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Keywords:

  • active metabolite;
  • clopidogrel;
  • monitoring;
  • pharmacokinetics;
  • platelet function test;
  • responsiveness

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Summary. Background: Multiple platelet function tests claim to be P2Y12-pathway specific and capable of capturing the biological activity of clopidogrel. Objectives: The aim of the present study was to determine which platelet function test provides the best reflection of the in vivo plasma levels of the active metabolite of clopidogrel (AMC). Patients/methods: Clopidogrel-naive patients scheduled for elective percutaneous coronary intervention (PCI) received a 600 mg loading dose of clopidogrel and 100 mg of aspirin. For pharmacokinetic analysis, blood was drawn at 0, 20, 40, 60, 90, 120, 180, 240 and 360 min after clopidogrel loading and peak plasma concentrations (Cmax) of the AMC were quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Platelet function testing was performed at baseline and 360 min after the clopidogrel loading. Results: The VASP-assay, the VerifyNow P2Y12-assay and 20 μmol L−1 adenosine diphosphate (ADP)-induced light transmittance aggregometry (LTA) showed strong correlations with Cmax of the AMC (VASP: R2 = 0.56, P < 0.001; VerifyNow platelet reactivity units (PRU): R2 = 0.48, P < 0.001; VerifyNow %inhibition: R2 = 0.59, P < 0.001; 20 μmol L−1 ADP-induced LTA: R2 = 0.47, P < 0.001). Agreement with Cmax of the AMC was less evident for 5 μmol L−1 ADP-induced LTA or whole blood aggregometry (WBA), whereas the IMPACT-R ADP test did not show any correlation with plasmalevels of the AMC. Conclusion: The flow cytometric VASP-assay, the VerifyNow P2Y12 assay and, although to a lesser extent, 20 μmol L−1 ADP-induced LTA correlate best with the maximal plasma level of the AMC, suggesting these may be the preferred platelet function tests for monitoring the responsiveness to clopidogrel.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Clopidogrel is administered as an inactive prodrug requiring metabolization by the hepatic cytochrome P450 system for its antiplatelet activity [1]. The formed active thiol metabolite of clopidogrel (AMC) irreversibly inhibits adenosine 5′-diphosphate (ADP)-mediated platelet activation and aggregation by antagonizing the P2Y12-receptor [2]. Although clopidogrel has proven its efficacy in reducing atherothrombotic events after percutaneous coronary intervention (PCI), a considerable interindividual variability in response has been reported [3]. Clinical, genetic and pharmacokinetic factors contribute to this wide variability in response and it has been demonstrated that the absolute magnitude of ADP-induced platelet reactivity at the time of PCI is associated with an increased risk of atherothrombotic events, including stent thrombosis [4–11]. It is therefore of utmost importance to have a confident parameter of the in vivo activity of clopidogrel.

Multiple platelet function tests claim to be P2Y12-pathway specific and be capable of capturing the specific biological activity of clopidogrel [12]. As the AMC is prone to rapid inactivation of its reactive thiol group [13], quantitative methods for the detection of the AMC require a sophisticated stabilization protocol. Data on the performance of different platelet function tests in relation to plasma levels of the AMC are therefore scarce.

In the present study we sought to investigate which of the currently available platelet function tests – if any – provides the best representation of the in vivo plasma levels of the active metabolite of clopidogrel.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Patient population

Consecutive patients with stable angina pectoris scheduled for PCI were eligible. All patients were on 80–100 mg of aspirin therapy daily. Exclusion criteria were a history of bleeding diathesis, presence of an acute coronary syndrome (ACS), a platelet count < 150 × 109 L−1, the use of a glycoprotein IIb/IIIa inhibitor or a coumarin within the last 14 days or any contraindication to clopidogrel or aspirin. The study protocol complied with the declaration of Helsinki and was approved by the ethical committee of our institution, and all patients gave written informed consent for participation.

Study protocol and blood sampling

All eligible patients visited the outpatient clinic for platelet function evaluation, physical examination and a standardized interview. All patients received a witnessed 600 mg loading dose of clopidogrel and 100 mg of aspirin.

Blood samples for platelet function evaluation were drawn from the antecubital vein with a loose tourniquet and collected in citrated (3.2%) non-vacuum tubes (Sarstedt, Nümbrecht, Germany) before and 6 h after the clopidogrel loading dose. All blood samples were processed within 2 h after collection.

