nevertheless,

HPR persisted from 6 to 12 month in 4 patients being on 75 mg and 1patient being on 150 mg clopidogrel in the MI group. Persisting HPRfrom 6 to 12 month was also observed in 3 stable angina patientsreceiving 75 mg clopidogrel.

By 12 month, newHPRwas observed in 8MI patients being on stan-dard clopidogrel therapy and no further incidence of HPR was observedon high dose clopidogrel therapy. The ratio of new HPR at 12 monthwas substantially lower in the SA group: only 1 patient on 75 mgclopidogrel returned with new HPR and no further incidence of HPRwas observed beside high dose clopidogrel therapy (Fig. 6).

Interestingly, all clopidogrel real non-responders switched over toticlopidine remained effectively inhibited during the whole follow-upperiod.

Clinical End-points During 12 Month Follow Up

During the study we documented clinical efficacy and safety end-points (Table 1). The 12 month cumulative event rate was 8,3% in theMI and 1,5% in the SA group. During the 12 month follow-up no bleed-ing events were observed.

p= 0,253 p= 0,005

p= 0,596

5 µM ADP baseline

5 µM ADP 6 month

5 µM ADP 12 month

p= 1.000 p= 0,110

p= 0,243

5 µM ADP baseline

5 µM ADP 6 month

5 µM ADP 12 month

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0

A B

Fig. 4. Long term follow-up results of platelet reactivity in MI (A) and SA (B) patients. In the MI group (N = 71), platelet function remained approximately the same throughout12 month follow-up period. In SApatients (N = 39), platelet function showedmodest oscillation during12 month follow-up. At 6 month platelet reactivity tended to be higher comparedto the baseline values and proved to be significantly higher than platelet reactivity at 12 month. Results are expressed asmedian (represented by squares), IQR (represented by boxes) andnon-outlier range (represented by bars). p b 0.05 was considered significant.

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Discussion

We examined the effectiveness of clopidogrel therapy with an“in vitro” platelet function test in order to identify MI or stable anginapatients with high on-clopidogrel-treatment platelet reactivity and togradually adjust antiplatelet therapy until laboratory proven plateletinhibition is achieved.

The LTA method is often criticized because it’s poor standardizationwhich makes the comparisons of the results difficult. However LTA is acheap and reliable method under well-established circumstances andis successfully and widely used to monitor platelet reactivity in numer-ous laboratories [24]. It proved to be one of the platelet function tests,which had predictive power regarding ischaemic events [12], moreoverits predictive power regarding MACE was somewhat higher or compa-rable with that of the recently introduced and validated, easily applica-ble point-of-care method, Verify Now [25,26]. Importantly, ourdefinition of HPR is in good agreement with the recommendation ofthe platelet expert panel (AGGRmax 5ADP = 46%) [26,27].

5 µM ADP baseline

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5 µM ADP 12 month

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Fig. 5. Individual platelet aggregation values induced by 5 μMADP at baseline, 6 and 12 molines. To declare high on-treatment platelet reactivity,we used the AGGRmax 5ADP = 50% cut-oftimes in both patient groups. The incidence of new HPR at 6 month is higher in the SA group, thHPR persisted from 6 to 12 month in both groups. However, there were HPR patients at 6 moadjustment.

In our study, we found that baseline platelet reactivity was signifi-cantly higher in patients with myocardial infarction than in patientswith stable angina. After adjusting for demographic differences thedifference remained significant with all concentrations of the ADPagonist. Our finding of higher platelet reactivity inmyocardial infarctionis in good agreement with the literary data [28–31]. The ratio ofclopidogrel real non-responders was also significantly higher amongthe MI patients, suggesting that these invasively treated acute cardiacpatients benefit from prospective measurement of platelet reactivitybecause of their higher subsequent atherothrombotic risk and widenedantiplatelet treatment possibilities. In the recent years many studylinked the high on-treatment ADP induced platelet reactivity to clinicaloutcomes in different cardiology patient groups [4–10]. Accepting HPRas a clinically relevant factor, we need to investigate its modifiabilityandwhether its optimization improves the clinical outcome in differentclinical settings of CAD. Large clinical trials investigating the effect of tai-lored antiplatelet therapy on clinical outcome closed with neutralresults [19–22]. Importantly, these studies used the same platelet

5 µM ADP baseline

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5 µM ADP 12 month

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nth in theMI (A) and in the SA (B) group. Patientswere represented by separate dots andf value. Therewere a remarkable number of patients returningwithHPR at bothmeasuringan in theMI group (in correlation with platelet function kinetics, Fig. 5). In some patients,nth who returned with normal platelet reactivity at 12 month without any therapeutical

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function test (Verify Now) and mostly only one agonist (ADP) to assessplatelet function. The subjects of these studies weremostly elective car-diac patients or partly patientswithNSTEMI or UAP, representing ratherlow and intermediate risk patient populations. However, we have totake notice of the heterogeneity of these studies in respect to study de-signs, patient population, definitions of clinical endpoints and follow-uptimes. Patientswith STEMIwere no subjects of such studies up to date. Itcannot be excluded, that other platelet function tests or the use of moreparallel agonists to compose a so-called “platelet reactivity index” couldbe more sensitive in discriminating patients with HPR.

