Thienopyridines such as clopidogrel inhibit P2Y₁₂, one of two platelet adenosine diphosphate (ADP) receptors (P2Y₁, P2Y₁₂) and have been shown to confer clinical benefit in a variety of conditions characterized by the risk of arterial thrombosis 123. Nonetheless, in the setting of coronary artery disease, about 1–1.9% of patients may experience acute or sub-acute stent thrombosis (ST) after implantation of a coronary stent 45 despite treatment with clopidogrel in combination with aspirin. Several mechanisms could explain a low platelet response to clopidogrel, including poor compliance to treatment, 6 variable absorption of the drug and/or variable generation of the active metabolite, and potential drug-drug interactions 7. Some studies contend that resistance to clopidogrel may be present in as many as 20% of subjects 8910. Patients with a low response to clopidogrel are known to have an increased risk of cardiovascular events 1112. Thus, there is a clinical need for a reliable test of platelet response to clopidogrel therapy as a guide to individualizing dosing regimens. However, the ideal method for quantifying inhibition of platelet function by clopidogrel has yet to be agreed upon by the European Society of Cardiology and American College of Cardiology. Current assays which might be considered to be the gold standard, such as light transmission aggregometry (LTA), flow cytometric evaluation of platelet activation markers and flow cytometric measurement of the vasodilator-stimulated phosphoprotein (VASP) phosphorylation status, are technically complex and restricted to specialized laboratories and therefore none stands out as the clear investigation of choice.

On the other hand, the accuracy of point-of-care assays is still unclear 1314. The VerifyNow-P2Y₁₂ test was designed to overcome the limitations of conventional optical platelet aggregation assays. It is a rapid test that uses ADP to stimulate platelets in the presence of prostaglandin (PG) E₁, which inhibits activation downstream of a second ADP receptor P2Y₁, thus making the assay more sensitive to the activity of P2Y₁₂. The test can be performed directly in the catheterization laboratory prior to percutaneous coronary intervention (PCI). However, there are minimal clinical data on which to define a suitable cut-off value for low responders 151617. The aim of our study was to compare the VerifyNow-P2Y₁₂ test with flow cytometric analysis of platelet activation to define a cut-off value for low responders to clopidogrel.

A simple test such as the VerifyNow-P2Y₁₂ test may be useful to monitor the response to antiplatelet therapy and offers the possibility of a point-of-care approach, e.g. to assess whether the response to a clopidogrel loading dose is adequate in patients undergoing coronary stenting. As relevant as the simplicity of its use is, the correct interpretation of results remains of paramount importance to make appropriate therapeutic decisions. According to the manufacturer, the initial proposed cut-off value to discriminate patients with a low response to clopidogrel using the VerifyNow-P2Y₁₂ test was 50% inhibition (not specified in the user manual, only suggested informally). Using such a value, we were surprised to find that an unexpectedly high 75% of our 52 patients treated with DAT were defined as low clopidogrel responders. We considered this as unlikely and sought to verify the validity of the proposed cut-off limit. Indeed, by using markers of platelet activation measured by flow cytometry to define low responders, we found by ROC curve evaluation that a % inhibition ≤ 15 and absolute PRU > 213 may be VerifyNow-P2Y₁₂ cut-off values that identify patients with low response to clopidogrel with maximum sensitivity and specificity. Using the ≤ 15% inhibition limit that we established, only 21% of our 52 patients receiving conventional clopidogrel treatment were classified as low responders (and 23% using the PRU > 213 limit). This result is in agreement with other studies that have shown resistance to clopidogrel may be present in as many as 20% of subjects 81022. Recently, Lordkipanidzé et al. 23 suggested that the assay-reported % inhibition of platelets fails to accurately quantify inhibition achieved by clopidogrel, as TRAP-induced aggregation may not adequately mimic off-drug ADP/PGE1-induced aggregation. We have therefore described the usefulness of both the PRU value and the % inhibition value for detecting 'low responders' in this manuscript.

In the present study, the reliability of both cut-off values was shown to be high on ROC curve analysis, with the value for % inhibition shown to have a 100% sensitivity for detecting low responders as defined by flow cytometry, a finding that contrasts with the results obtained in the work by Lordkipanidzé et al. 23. This emphasises the importance of further published studies in this area, as establishing the reliability of each assay may not be as straight forward as first appears. Whilst the accuracy of the % inhibition value quoted by the VerifyNow-P2Y12 test remains to be clarified, there are now several clinical studies which show that a high PRU obtained with this assay is associated with adverse clinical consequences. For example, in a large clinical study of 380 PCI patients, a high PRU was strongly associated with adverse clinical outcomes, including stent thrombosis (p = 0.004) and furthermore was associated with the hardest of clinical endpoints – cardiovascular death (p = 0.04) 15. Subsequent publications have confirmed the usefulness of the VerifyNow assay in predicting adverse clinical events 1617. The usefulness of our lab cut-off value of PRU > 213 should be considered in conjunction with other published cut-offs proven be of clinical significance, such as those identified by Price et al. (PRU > 235) 15 and Patti et al. (PRU > 240) 16. From these data, we can infer that whilst the % inhibition value remains of interest, the PRU value (a direct measure of platelet aggregation induced by either ADP/PGE1) may be a more reliable method of identifying subjects who are hyporesponsive to clopidogrel therapy and subsequently at risk of clinical events.