Blood samples for determining the AMC plasma concentration were collected from the antecubital vein in tubes containing K3-EDTA at 0, 20, 40, 60, 90 120, 180, 240 and 360 min after the clopidogrel loading dose. Samples were immediately centrifugated at 1500 ×g for 10 min and plasma was pipetted into tubes containing a stabilizing agent (Pat. No. DE 10 2004 046 159.7) [14] in order to prevent degradation of the AMC. After vortexing for 60 s, samples were stored at −80°C until analysis of the AMC plasma concentration with liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Platelet function testing

Platelet function was evaluated using five different ADP-induced platelet function tests. All tests were performed according to the manufacturer’s recommendations or according to generally accepted standard procedures.

Light transmittance aggregometry  Light transmittance aggregometry (LTA) was quantified in non-adjusted platelet-rich plasma on a four-channel APACT 4004 aggregometer (LABiTec, Arensburg, Germany). Platelet-poor plasma was set as 100% aggregation and after stimulation of platelet aggregation with ADP in final concentrations of 5 and 20 μmol L−1, both peak (maximal) and late (at 360 s) aggregation (%) were measured [15,16].

Whole blood aggregometry  Whole blood aggregometry (WBA) was performed in citrated whole blood as described previously [17]. In brief, citrated whole blood was diluted 1:1 with pre-warmed (37 °C) saline. Platelet aggregation was induced by the addition of 10 μmol L−1 ADP and the increase in impedance caused by aggregated platelets between the two electrodes was recorded on a ChronoLog 700 model Aggregometer (ChronoLog, Havertown, PA, USA). The results are reported as the maximal amplitude of impedance (Ω) after 10 min.

IMPACT-R test  The Impact-R device (DiaMed, Cresier, Switzerland) is based on the cone and plate(let) analyzer technology [18]. Citrated whole blood samples were incubated for 1 min with 1.38 μmol L−1 ADP, causing the formation of micro platelet aggregates in non-responsive samples. A volume of 130 μL of this ADP-pre-incubated whole blood sample was pipetted on a polystyrene well and subjected to shear (1800 s−1) for 2 min, using a rotating cone. The wells were washed and stained followed by measurement of the percentage of surface coverage (SC) from shear-induced platelet adhesion and aggregation with an image analysis system. The percentage SC is inversely correlated with the magnitude of ADP-induced platelet activation.

The VerifyNow® P2Y12 assay  The VerifyNow® P2Y12 assay (Accumetrics, San Diego, CA, USA) is based on optical detection of platelet aggregation in whole blood [19]. The assay contains 20 μmol L−1 ADP to induce platelet aggregation and 22 nmol L−1 prostaglandin E1 to suppress the undesirable contribution from ADP-agonism of P2Y1-receptors to platelet aggregation. The magnitude of ADP-induced platelet activation is expressed as P2Y12 reaction units (PRU). In addition, the device calculates the percentage of P2Y12-inhibition, based on thrombin receptor-activating peptide (TRAP)-induced platelet aggregation (BASE value) and PRU.

Flowcytometric vasodilator-stimulated phosphoprotein (VASP) analysis  Flowcytometric analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation was performed using a commercially available kit from Biocytex (Marseille, France) [10]. In brief, citrated blood was incubated with either PGE1 or PGE1 + ADP and fixed with paraformaldehyde, after which platelets were permeabilized followed by immunolabeling with a CD61 phycoerythrin-labeled platelet specific antibody and a FITC-labeled VASP-P specific mouse monoclonal antibody or a negative isotopic control antibody. Samples were analyzed on a 500 MPL flowcytometer (Beckman Coulter, Brea, CA, USA). The magnitude of platelet activation was expressed as the platelet reactivity index (PRI), which can be calculated from the mean fluorescence intensity (MFI) of samples incubated with PGE1 or PGE1 + ADP using the following formula:

  • image

Liquid chromatography-tandem mass spectrometry

Analysis and detection of the active thiol metabolite were performed as previously described on a triple-quadruple tandem mass spectrometer (TSQ Quantum, Thermo Electron, Dreieich, Germany) [20]. Measurements were performed in triplicate using a Surveyor HPLC system. Plasma concentration versus time data of each patient were fitted by a one-compartment first order model (Bateman function r > 0.94) using WinNolinTM Software (Pharsight, Palo Alto, CA, USA). The maximal plasma concentration (Cmax, ng mL−1) was calculated from the individual regression fits.

