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Relation of Endothelial Function to Residual Platelet ReactivityAfter Clopidogrel in Patients With Stable Angina Pectoris

Undergoing Percutaneous Coronary Intervention

Olivier Muller, MD, PhDa,†, Michalis Hamilos, MD, PhDa,†, Jozef Bartunek, MD, PhDa,Hans Ulrichts, PhDb, Fabio Mangiacapra, MDa, Josefin-Beate Holz, MD, PhDb,

Argyrios Ntalianis, MD, PhDa, Catalina Trana, MDa, Karen Dierickx, PhDa,Kristof Vercruysse, BScb, Bernard De Bruyne, MD, PhDa, William Wijns, MD, PhDa, and

Emanuele Barbato, MD, PhDa,*

Platelet reactivity is greater in patients with stable angina and with more extensiveperipheral vascular atherosclerosis. We sought to evaluate whether impaired peripheralmicrocirculatory endothelial function might correlate with platelet reactivity after clopi-dogrel and therefore predispose to an unfavorable outcome after percutaneous coronaryintervention (PCI). In 52 consecutive patients with stable angina undergoing elective PCI,endothelial function was assessed by (1) endothelial peripheral arterial tonometry (mea-suring the “Endoscore”); (2) the von Willebrandt factor antigen level and ristocetinco-factor activity. Basal platelet reactivity was assessed by soluble P-selectin. Patients thenreceived a 600-mg clopidogrel loading dose >12 hours before PCI. A blood sample waswithdrawn 12 hours later, but before PCI, to assess platelet reactivity using the P2Y12reaction unit and percentage of P2Y12 inhibition with the point-of-care VerifyNow P2Y12assay. Troponin T was assessed 24 hours after PCI. The Endoscore inversely correlatedwith von Willebrandt factor antigen activity (r � �0.52, p � 0.0001) and soluble P-selectinconcentration (r � �0.36, p � 0.021), suggesting greater platelet reactivity with increasedimpaired endothelial function. After clopidogrel, the Endoscore correlated directly with thepercentage of P2Y12 inhibition (r � 0.36, p � 0.009) and inversely with the P2Y12 reactionunit (r � �0.41, p � 0.002), suggesting greater residual platelet reactivity with moreimpaired endothelial function. The average Endoscore was significantly lower in patientswith troponin T elevation (troponin positive group 0.267 � 0.091) than in patients withouttroponin T elevation (troponin negative group 0.508 � 0.041, p � 0.015 vs troponinpositive). In conclusion, an impaired endothelial response before clopidogrel was associatedwith greater platelet reactivity after clopidogrel. This link might explain the unfavorablePCI outcomes in patients with more severe endothelial impairment. © 2010 Elsevier Inc.

All rights reserved. (Am J Cardiol 2010;105:333–338)

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Dual antiplatelet therapy with aspirin and clopidogrel iseing widely administered to patients undergoing percuta-eous coronary intervention (PCI).1 However, high residuallatelet reactivity can limit the benefit of clopidogrel in21% of patients,2 resulting in increased cardiovascular

vents both during the procedural3 and during long-termollow-up.4 Genetic, cellular, and clinical factors might ac-ount for the suboptimal platelet response to clopidogrel.5–7

n addition, basal platelet reactivity is a strong predictor of

aCardiovascular Center Aalst, OLV Hospital, Aalst, Belgium; andAblynx NV, Zwijnaarde, Belgium. Manuscript received July 20, 2009;evised manuscript received and accepted September 9, 2009.

This study was supported by the Meijer Lavino Foundation for Car-iovascular Research, Aalst, Belgium.

*Corresponding author: Tel: (��32) 53-72-4439; Fax: (��32) 53-2-4185.

E-mail address: emanuele.barbato@olvz-aalst.be (E. Barbato).

s† Both authors contributed equally to the study.