The validity of our conclusions is entirely dependent on the definition of resistance to anti-platelet therapy, for which we relied on the measurement of two platelet activation markers by flow cytometric analysis. Flow cytometry, similarly to light transmission aggregometry (LTA), is used by specialized laboratories and represents a reference standard for the assessment of platelet function 1314.

In this regard, LTA is the most widely used method but has well-known limitations, including poor reproducibility, high sample volume, requirement for sample preparation, length of assay time, requirement for a skilled technician, and cost 13. Therefore, new options for platelet function testing have been developed to address these disadvantages and to meet the need for point-of-care testing that can be performed at or near a patient's bedside without requiring a high degree of technical expertise. The new tests include VerifyNow (Accumetrics, San Diego, CA); Plateletworks (Helena Laboratories, Beaumont, TX); Thrombelastograph PlateletMapping System (Haemoscope Corporation, Niles, IL); Impact cone and plate(let) analyzer (DiaMed, Cressier, Switzerland); and Platelet Function Analyzer 100 (PFA-100; Dade Behring, Newark, DE) 13. However, the relationship of in vivo platelet function and adverse clinical events to results of ex vivo platelet function tests remains largely unknown 24.

Aggregation assays are influenced by GP IIb-IIIa function downstream of the outside-in signals generated by platelet agonists, including ADP, and are dependent as well on inside-out signals required for activation of the integrin. For these reasons, which may confound the interpretation of the functional state of P2Y₁₂, we elected to measure surface translocation of P-selectin as an activation marker possibly more directly linked to ADP-induced platelet stimulation, as well as PAC-1 binding that, by reflecting GP IIb-IIIa activation, provides information consistent with that of aggregation assays. In order to obtain strong platelet stimulation, a desirable condition to study the effect of anti-platelet drugs, we used a combination of ADP and collagen to activate two distinct signalling pathways. Moreover, we stimulated platelets in the presence of PG E₁, a condition occurring in the VerifyNow-P2Y₁₂ test as well, with the intent of inhibiting signalling downstream of the P2Y₁ ADP receptor and highlighting the functional state of P2Y₁₂, which is the specific target of thienopyridines such as clopidogrel 25. Of note, the use of PG E₁ is not compatible with aggregation studies. CD62P expression and PAC-1 binding appeared equally useful as indicators of the effects of anti-platelet treatment. Because we collected blood specimens with the addition of PG E₁ (to prevent post-sampling artefacts) we could verify that, in spite of the presence of this inhibitor, dual agonist-induced activation still caused an enhancement of activation markers, and the difference between normal controls and patients with stable coronary artery disease receiving anti-platelet drugs remained highly significant.

In conclusion, these results provide information on the cut-off values, using the VerifyNow-P2Y₁₂ test, that identify patients with a low response to antiplatelet therapy. A % of inhibition ≤ 15% and absolute PRU > 213 appear to reflect low platelet inhibition.

ADP: adenosine diphosphate; PG: prostaglandin; PCI: percutaneous coronary intervention; PRU: P2Y₁₂ reactive units; CD62P: P-selectin; mAb: monoclonal antibody; GP: glycoprotein; PRP: platelet-rich plasma; FITC: fluorescein isothiocyanate; PE: R-phycoerythrin; FS: forward light scatter; SS: side light scatter; CD42b: GP Ibα; TRAP: thrombin receptor activating peptide; IQR: interquartile range; ROC: receiver operating characteristic; LTA: light transmission aggregometry.

The authors do not have any financial associations to disclose that might pose a conflict of interest in connection with the submitted article. The Endotech S.PA., Italy, provided VerifyNow device and kit support for this study. The sponsor had no role in the study design, data analysis, and manuscript writing.

Each author have contributed significantly to the submitted work, specifically: Dr CG wrote the manuscript and Drs LM, FF, MC, AC, AC were responsible for the collection analysis and interpretation of the data. Drs AC, AL, AS, JC revised the manuscript for intellectual content and Dr GC revised the manuscript for the statistical methods. Drs CG, Prof. AM, Prof. ZR and Dr. AC designed the study and gave final approval of the manuscript. All the authors have read and approved this paper.