Sample size calculation and statistics

The t-test for point biserial correlation based on the non-centrality parameter δ was used for a priori sample size calculation assuming a two-sided significance level of α = 0.05 and a power 1 − β = 0.95. From previous assessments (and from the clinical perspective that a diagnostic test should explain at least 50% of the variance of a variable) a determination coefficient of R2 = 0.5 was deemed relevant [20]. The total required sample size was calculated to be 16. Platelet function testing results were related to the Cmax of the AMC, as this appeared to be the pharmacokinetic parameter correlating best with inhibition of platelet aggregation (IPA) in a previous study [20]. Continuous variables are expressed as mean ± SD and categorical variables as frequencies (%). The individual clopidogrel responsiveness, defined as the absolute decrease in platelet reactivity from baseline to 6 h after clopidogrel loading, was tested using a paired t-test. The coefficient of determination (R2) was calculated to test the linear association between the Cmax of the AMC and the absolute inhibition of platelet aggregation (IPA) at 6 h after clopidogrel loading. Additionally, a normalized anova was performed on the ratio ‘% platelet inhibition [(baseline-T6)/baseline]/cmax of the AMC’, to determine the statistical significance of the observed differences between platelet function tests with regard to their correlation with plasma levels of the AMC. All statistical analyses were performed with spss (version 15.0, SPSS Inc., Chicago, IL, USA) and P-values < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

A total of 20 consecutive patients were enrolled. Clinical characteristics of the studied population are summarized in Table 1. All patients were on aspirin therapy (80−100 mg day−1) at the time of inclusion.

Table 1.   Baseline characteristics (n = 20)
  1. Continuous variables are presented as mean ± SD, and categorical variables as counts (%). BMI, body mass index; CAD, coronary artery disease; MI, myocardial infarction; PCI, percutaneous coronary intervention; PRP, platelet-rich plasma.

Age (years)60.2 ± 10.3
Men19 (95%)
Risk factors
 Hypertension13 (65%)
 BMI (kg m−2)27.90 ± 3.31
 Diabetes Mellitus2 (10%)
 Dyslipidemia13 (65%)
 Current smoker12 (60%)
 Familial history of CAD12 (60%)
 Previous MI9 (45%)
 Previous PCI9 (45%)
Concomitant medication
 Aspirin20 (100%)
 ACE inhibitor8 (40%)
 Beta-blocker14 (70%)
 Statin18 (90%)
Laboratory parameters
 Platelet count in whole blood (× 109 L−1)242 ± 53
 Platelet count in PRP (× 109 L−1)356 ± 96
 Hemoglobin (mmol L−1)8.9 ± 0.5

Responsiveness to a 600-mg loading dose of clopidogrel

All platelet function tests were able to detect a significant reduction in platelet function 6 h after clopidogrel loading dose administration (P < 0.001 for all), except the IMPACT-R ADP-test (P = 0.13). Despite the observed inhibitory shift in test results after clopidogrel administration, some overlap between baseline and post-clopidogrel values was notable for all platelet function tests.

Correlation between platelet function tests

Results of the VASP, VerifyNow P2Y12 assay and 20 μmol L−1 ADP-induced LTA showed moderate to good correlation with one and another, but lacked significant correlation with WBA and the IMPACT-R ADP-test (Table 2). Agreement between LTA induced by 5 μmol L−1 ADP and the other tests was less explicit, showing a moderate correlation with 20 μmol L−1 ADP-induced LTA and the VerifyNow P2Y12 assay, but no correlation at all with the VASP-assay, or with the WBA and the IMPACT-R ADP-test.

Table 2.   Correlation between different platelet function tests
 LTA 5 ADP peakLTA 5 ADP lateLTA 20 ADP peakLTA 20 ADP lateWBAVerifyNowPRUVerifyNow % inhibitionImpact- R ADPVASP PRI
  1. The correlation coefficient (r, above the diagonal axis) and the coefficient of determination (R2, below the diagonal axis) are presented for each mutual comparison, *P < 0.05, P < 0.01, P < 0.001. ADP, adenosine diphosphate; LTA, light transmittance aggregometry; PRI, platelet reactivity index; PRU, P2Y12 reaction units; VASP, vasodilator-stimulated phosphoprotein; WBA, whole blood impedance aggregometry.

