Introduction
The combination of aspirin with P2Y 12 inhibitors, which is also known as dual antiplatelet therapy, is the standard of care for oral antiplatelet treatment in patients presenting with an acute coronary syndrome (ACS) and/or who are undergoing percutaneous coronary intervention (PCI). The widely variable pharmacological effects of clopidogrel and the heterogeneous relationship between the extreme value of adenosine diphosphate (ADP)–induced platelet aggregation and the occurrence of ischemic and bleeding events are limitations of this prodrug. In contrast, second-generation oral P2Y 12 inhibitors (i.e., prasugrel and ticagrelor) display a more consistent, rapid, and profound inhibition of the P2Y 12 receptor and produce further reductions in the risk of ischemic events, albeit with more bleeding complications (see Chapter 19 ). Clopidogrel is the second-leading drug sold worldwide, and its nonselective administration is counterintuitive when it is possible to assess for a measurable drug effect and when there is the possibility of identifying patients at risk of developing adverse outcomes. Individualized treatment based on point-of-care assays is now technically possible with the potential to improve the risk and/or benefit of oral P2Y 12 therapy by identifying extreme responses and adjusting treatment. There is a consensus that high on-treatment platelet reactivity (HPR) or inhibition to ADP is a major risk factor for post-PCI ischemic and/or bleeding events, respectively. However, guidelines have given a class IIb recommendation for platelet function testing or genotyping if the results of testing may alter management. In this chapter, we develop the concept that individualization of oral antiplatelet therapy reflects multifaceted influences with a variety of potential clinical implications.
Clopidogrel Metabolism and Biological Response
Clopidogrel is a second-generation thienopyridine derivative that binds specifically and irreversibly to the platelet P2Y 12 purinergic receptor, inhibiting ADP-mediated platelet activation and aggregation (see Chapter 19 ). It is an inactive prodrug that requires oxidation by the hepatic cytochrome P450 (CYP) system to generate clopidogrel H4-thiol, the putative-only active metabolite that selectively binds the P2Y 12 receptor. Platelet aggregation is affected not only when triggered by ADP, but also by other substances that require released ADP as an amplifier ( Figure 20-1 ).
The wide interindividual variability of the biological response to clopidogrel is an established limitation of clopidogrel, and it has multiple determinants, including environmental, cellular, clinical, and genetic factors ( Figure 20-2 ). HPR and low-on treatment platelet reactivity (LPR) have been associated with recurrent ischemic and bleeding events. The aim of platelet function testing is to measure individual responses to the drug to avoid HPR and LPR. Multiple methods exist, without consensus with regard to the best method to use.
Methods for Platelet Function Testing
There are currently four ADP-stimulated assays (vasodilator-stimulated phosphoprotein [VASP] phosphorylation: VASP-P assay, [Diagnostica Stago, Biocytex, Asnières, France]; Multiplate impedance aggregometry, [Dynabyte Medical, Munich, Germany]; VerifyNow [Accumetrics, San Diego, California]; and light transmission aggregometry [LTA]) that are validated for the prediction of stent thrombosis and bleeding in patients with ACS. There are substantial methodological differences that explain the imperfect agreement among ADP-stimulated assays and the heterogeneity in classification of subjects at risk for thrombotic events. The global aggregation measure approach (platelet aggregation) is usually less specific to the drug action, whereas analysis of the drug effect with high specificity at subcellular levels (such as VASP phosphorylation) provides less information with regard to the overall state of the activation–aggregation cascade. LTA with ADP stimulation is only recommended when no standardized assays are available. Based on the currently available evidence, the best preliminary cutoffs for risk stratification include 95 and 208 P2Y 12 reaction units (PRUs) for VerifyNow, 19 and 46 U for Multiplate, and 10% and 50% platelet reactivity index (PRI) for VASP-P for bleeding and stent thrombosis, respectively ( Table 20-1 ). These suggested cutoffs might be different according to clinical presentation, timing from PCI, procedural success, and ethnicity and therefore need further validation.
| Assays | Stent thrombosis | Bleeding | ||
|---|---|---|---|---|
| Cutoff | n | Cutoff | n | |
| VerifyNow | >208 PRU | 11,245 | <95 PRU | 8449 |
| Multiplate | >46 U | 1608 | <19 U | 2533 |
| VASP-P | >50% PRI | 640 | <10% PRI | 1542 |
Vasodilator-Stimulated Phosphoprotein Phosphorylation
Measuring the phosphorylation state of VASP using flow cytometry is a completely P2Y 12 receptor–specific method for the evaluation of ADP-receptor inhibition. VASP is a second messenger in the signaling pathway of the P2Y 12 receptor. The ratio of dephosphorylated and phosphorylated VASP is a selective measure of P2Y 12 inhibition. The measurement is not influenced by the presence of glycoprotein IIb/IIIa receptor inhibitors (GPIs), and it is the only assay that is able to evaluate the extent of P2Y 12 receptor inhibition without the influence of the P2Y 1 receptor. It requires a special laboratory environment and staff experienced in flow cytometric analysis, making the method inappropriate for routine clinical purposes, but ideal for platelet function research.