002-9149/10/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.amjcard.2009.09.033

he platelet response to clopidogrel.8 Platelet reactivity haseen related to systemic atherosclerotic disease.9 In partic-lar, greater platelet reactivity has been reported in theresence of more severe and diffuse peripheral vasculartherosclerosis.10 Whether the latter also has an effect onostclopidogrel platelet reactivity has not been reported. Weested the hypothesis that impaired peripheral endothelialunction, an early marker of vascular atherosclerosis, cor-elates with platelet reactivity after the administration oflopidogrel.

ethods

A total of 89 patients with stable angina who werecheduled to undergo elective coronary angiographyere consecutively and prospectively assessed for pe-

ipheral endothelial function. Only those patients forhom PCI was electively staged (n � 52) were finally

ncluded in the study protocol. The reasons to not stageCI were (1) ad hoc PCI (n � 13); (2) the absence of

ignificant coronary stenosis at angiography (n � 12); (3)

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referral for coronary bypass surgery (n � 9); and (4)reatment at another hospital (n � 3). The exclusionactors were the presence of acute coronary syndrome, anown allergy to clopidogrel, a history of gastrointestinalleeding, or thrombocytopenia with a platelet count of100/nl, pretreatment with clopidogrel, chronic renal

ailure (glomerular filtration rate �60 ml/min), inflam-atory disease, serious co-morbidities, or a left ventric-

lar ejection fraction �50%. All patients were receivinghronic aspirin treatment (160 to 325 mg) at recruitment.

The study protocol is shown in Figure 1. All vasoactiveedications were withheld for �24 hours before the mea-

urements were taken. Peripheral endothelial function as-essment and blood sample withdrawal (for endothelial andlatelet biomarker measurement) were performed in theorning, after a 12-hour fasting and before coronary an-

iography. Thus, the investigators were unaware of theatients’ coronary artery disease and the possible treatmenttrategy. In patients with significant coronary lesions, PCIas scheduled such that a 600-mg loading dose of clopi-ogrel was given uniformly �12 hours before PCI. Exceptor clopidogrel, no changes in the ongoing medical therapyere allowed between the diagnostic and interventional

igure 1. Flow chart of study. sP-selectin, soluble P-selectin; vWF:Ag �on Willebrand factor antigen level; PRU � P2Y12 reaction unit; ARU �spirin reaction unit.

rocedures. Blood samples were withdrawn at PCI for as- p

essment of platelet inhibition by clopidogrel. The PCIsere uncomplicated and performed according to the oper-

tor’s discretion. During catheterization, all patients re-eived intravenous heparin to achieve a target activatedlotting time of 250 to 350 seconds. No glycoprotein IIb/IIIalockers were administered. An additional blood with-rawal was performed 24 hours after PCI for troponin Teasurement. The local ethics committee approved the

tudy protocol, and all patients provided written informedonsent.

Peripheral endothelial function was measured by digitalulse amplitude with the Endothelial Peripheral Arterialonometry (Endo-PAT2000, Itamar Medical, Caesarea, Is-

ael), as previously described.11,12 Figure 2 shows an ex-mple of the tracings obtained with the Endo-PAT2000rom 2 patients, 1 with good and 1 with poor endothelialunction. In brief, the device measures the distal fingerlood volume changes that accompany pulse waves. Aeripheral arterial tonometry finger probe was placed at theip of each index finger, and a blood pressure cuff waslaced at the level of the study arm. After a 5-minute restingeriod (baseline), the blood pressure cuff was inflated to 20m Hg greater than the systolic pressure for 5 minutes

occlusion). Next, the blood pressure cuff was deflated, andhe peripheral arterial tonometry recording was continuedor an additional 5 minutes (hyperemia; Figure 2). Thendothelial responses were assessed using the recentlyalidated Framingham Reactive Hyperemia Index (En-oscore),12 according to the following formula: Endoscore �an[RH occluded (90 to 120 seconds)/RH control (90 to 120econds)] �

lan�PWAocc(90s � 120s)

PWAocc(BL) � PWAcont(90s � 120s)

PWAcont(BL) �here lan is the natural base log; RH is reactive hyperemia

which equals the mean pulse wave amplitude [PWA] of theostocclusion section divided by the mean pulsewavemplitude of the baseline region of interest; occ isccluded and indicates the test arm (eg, the arm on whichhe cuff was placed; and cont is the control and indicateshe control arm.

The von Willebrand factor (vWF) antigen level and ris-ocetin co-factor activity (vWF:RiCo) were assessed asarkers of endothelium impairment. The vWF antigen levelas assessed using a sandwich enzyme-linked immunosor-ent assay with polyclonal anti-vWF immunoglobulin andnti-vWF immunoglobin labeled with horseradish peroxi-ase (Dako, Glostrup, Denmark) for immobilization andetection, respectively. The vWF:RiCo explores the inter-ction of vWF with the platelet receptor glycoprotein Ib�nd subendothelial collagen. It is based on the property ofhe antibiotic ristocetin to agglutinate formalin-fixed normallatelets in the presence of plasma vWF. vWF:RiCo waseasured using the vWF ristocetin co-factor assay (Trinityiotech, Bray, Ireland).

