Response Variability and the Role of Platelet Function Testing

From the Sinai Center for Thrombosis Research, Baltimore Maryland. Disclosure: One or more authors has disclosed a potential conflict of interest regarding the content herein. Dr. Gurbel has received research grants and honoraria from Schering Plough, Haemoscope, Astra Zeneca, Medtronic, Lilly/Sankyo, Sanofi, Boston Scientific, Portola Pharmaceuticals and Bayer Healthcare. Manuscript submitted February 18, 2009 and accepted February 20, 2009. Address for correspondence: Paul A. Gurbel, MD, Sinai Center for Thrombosis Research, Sinai Hospital, 2401 West Belvedere Avenue, Baltimore, MD 21215. E-mail: pgurbel@lifebridgehealth.org ____________________________________ Oral antiplatelet therapy with clopidogrel and aspirin is an effective strategy to attenuate ischemic event occurrence in patients treated with coronary artery stents in the presence or absence of an acute coronary syndrome (ACS). Although this strategy has improved clinical outcomes, the frequency of recurrent events remains substantial (~10%), and bleeding in selected patients is a persistent and important concern. Current practice guidelines for antiplatelet therapy are mainly based on the clinical endpoints of large-scale multicenter trials that have employed a “one-size-fits-all” dosing strategy. Moreover, these clinical trials have largely been conducted without a concomitant evaluation of the antiplatelet response in the individual patient.1 Platelet activation and aggregation are the most critical factors in the generation of ischemic events, including stent thrombosis and myocardial infarction (MI). Unlike the routine measurement of blood glucose, cholesterol and C-reactive protein performed during the management of patients with atherosclerosis, the measurement of platelet function is largely ignored during the management of cardiovascular patients, even in those at the highest risk. Therein lies the fundamental paradox: blind administration of the most important drugs without confirmation of an adequate response based on an objective laboratory measurement. The limitations of current oral antiplatelet treatment are a persistent concern as evidenced by the development of four new P2Y12 inhibitors. Pharmacodynamic studies of antiplatelet drug responsiveness have exposed the various limitations of current dual antiplatelet strategy. These limitations include delayed onset, overall modest and unpredictable inhibition with wide response variability and resistance, as well as irreversible platelet inhibition.1,2 Moreover, translational research studies in percutaneous coronary intervention (PCI) patients have established the relationship between antiplatelet drug nonresponsiveness and high on-treatment platelet reactivity to the occurrence of clinical ischemic events.3,5 Other research has focused on the development of simple, reproducible and user-friendly point-of-care methods to measure platelet reactivity during aspirin and clopidogrel therapy.5 The latter methods may assist clinicians in tailoring antiplatelet therapies for the individual patient in the future. Antiplatelet drug resistance. Since multiple signaling pathways mediate platelet activation, a treatment strategy that inhibits a single pathway cannot be expected to prevent the occurrence of all thrombotic events.5 Therefore, antiplatelet treatment failure alone is not sufficient evidence for antiplatelet drug resistance. The optimal definition of resistance or nonresponsiveness to an antiplatelet agent is persistent activity of the specific target of the antiplatelet agent that is unchanged from baseline. Because the active metabolite of clopidogrel irreversibly inhibits the P2Y12 receptor, unchanged P2Y12 reactivity compared to baseline has been considered by some investigators as evidence of clopidogrel nonresponsiveness. Similarly, residual activity of the cyclo-oxygenase (COX)-1 enzyme has been a criterion for aspirin nonresponsiveness. However, a standardized laboratory method that measures platelet reactivity and indicates nonresponsiveness has not yet been established. Antiplatelet drug nonresponsiveness will be more meaningful when a level of platelet reactivity associated with ischemic risk has been established. A confirmatory large-scale prospective study using a reproducible and simple method is needed. Optimally, a threshold of platelet reactivity should be established based on a receiver/operator characteristics curve analysis that has both high sensitivity and specificity. Aspirin. The antiplatelet effect of aspirin has been attributed primarily to irreversible acetylation of a serine residue (Ser530) in COX-1 present in platelets that prevents the binding of arachidonic acid to the catalytic site.1 Subsequent generation of thromboxane (Tx)A2 and TxA2-induced platelet aggregation are inhibited for the lifespan of the platelet. Aspirin also attenuates platelet aggregation induced by low concentrations of ADP and collagen in ex-vivo laboratory methods that may be attributed to COX-1-independent effects. In addition, it has been demonstrated that aspirin therapy is also associated with attenuation of thrombin generation at the site of microvascular injury and acetylation of fibrinogen, resulting in increased clot permeability. Finally, other evidence suggests that aspirin directly affects