Multiplate Impedance Aggregometry
Muliplate impedance aggregometry is a semi-automated, standardized aggregometry that evaluates the efficacy of platelet inhibition in whole blood. The assessment is significantly faster and more reliable than conventional aggregometry. It uses an impedance aggregometer that detects changes in electric impedance over time between two electrodes immersed in hirudin-anticoagulated whole blood diluted with saline. Changes in impedance are plotted over time, resulting in an aggregation curve that is similar to LTA. This technique requires sample preparation and pipetting throughout the assessment. The cost of testing is between the costs of LTA and VerifyNow.
VerifyNow
The VerifyNow System is a point-of-care assay that measures agonist-induced platelet aggregation by turbidimetric-based optical detection. Platelets are activated by the presence of agonists and bind to fibrinogen-coated beads, causing agglutinates to drop out of solution. Results are reported as PRUs, with a lower PRU value corresponding to a higher degree of P2Y 12 receptor inhibition. Advantages of the VerifyNow system include simplicity, sensitivity, speed, and user-friendliness.
Light Transmission Aggregometry
Infrared light transmittance passing through platelet-poor plasma is used to represent 100% aggregation, and the optical changes from 0%, set by the unstimulated platelet-rich plasma, is evaluated in response to inductors. As activated platelets aggregate after the inductors, optical density decreases in the absence of antiplatelet medication. It is inexpensive and the historical gold standard tool for platelet function studies, with widespread use and with significant clinical experience in both pharmacodynamic and clinical studies. However, LTA is time-consuming and requires trained laboratory personnel, which precludes testing 24-hour/7-day service at the bedside. The lack of standardization caused by the diversity in the concentration of agonist used (5, 10, 20 μM), the preferred estimate for evaluation (peak aggregation, late aggregation), the choice of anticoagulants (citrate, hirudin, or phenylalanyl-prolyl-arginyl chloromethyl ketone [PPACK] anticoagulation), and the different specifications in sample preparation techniques (centrifuging time and speed) are other important limitations.
Rationale for Platelet Function Testing
Prognostic Usefulness of Platelet Function Testing for Thrombotic Events
Platelet function testing has confirmed that P2Y 12 -receptor signaling is a major component of pathophysiological thrombus formation in patients with ACS treated with PCI. In particular, HPR has emerged as an independent predictor for stent thrombosis. In the ADAPT-DES (Assessment of Dual AntiPlatelet Therapy with Drug-Eluting Stents) trial, the largest observational platelet function study conducted to date, approximately 50% of 30-day post-PCI stent thrombosis was attributable to high platelet reactivity (propensity-adjusted hazard ratio [HR], 3.0; 95% confidence interval [CI], 1.39 to 6.49; P = .005), defined as a PRU value of more than 208 when using the bedside test VerifyNow. HPR was also independently correlated with a 1-year definite and/or probable stent thrombosis (adjusted HR, 2.49; 95% CI, 1.43 to 4.31; P = .001) and myocardial infarction (MI) (adjusted HR, 1.42; 95% CI, 1.09 to 1.86; P = .01). However, the risk associated with HPR is modulated by the clinical characteristics and procedural results of the studied individual. For example, the predictive accuracy of HPR to ADP was higher in patients with ACS (adjusted HR, 3.91; 95% CI, 1.51 to 10.11; P = .005) than in patients with stable coronary artery disease (adjusted HR, 1.49; 95% CI, 0.35 to 6.36; P = .59). In the TRILOGY-ACS (Targeted Platelet Inhibition to Clarify the Optimal Strategy to Medically Manage Acute Coronary Syndromes) trial, HPR in patients treated by clopidogrel or prasugrel was not an independent predictor of adverse events. Finally, HPR is much less prevalent in prasugrel- or ticagrelor-treated patients than in patients treated with clopidogrel, and its correlation with outcome in patients treated with these third-generation ADP antagonists has still not been established.