The plasma P-selectin level was assessed as a marker of

latelet activation and measured using the enzyme-linked

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335Coronary Artery Disease/Impaired Endothelial Function and Platelet Reactivity After Clopidogrel

mmunosorbent assay method with a commercially avail-ble kit (CD62E, R&D Systems, Minneapolis, Minnesota).

At PCI, blood was taken from the femoral arterymmediately after sheath placement; the first 5 ml oflood was discarded, and then samples were collected in-ml tubes containing 3.2% sodium citrate. The point-of-are VerifyNow assay (Accumetrics, San Diego, California)as used to assess the platelet response to aspirin and

lopidogrel, as previously described.13 VerifyNow is a rapidlatelet-function cartridge-based assay with specific car-ridges for the P2Y12 pathway (clopidogrel) and aspirin.he VerifyNow P2Y12 cartridge is designed to directlyeasure the effects of drugs on the P2Y12 receptor, using

rostaglandin E1, in addition to adenosine diphosphate toncrease intraplatelet cyclic adenosine monophosphate. TheerifyNow aspirin cartridge uses arachidonic acid. The

esults provided are expressed in P2Y12 reaction units andspirin reaction units. The percentage of inhibition of2Y12 from baseline activation by thrombin receptor acti-ating peptide was calculated as (1 � P2Y12 reaction unit/aseline) � 100. Residual high platelet reactivity was de-ned as P2Y12 reaction units �240. This P2Y12 reactionnit value has been previously proposed as the optimumutoff to discriminate patients undergoing PCI at greaterisk of 30-day major adverse cardiovascular events.3,4

The troponin T levels were assessed 24 hours after PCIo detect periprocedural myocardial necrosis. The troponinevels were measured using a 4-generation electrochemilu-inescent immunoassay (Elecsys troponin T, Roche Diag-

ostics, Basel, Switzerland).Statistical analysis was performed using GraphPad

rism, version 5.00 (GraphPad Software, San Diego, Cali-ornia). Variability between serial measurements was as-essed in 6 normal volunteers by performing peripheralndothelial function assessment with Endo-PAT 2000 on 2onsecutive days in the morning after 12 hours of fasting.he average coefficient of variation between the 2 consec-tive measurements was 15%, in line with that from previ-us reports.14 The hypothesis of the present study was thatatients with impaired endothelial function would demon-trate high platelet reactivity after clopidogrel administra-

igure 2. Peripheral endothelial function measured by digital pulse amplituf patients with normal and abnormal reactive hyperemic response. Normaluff occlusion (at bottom), corrected with contralateral finger signal (at topmplitude.

ion compared to patients with better or preserved endothe- E

ial function. In the present study, we determined the sampleize calculation from the previously reported incidence ofigh platelet reactivity (25%) in our patient population.13

e calculated that 52 patients should be included to detect50% difference in the Endoscore between patients withoutigh platelet reactivity and those with high platelet reactiv-ty (� � 0.05, statistical power � 0.80). Continuous vari-bles are presented as the mean � SEM and, if not normallyistributed, as the median with the interquartile range. Cat-gorical variables are reported as frequencies and percent-ges. The correlation among variables was determined byearson’s or Spearman’s correlation tests, accordingly. The

endothelial peripheral arterial tonometry (Endo-PAT2000): 2 recordingse (left recording) resulted from increase of signal amplitude after unilateralrmal response (right recording) characterized by lack of increase in signal

able 1atient characteristics (n � 52)

haracteristic Value

ge (years) 68 � 10en 34 (66%)ody mass index (kg/m2) 27 � 4iabetes mellitus 17 (33%)ypertension 29 (56%)yperlipidemia 37 (71%)moker 9 (17%)-Blocker therapy 29 (56%)itrate therapy 9 (17%)ngiotensin-converting enzymeinhibitor/receptor blocker therapy