the function of the glycoprotein (GP) IIb/IIIa receptor (Figure 1).6,7 A laboratory method to identify aspirin nonresponsiveness has not yet been uniformly accepted by investigators. Point-of-care methods using agonists such as ADP-, epinephrine- or collagen-induced platelet aggregation do not solely indicate COX-1 activity. Methods that more specifically measure the extent of COX-1 blockade employ arachidonic acid as the agonist to induce platelet aggregation. In addition, the measurement of the stable metabolite of TxA2, serum TxB2, has been used as a measure of COX-1 activity during aspirin therapy, but it is important to note that TxA2 also originates from COX-2 activity in leukocytes. The VerifyNow aspirin assay measures arachidonic acid-induced aggregation of platelets to fibrinogen-coated beads in whole blood where leukocyte COX-2 may play a role. Similarly, the thrombo-elastrography (TEG) platelet mapping assay measures arachidonic acid-induced platelet-fibrin clot strength in anticoagulated whole blood. The Multiplate® analyzer (Dynabyte, Munich, Germany) is an impedance aggregometer that employs arachidonic acid as an agonist in whole blood. Finally, the least specific indicator of COX-1 activity is the Platelet Function Analyzer (PFA)-100™ assay (Dade-Behring, Eschborn, Germany) that employs stimulation with collagen and epinephrine. The definition of aspirin resistance is arbitrary and varies between methods and studies. Based on the various methods used to measure responsiveness to aspirin, it is not surprising that the prevalence reported varies greatly. We have shown that when methods employ arachidonic acid to stimulate platelet aggregation, the prevalence of resistance is low to nil.7,8 However, when shear, adenosine diphosphate (ADP) or collagen are used to stimulate platelet aggregation, or when urinary dehydro-TxB2 is measured, the prevalence of resistance is higher and a dose effect is observed. For example, aspirin nonresponsiveness was found to be uncommon in compliant PCI patients treated with high-dose aspirin (325 mg/day) when measured by turbidometric aggregation, and TEG, where arachidonic acid was used as the agonist.8 In a recent prospective, randomized, double-blind, double-crossover investigation studying aspirin dosing (81 mg, 162 mg, 325 mg) using multiple assays in patients with stable coronary artery disease (n = 120), it was found that aspirin nonresponsiveness was rare (1–6%) using methods that employed arachidonic acid stimulation (light transmittance aggregometry, VerifyNow® aspirin assay, TEG) (Accumetrics; San Diego, California) at all doses of aspirin. When other agonists were used, the prevalence of resistance was 1–27%. Moreover, a dose-dependent response to aspirin treatment was observed when collagen, ADP and shear were used to activate platelets. The latter occurred in the presence of near complete inhibition of the COX-1 enzyme activity as measured by arachidonic acid-induced platelet aggregation, indicating that aspirin may have non-COX-1 mediated dose-dependent effects in platelets.7 More importantly, these dose-dependent effects were more pronounced in patients with diabetes where treatment with higher doses was found to be more effective in inhibiting platelet function.9 Similar results reporting a low prevalence of aspirin resistance using COX-1 specific assays, including the VerifyNow Aspirin assay, have been reported.10–12 Thus, taken together, all of these studies indicate that noncompliance, various assessment methods and underdosing may all be important factors responsible for the reported variability in aspirin nonresponsiveness estimates in clinical studies. Aspirin nonresponsiveness: Relationship to clinical outcomes. There are no definitive data demonstrating a threshold value of laboratory aspirin nonresponsiveness and its association with adverse clinical outcomes based on a receiver operator characteristic (ROC) analysis in a large-scale investigation. However, numerous studies utilizing various methods have correlated adverse clinical events to aspirin nonresponsiveness. Studies employing the PFA-100 have been most widely reported13–26 (Table 1). Since the PFA-100 is not a specific method to measure COX-1 activity, these observations suggest that other signaling pathways play an important role in the genesis of post-PCI ischemic events. Clopidogrel. Most of the pharmacodynamic studies demonstrating clopidogrel response variability were conducted in patients undergoing PCI.27 Similar to aspirin nonresponsiveness, definitions of clopidogrel nonresponsiveness or resistance also vary and include: 1) ≤ 10% absolute difference between pre- and post-treatment maximal ADP-induced aggregation; and 2) 70% was associated with an increased risk of recurrent ischemic events in non-ST-elevation MI-ACS patients undergoing PCI.53 Similarly, in a study by Frere et al, 20 µM ADP-induced platelet aggregation ≥ 70% and VASP-platelet reactivity index > 53% were associated with 30-day recurrent ischemic events.56 A cutpoint of ≥ 240 VerifyNow P2Y12 reaction units has been associated with 12-month ischemic events in patients with ACS.57 Finally, long-term post-PCI risk has been associated with high on-treatment platelet reactivity measured at discharge. In a recent study of patients undergoing stenting, measurement of periprocedural platelet reactivity