LTA 5 ADP peak10.840.810.780.130.56*0.56−0.190.16
LTA 5 ADP late0.7110.590.56*−0.010.360.34−0.030.02
LTA 20 ADP peak0.660.3510.960.210.820.80−0.360.50*
LTA 20 ADP late0.610.31*0.9210.320.830.79−0.250.49*
WBA0.020.000.040.1010.200.230.160.08
VerifyNow PRU0.32*0.130.670.690.0410.94−0.300.55*
VerifyNow % inhibition0.320.110.640.620.050.891−0.380.64
Impact-R ADP0.040.000.130.060.030.090.141−0.48*
VASP PRI0.030.000.25*0.24*0.000.31*0.410.23*1

Correlation between IPA and plasma levels of the AMC

Linear regression analysis revealed major differences between the various platelet function tests regarding correlations between IPA and the Cmax of the AMC. The highest correlation coefficients were observed for the flow cytometric VASP-assay (R2 = 0.56, P < 0.001; Fig. 1) and the VerifyNow P2Y12 assay (R2 = 0.48, P < 0.001 for PRU and R2 = 0.59, P < 0.001 for % inhibition). The absolute change in platelet reactivity when measured with 20 μmol L−1 ADP-induced LTA also showed strong correlations with the Cmax value of the AMC (R2 = 0.46, P = 0.001 for peak and R2 = 0.47, P < 0.001 for late IPA; Fig. 2). In contrast, no significant association was observed between the Cmax value of the AMC and IPA when measured with LTA using a lower concentration of 5 μmol L−1 ADP (peak: R2 = 0.19, P = 0.06; late: R2 = 0.02, P = 0.56) or WBA (R2 = 0.07, P = 0.28; Figs 2 and 3). The IMPACT-R ADP-test did not show any correlation with Cmax of the AMC (Fig. 3). The normalized anova revealed that the IMPACT-R ADP-test was significantly inferior compared with all other platelet function tests (P-values for comparisons with all other tests were < 0.05), while no statistical differences were observed between all other platelet function tests (P > 0.05).

image

Figure 1.  Correlation between the peak plasma concentrations (Cmax) of the active metabolite of clopidogrel (AMC) (ng mL−1) and inhibition of platelet aggregation (IPA) assessed using the flow cytometric VASP-assay (upper graph) and the VerifyNow P2Y12 assay, both platelet reactivity units (PRU) (middle graph) and % inhibition (lower graph). IPA was calculated as Δ (baseline-T6) of the results for each platelet function test. Solid lines represent the linear regression fit to the data, dashed lines indicate 95% confidence intervals. The coefficient of determination (R2) and the corresponding P-values are represented in the separate graphs.

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image

Figure 2.  Correlation between the peak plasma concentrations (Cmax) of the active metabolite of clopidogrel (AMC) (ng mL−1) and inhibition of platelet aggregation (IPA) assessed using the adenosine 5′-diphosphate (ADP)-induced light transmittance aggregometry (LTA) using 5 μmol L−1 and 20 μmol L−1 (upper and lower panels, respectively) ADP-induced peak and late (at 360 s) aggregation (left and right panels, respectively). IPA was calculated as Δ (baseline-T6) of the results for each platelet function test. Solid lines represent the linear regression fit to the data, dashed lines indicate 95% confidence intervals. For each test, the coefficient of determination (R2) and the corresponding P-values are represented in the separate graphs.

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image

Figure 3.  Correlation between the peak plasma concentrations (Cmax) of the active metabolite of clopidogrel (AMC) (ng mL−1) and inhibition of platelet aggregation (IPA) assessed using the adenosine 5´-diphosphate (ADP)-induced whole blood impedance aggregometry (WBA) and the IMPACT-R ADP test. IPA was calculated as Δ (baseline-T6) of the results for each platelet function test. Solid lines represent the linear regression fit to the data, dashed lines indicate 95% confidence intervals. For each test, the coefficient of determination (R2) and the corresponding P-values are represented in the separate graphs.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

The magnitude of platelet inhibition varies widely according to the platelet function assay used in monitoring responsiveness to clopidogrel. The current recommendations issued by the American College of Cardiology (ACC), the American Heart Association (AHA) and the Society for Cardiovascular Angiography and Interventions (SCAI) state the following: ‘In patients in whom stent thrombosis may be catastrophic or lethal (unprotected left main, bifurcating left main, or last patent coronary vessel), platelet aggregation studies may be considered and the dose of clopidogrel increased to 150 mg per day if < 50% inhibition of platelet aggregation is demonstrated’ [21]. However, these guidelines do not further specify any details on which test or test conditions should be used. This puts the clinician in a difficult situation as multiple platelet function tests are now widely available but little is known about their ability to provide a reliable reflection of the in vivo biological activity of clopidogrel.