20 (38%)

alcium blocker therapy 4 (8%)tatin therapy 40 (77%)roton pump inhibitor therapy 5 (10%)latelet count (/nl) 213 � 41-reactive protein (mmol/L) 1.9 (0.82–3.22)otal cholesterol (mg/dl) 179 � 45eft ventricular ejection fraction 65% � 15%eft ventricular end-diastolic pressure (mm Hg) 14 � 6arrowed coronary arteries (n) 1.75 � 0.67yntax score 10.9 � 4.9reated coronary arteries (n) 1.38 � 0.57otal stent length (mm) 29.8 � 14.3

Values are presented as mean � SD for quantitative variables, medianith interquartile range for C-reactive protein, or numbers (%) for cate-orical variables.

de withrespons). Abno

ndoscore between troponin groups was compared using an

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npaired t test. A p value of �0.05 was considered statis-ically significant.

esults

The patient characteristics are listed in Table 1. Periph-ral endothelial function was assessed in all patients withoutny discomfort. The average duration of the test was 19 �minutes. The average Endoscore was 0.47 � 0.30. The

WF antigen level and activity (vWF:RiCo) were, on aver-ge, 1.40 � 0.81 U/ml and 103 � 28%, respectively. Theverage soluble P-selectin level was 30.35 � 6.63 ng/ml.

The Endoscore correlated significantly with vWF:RiCor � �0.52, p � 0.0001), suggesting greater vWF activityn the presence of more impaired peripheral endothelialunction. No correlation was found between the Endoscorend the vWF antigen level (r � �0.006, p � 0.967; Figure). The Endoscore correlated significantly with baselineoluble P-selectin (r � �0.36, p � 0.021), suggestingreater platelet reactivity in the presence of more impairedndothelial function (Figure 4).

The average aspirin reaction unit value was 467 � 67.fter 600 mg of clopidogrel, the average P2Y12 reactionnit value was 263 � 79 and the average percentage of2Y12 inhibition was 20 � 18%. No significant correlationas observed between the aspirin reaction units and En-oscore (r � �0.20, p � 0.159; Figure 5). The Endoscoreorrelated inversely with the P2Y12 reaction unit (r �

igure 3. Relation between (A) vWF antigen level (vWF:Ag; r � �0.006,� 0.967) and (B) vWF:RiCo (r � �0.52, p � 0.0001) with Endoscore.

igure 4. Soluble P-selectin (sP-selectin) in function of Endoscore tertilesanalysis of variance, p for trend � 0.021).

0.41, p � 0.002) and directly with the percentage of 0

2Y12 inhibition (r � 0.36, p � 0.009), suggesting greateresidual platelet reactivity after clopidogrel in the presencef more impaired endothelial function (Figure 5). In addi-ion, the average Endoscore was greater in patients with lowlatelet reactivity (0.76 � 0.048 in the first quartile of2Y12 reaction units vs 0.39 � 0.049 in the fourth quartile;�0.0001).At 24 hours after PCI, troponin T was significantly ele-

ated (�3 times the upper limit of normal) in 10 patients.he average Endoscore was significantly lower in patientsith troponin T elevation (troponin T positive 0.267 �

igure 5. Relation between (A) aspirin reaction unit (ARU; r � �0.20,� 0.159), (B) P2Y12 reaction unit (PRU; r � �0.41, p � 0.002), and

C) percentage of P2Y12 inhibition (r � 0.36, p � 0.009) withndoscore.

.091) than in patients without troponin T elevation (tropo-

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337Coronary Artery Disease/Impaired Endothelial Function and Platelet Reactivity After Clopidogrel

in T negative 0.508 � 0.041, p � 0.015 vs troponin Tositive; Figure 6).

iscussion

In the present prospective study, conducted in patientsith stable angina who were undergoing elective PCI, we

ound a close correlation between peripheral endothelialunction and platelet reactivity. In addition, we have dem-nstrated a direct correlation between impaired endothelialunction and greater residual platelet reactivity after clopi-ogrel administration, potentially contributing to an unfa-orable periprocedural outcome.