Prognostic Usefulness of Platelet Function Testing for Bleeding Events
Among patients treated with P2Y 12 inhibitors, LPR, defined in Table 20-1 , is associated with an increased risk of major bleeding complications compared with optimal platelet reactivity (relative risk [RR] 1.74; 95% CI, 1.47 to 2.06; P <.00001). Of interest, the increase in the risk of bleeding using current generation potent P2Y 12 inhibitors is becoming closer to the magnitude of reduction in stent thrombosis or MI. The lack of standardization in the definition of bleeding events, duration of follow-up, adjudication of bleeding events in many studies, and predefined cutoff value of LPR are recognized limitations. In addition, evidence with the current generation P2Y 12 inhibitors is much less compared with clopidogrel, and the relevance of clopidogrel thresholds to define LPR with prasugrel and ticagrelor needs to be verified. A therapeutic window for P2Y 12 inhibition has been suggested and has raised the possibility of tailoring antiplatelet therapy for optimization of the benefit-to-risk ratio in clinical practice ( Figure 20-3 ).
Platelet Function Test-Guided Treatment Strategy
Even if on-treatment platelet reactivity appears to be a reliable and independent measure of the risk of future events, the concept of selective intensive antiplatelet therapy based on a measured drug effect has never been successfully proven. Randomized trials that have examined the platelet function testing hypothesis have been limited by low event rates, insufficient pharmacological interventions, bias toward low-risk patient recruitment, and intervention in patients who are considered to be nonresponders after stent placement.
The GRAVITAS (Gauging Responsiveness with A VerifyNow assay-Impact on Thrombosis And Safety) trial tested a strategy of a fixed regimen of high-dose clopidogrel (600 mg followed by 150 mg/day for 6 months) in patients with high on-treatment reactivity (defined as 230 PRUs according to the VerifyNow P2Y 12 test). This strategy of a fixed higher dose, regardless of the achieved level of platelet inhibition, did not reduce cardiovascular death, MI, and stent thrombosis after PCI compared with a standard dose of clopidogrel (75 mg/day). Interpretation of these results should take into account the following limitations: a stringent definition of clopidogrel poor response (PRU >230); a low-intensity intervention to overcome poor response; a small proportion of ACS patients (15.5% with positive biomarkers); and a low event rate (observed event rate was 2.3% compared with the expected 5%) because of randomization after coronary intervention. A post hoc analysis of the study showed that PRU values less than 208 were independently associated with the 60-day primary endpoint, which was a composite of death, MI, and stent thrombosis (HR, 1.68; 95% CI, 0.76 to 1.32).
In Testing Platelet Reactivity in Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy with Prasugrel (TRIGGER-PCI), 2150 low-risk stable coronary artery disease patients with successfully implanted drug-eluting stents and more than 208 PRUs on VerifyNow were planned to be randomized to receive either prasugrel administered as a 60-mg loading dose, followed by 10-mg/day clopidogrel administered with a 600-mg loading dose and followed by 75 mg/day. Although TRIGGER-PCI was terminated early because of futility, the active strategy was effectively reduced HPR, which was 6% on prasugrel. Only 1 occurrence of the primary endpoint was reported among 236 patients who completed 6 months of follow-up. Of importance, 30% of the enrolled patients declined randomization after being identified as having HPR, which underscored the limitation of a trial strategy based on identification of deemed nonresponders instead of randomizing the use of platelet function testing and adjusting treatment.
In the ARCTIC (Assessment by a Double Randomization of a Conventional Antiplatelet Strategy Versus a Monitoring Guided Strategy for Drug-Eluting Stent Implantation and of Treatment Interruption Versus Continuation One year After Stenting) trial (n = 2440), HPR was identified in patients allocated by randomization to the strategy of platelet function monitoring, and treatments were adjusted to control this risk factor as much as possible both before and after hospital discharge ( Figure 20-4 ). However, this strategy failed to show a benefit on ischemic events that occurred during the first year after hospitalization for revascularization with coronary drug-eluting stents ( Figure 20-5 ). High-platelet reactivity during treatment with aspirin was defined as ≥550 aspirin reaction units. High-platelet reactivity during treatment with thienopyridine was defined as ≥235 PRU and 15% or less inhibition compared with a baseline measurement of aggregation, or both. Drug adjustment consisted of a new loading dose of clopidogrel or use of prasugrel, infusion of GPI, and an increased maintenance dose of clopidogrel or use of prasugrel. The low ACS rate (30%) and the inability of the intervention to overcome HPR in 15% of patients were the major drawbacks of the ARCTIC study. The same results were confirmed after excluding all in-hospital events, refuting the hypothesis that HPR is not only a marker of risk, but is also a risk factor that can possibly be modified with the antiplatelet drugs available. This study was appropriately powered with a significant more aggressive pharmacological intervention in nonresponders, leading to a twofold reduction in the rate of nonresponders. Of interest, patients with major bleeding were more likely to have HPR than those who did not bleed (34.4% vs. 15.2%; P = .001). This demonstrates that HPR is a complex trait that solely integrates treatment response, and it furthermore explains why it was not an independent predictor of death in the ADAPT-DES registry.