The present study demonstrates that the flowcytometric VASP-assay and the VerifyNow P2Y12-assay are the most appropriate platelet function tests to monitor peak plasma levels of the AMC, achieved by a 600 mg loading dose of clopidogrel. Therefore, these platelet function tests are likely to be the most accurate in measuring the actual in vivo biological activity of clopidogrel.

LTA is commonly recognized as the ‘gold standard’ platelet function test and showed fine correlations with peak plasma levels of the AMC as well, although only when platelet aggregation was stimulated with a high concentration of ADP (20 μmol L−1). LTA induced by 5 μmol L−1 lacks significant correlation with peak plasma levels of the AMC, and should therefore not be used to determine clopidogrel responsiveness. Both the flow cytometric VASP-assay and the VerifyNow P2Y12-assay incorporate PGE1 to decrease the contribution of ADP-agonism at the P2Y1-receptor to platelet aggregation, thereby increasing the selectivity of these tests for the P2Y12-pathway [22,23]. This might indeed be reflected by the high agreement between IPA and the Cmax of the AMC when measured with these tests. Using a higher concentration of the agonist ADP (20 μmol L−1) appears to improve the assessment of the responsiveness to clopidogrel as well, as 20 μmol L−1 ADP-induced LTA showed a higher degree of correlation with peak plasma levels of the AMC than 5 μmol L−1 ADP-induced LTA.

Although the VASP-assay has the advantage of providing stable test results until 24 h after blood drawing, both the VASP-assay and LTA are relatively labour intensive [24]. In contrast, the point-of-care VerifyNow P2Y12 assay is suitable for use in daily clinical practice, as the semi-automated technique allows rapid assessment of platelet inhibition in whole blood without the need for expert laboratory personnel. The accuracy of the Impact-R ADP-test in monitoring clopidogrel responsiveness might be seriously hampered by the requirement of multiple sample preparation proceedings that are sensitive to the introduction of variation in test results by the technician performing the test. Furthermore, the Impact-R ADP-test relies on a mechanism of platelet activation induced by shear stress, which is a different aspect of platelet reactivity, and apparently not a very suitable method for monitoring inhibition of the P2Y12-pathway.

The present study shows that the flowcytometric VASP, the VerifyNow P2Y12-assay and 20 μmol L−1 ADP-induced LTA are the most appropriate tests for determining the in vivo plasma levels of the AMC. In addition, these tests have previously shown to predict clinical outcome in patients treated with clopidogrel [5–10]. Poor responsiveness to clopidogrel is, however, one out of multiple factors contributing to the development of atherothrombotic events. Furthermore, the magnitude of post-treatment platelet reactivity is a composite of both clopidogrel responsiveness as well as pretreatment (baseline) platelet reactivity, resulting in a high on-treatment platelet reactivity in some patients showing a sufficient response to clopidogrel [25,26]. Hence, other platelet function tests evaluating different aspects of platelet function may also be capable of predicting clinical outcome in patients on clopidogrel, despite the lack of correlation with plasma levels of the AMC.

The present study describes the level of correlation of different ADP-induced platelet function tests with peak plasma levels of the AMC. This important message might resolve the ongoing debate on which platelet function test is suitable for monitoring clopidogrel responsiveness. Two aspects have to be addressed that need to be explored to further improve the clinical applicability of this message. First, the present study does not cover the complete subset of platelet function tests available for monitoring clopidogrel responsiveness. Additional tests include the Multiplate and the thromboelastography, which might show correlation with the plasma levels of the AMC as well. Second, although the present study contains a sufficient number of patients for a pharmacokinetic analysis, it does not allow an answer to the question whether patients with low plasma levels of the AMC are the ones to develop atherothrombotic complications after PCI. Nonetheless, combining the results of the present study with the already available evidence on the clinical efficacy of clopidogrel in reducing atherothrombotic events after PCI suggests a positive answer to that question.

In conclusion, the flow cytometric VASP-assay, the VerifyNow P2Y12 assay and, although to a lesser extent, 20 μmol L−1 ADP-induced LTA correlate best with the maximal plasma level of the AMC, suggesting these may be the preferred platelet function tests for monitoring the responsiveness to clopidogrel. Further distinction between these tests should be based on their labor intensiveness, costs and most importantly their sensitivity and specificity in predicting the occurrence of atherothrombotic events after PCI.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References
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