Platelets are actively involved in endothelial functionmpairment and vascular atherosclerosis.9 We assessed im-aired peripheral endothelial function using endothelialulse amplitude tonometry (Endo-PAT), a reliable noninva-ive bedside test of peripheral endothelial function15 thatas been validated in large cohort of patients,12 and theWF antigen level and activity (vWF:RiCo), markers ofndothelial damage. Soluble P-selectin was measured tossess preclopidogrel platelet reactivity.16 Our resultshowed greater vWF activity (greater vWF:RiCo) in pa-ients with more impaired endothelial function (lower En-oscore); however, no correlation was observed with theWF antigen level. This finding agrees with that from pre-ious reports.17,18 vWF is the first promoter of plateletdhesion to damaged arterial walls, activating platelets un-er shear stress.19 Accordingly, we found greater plateleteactivity (greater soluble P-selectin) in patients with morempaired endothelial function (lower Endoscore), confirm-ng the association between the degree of endothelium im-airment and platelet aggregability.16,20,21

Dual antiplatelet therapy with aspirin and clopidogrel isidely administered in patients with acute coronary syn-rome and patients undergoing PCI.1 However, the variabil-ty in platelet response to clopidogrel hampers its protectiveffect and has been associated with increased cardiovascularvents both during the procedure and long term.7,22 Resid-al platelet reactivity after clopidogrel was assessed in ourtudy with the point-of-care VerifyNow P2Y12 assay

igure 6. Endoscore in patients with periprocedural negative and positiveroponin T release (�3 times upper limit of normal) (p � 0.015).

P2Y12 reaction unit; percentage of P2Y12 inhibition),

hich has been demonstrated to significantly predict car-iovascular adverse events in patients undergoing PCI.3,4

Several mechanisms have been described for the subop-imal platelet response to clopidogrel, including genetic,ellular, and clinical factors.5–7 However, basal platelet re-ctivity, irrespective of other factors, is a strong predictor oflatelet response to clopidogrel.8 Basal platelet activation isncreased in patients with peripheral and coronary arteryisease10,23,24 and might potentially limit the degree oflatelet inhibition induced by clopidogrel. In the presenttudy, we showed that impaired endothelial function (lowerndoscore), an early marker of vascular atherosclerosis,orrelated significantly with greater residual platelet reac-ivity after clopidogrel (more P2Y12 reaction units andower percentage of P2Y12 inhibition). We did not observeny correlation between the aspirin reaction unit amountnd impaired endothelial function. This could be becauseost of our patients had an optimal response to aspirin.nly 6 patients had an aspirin reaction unit value of �550,threshold previously used to define aspirin resistance.25

Periprocedural troponin elevation is relatively commonfter PCI in patients with stable angina, although it has andverse effect on the patient’s prognosis. A troponin Tlevation �3 times the reference value26 has been associ-ted with adverse mid- and long-term prognoses.27 Coro-ary manipulation and side branch occlusion are knowneterminants of periprocedural myonecrosis in patients un-ergoing elective PCI.28 In addition, greater platelet reac-ivity after clopidogrel has been found to be significantlyssociated with periprocedural myonecrosis.3,13 We ob-erved a periprocedural significant troponin T elevation (�3imes the reference value) in those patients with more im-aired endothelial function before the procedure. Endothe-ial function has been shown to be a predictor of cardiovas-ular adverse events in several studies.29 In unstable angina,WF activity was significantly increased in patients withositive troponin T levels compared to those with negativeroponin T values.30 We can speculate that the greater vWFctivity observed in patients with more impaired endothelialunction might increase platelet adhesion and aggregation,redisposing to a thrombotic status eventually responsibleor the distal microcirculatory embolism and myonecrosisbserved during PCI.

Our pilot investigation was limited by its small sampleize, and the present findings require additional confirma-ion in larger clinical studies. Genetic polymorphisms haveecently been described in the subtype 2C19 of cytochrome450 enzymes that result in a reduced transformation oflopidogrel in its active metabolite.5 The presence of thisenetic variant in our patients might have been a potentialonfounding factor. However, we believe this played only aimited role considering the significant correlation observedetween endothelial impairment and basal platelet reactivitys assessed by soluble P-selectin.

1. Mehta SR, Yusuf S, Peters RJ, Bertrand ME, Lewis BS, NatarajanMK, Malmberg K, Rupprecht H, Zhao F, Chrolavicius S, et al. Effectsof pretreatment with clopidogrel and aspirin followed by long-termtherapy in patients undergoing percutaneous coronary intervention: thePCI-CURE study. Lancet 2001;358:527–533.

2. Snoep JD, Hovens MM, Eikenboom JC, Van Der Bom JG, JukemaJW, Huisman MV. Clopidogrel nonresponsiveness in patients under-

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