In our patient population the initial ratio ofHPR in the overall patientpopulation (34/200 = 17%) was lower than generally known from theliterature (even up to 45%) [32,33]. The cause in its background mighthave been the high proportion of MI patients being on 150 mgclopidogrel initially. Unexpectedly, the platelet reactivity in MI patientsreceiving 150 mg clopidogrel was approximately on the same level asthat of the patients on standard clopidogrel dose. As indicated bythese data, we can identify those patients inwhom standard clopidogreldose is presumably less effective by considering the clinical influencingfactors (acute form of cardiac disease, younger age, higher BMI, higherfrequency of comorbidities [32,34–37]) and successfully treat this pop-ulation with the double dose. In a large clinical study initial 150 mgclopidogrel regimen was associated with a reduction in cardiovascularevents and stent thrombosis compared with the standard dose [38]. Italso supports ourfinding, that in absence of possibility to performplate-let function tests or to give prasugrel/ticagrelor it seems to be beneficialto give 150 mg clopidogrel initially in high risk, acute cardiac patientsselected by certain clinical parameters, because a portion of thesepatients have HPR beside 75 mg clopidogrel and need high doseclopidogrel to be effectively inhibited. 150 mg clopidogrel might alsobe reasonable in the gradual therapy intensification of MI patients ifmore potent drugs are contraindicated, as it may overcome HPR with-out increasing bleeding risk [19,38].

Our data also suggest that with switch of therapy we can converteven real non-responders to responders according to platelet functiontests. Ticlopidine induction in clopidogrel real non-responders provedto be laboratory effective during long-term follow up, however appear-ance of new HPR was observable even besides high dose clopidogrel.There are relatively few and controversial data about long term plateletfunction kinetics [19–21]. Amongmedically treated ACS patients almost50% of the patients on clopidogrel had HPR at the subsequent controlpoints during 30 months follow up and the ratio of HPR patients provedto be stable over the time [22]. According to our results, platelet reactiv-ity remained on the same level in the MI group throughout 12 monthand proved to be modestly oscillating in the SA group. However, anew appearance of HPR at 6 and 12 months was observed in both pa-tient groups independently from the clopidogrel maintenance dose.This finding may have clinical consequences suggesting, that onclopidogrel treatment platelet reactivity - beside wide-scale inter-individual variability - is not a stable parameter. It is determined by sev-eral clinical, genetic and cellular factors showing considerable alterna-tion during follow-up [32,34]. Interestingly, platelet function inpatients on clopidogrel altered in both directions spontaneously duringlong term follow up. It also supports that clopidogrel’s effectiveness isvery vulnerable and thienopyridines using other metabolization path-ways may exert not only more extensive but more stable long-termplatelet inhibitory effect [39]. However appearance of new HPR andinter-patient variability of platelet function was also described recentlyin prasugrel treated patients, particularly among patients on lowerprasugrel maintenance dose [40,41]. Appearance of HPRmay have clin-ical significance in patients with DES as their endothelial regenerationmay be prolonged resulting in higher risk for late and very late stentthrombosis [42]. Therefore re-evaluation of platelet reactivity may beuseful in patients with DES or before the performance of a subsequentpercutaneous coronary intervention or to assess the efficacy of themaintenance dose of an antiplatelet drug. The right approach to platelet

function testing may include early assessment to exclude drugineffectivity and re-evaluation to exclude dose ineffectivity or to adjustproper maintenance dose of an antiplatelet agent.

In our patient population no bleeding consequences were observedassociated with tailored antiplatelet therapy, which may suggest, thattherapy intensificationmight be applied safely in a selected, well deter-mined patient population. However re-evaluation of platelet functionafter modification of therapy seems to be desirable. Moreover, a recentstudy identified strong inverse relationship between high on-treatmentplatelet reactivity and clinically relevant bleeding aswell as strong asso-ciation of bleeding consequences withmortality; similar to that of stentthrombosis or myocardial infarction [11]. Therefore, possible predictionof bleeding riskwith platelet testingmay also gain importance in the fu-ture to maximize the net clinical benefit of cardiac patients besides theexpanding use of more potent antithrombotic agents. Overcoming HPRat any price may not improve survival because of the counter balancingeffect of haemorrhagic complications – especially in low risk cardiacpatients.

Themain limitation of our studywas the lownumber of clinical end-points, which prevented us from the correct statistical analysis of thecorrelation of clinical outcomes and antiplatelet therapy adjustmentbased on platelet function testing.

Platelet function is clearly an influencable laboratory marker, never-theless, purely on our data, effective inhibition cannot be linked to bet-ter outcomes. Due to low number of adverse events, we can onlyassume the improvement of patient outcomes based on previously re-ported large clinical studies linking effective platelet inhibition to betterclinical outcomes. Therefore, deciding whether adjusted antiplatelettherapy has beneficial effect on clinical outcomes regarding ischemicand bleeding events in high risk, acute cardiac patients warrants furtherlarge scale randomized, controlled trials. However the results of thesestudies are hardly applicable at individual levels. Widening supply ofantiplatelet agents made the question of individual antiplatelet therapymore complex. Our results suggest that platelet function is a well deter-minable and influenceable clinical factor and its monitoring may con-tribute to select the adequate individual therapy of a cardiac patient ina complex clinical setting. Applying proper therapy at the individuallevel may be the fair way to improve the clinical outcome of a largepopulation.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.thromres.2013.11.029.

Conflict of Interest Statement

The corresponding author has no conflict of interest to declare.

Acknowledgements

This study was supported by research grants from the HungarianMinistry of Health (ETT-085-06 and ETT 036/2009).

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