to ADP was associated with 2-year clinical outcomes. Using ROC curve analysis, cutpoints of > 46% aggregation following 5μµM ADP stimulation and > 59% aggregation following 20μµM ADP stimulation were associated with 58% and 54% of ischemic events, respectively.58 High post-procedural platelet reactivity to ADP was independently associated with a nearly four-fold increased risk of ischemic events within 2 years of non-emergent PCI.58 All of these studies may provide a “testable” level of platelet reactivity for future investigations, similar to the international normalized ratio (INR) used for warfarin therapy. Strategies to address high on-teatment platelet reactivity. Higher clopidogrel loading doses have been associated with enhanced overall platelet inhibition and a lower prevalence of nonresponsiveness. Recent studies have also demonstrated a modestly enhanced inhibitory effect of a 150 mg maintenance dose. Bonello examined VASP-phosphorylation levels in patients prior to stenting and identified high-risk patients using a cutoff PRI > 50%. In a randomized fashion, half of the patients were repeatedly loaded with clopidogrel up to a total of 2,400 mg in order to achieve a PRI below the cutpoint. The latter strategy was successful in bringing 86% of the patients below the cutpoint. None of the patients who were treated with personalized therapy had a 30-day post-discharge thrombotic event, whereas 10% of the patients in the group without dose escalation experienced events.67 Valgimigli et al demonstrated that treatment with the GP IIb/IIIa inhibitor tirofiban in patients identified as nonresponders by VerifyNow aspirin and P2Y12 receptor assays during PCI is associated with a significant decrease in periprocedural MI compared to nonresponders treated with placebo.68 Studies are currently underway employing point-of-care testing to tailor antiplatelet therapy with clopidogrel (Table 3).69 Finally, the OASIS trial will examine the effect of 300 mg and 600 mg loading doses and the effect of a 150 mg maintenance dose compared to the standard maintenance dose in patients with ACS.70 Prasugrel, a third-generation thienopyridine, is a more potent P2Y12 inhibitor associated with less response variability than clopidogrel. In the TRITON-TIMI-38 (TRial to assess Improvement in Therapeutic Outcomes by optimizing platelet InhibitioN with prasugrel Thrombolysis In Myocardial Infarction 38) trial, prasugrel treatment was associated with a reduction in long-term ischemic events in patients with ACS undergoing PCI as compared to clopidogrel. Although bleeding events were more pronounced during prasugrel therapy, selected cohorts of patients derived net clinical benefit from treatment with the latter agent.71 Since a “one-size-fits-all” approach without the guidance of platelet function testing was used in TRITON-TIMI-38, as in all previous large-scale investigations of antiplatelet agents, it is impossible to determine whether insufficient blockade of P2Y12 was the cause of treatment failure (~10%), and conversely, whether excessive blockade was the cause of bleeding. In order to optimally determine the mechanism of treatment failure it is mandatory that platelet function testing be implemented in future trials. In the ongoing TRILOGY study, platelet function testing using the VerifyNow P2Y12 assay will be performed in a large number of patients in order to better understand the relationship between treatment effects of clopidogrel or prasugrel and the occurrence of ischemic and bleeding events. Bleeding and the therapeutic window for P2Y12 inhibitors. In the CURE trial, dual antiplatelet therapy was associated with a 38% increased relative risk of major bleeding compared to aspirin monotherapy.72 In the TRITON-TIMI-38 trial, prasugrel treatment was associated with a 32% and 52% increased relative risk of non-coronary artery bypass graft (CABG)-related thrombolysis in myocardial infarction (TIMI) major and life-threatening bleeding, respectively.71 The discordance between lower ischemic endpoints and higher bleeding rates in the prasugrel group compared to the clopidogrel group suggests that clinical efficacy of superior platelet inhibition translates into reduced safety. Therefore, the ultimate goal would be the potential identification of a therapeutic window for P2Y12 receptor blockade, as demonstrated in Figure 2. The current available data suggest that post-stenting ischemic events are associated with a P2Y12 reactivity cutpoint. However, there is a very large gap in our understanding of the relationship between P2Y12 reactivity and bleeding. Clopidogrel, in combination with aspirin, is known to augment bleeding, transfusion requirements and re-operation rates after CABG, and the greatest risk occurs when the drug is given within 48 hours prior to surgery.73,74 Although there is indisputable evidence that clopidogrel therapy is associated with superior outcomes in patients with unstable coronary syndromes,73 the favorable effects on preventing thrombus formation have to be balanced against the hazards of increased bleeding associated with surgery, since bleeding, allogenic blood transfusion and re-thoracotomy have been associated with increased morbidity, mortality and costs.75,76 The impact of clopidogrel combined with aspirin on bleeding in patients undergoing CABG was assessed within the CURE trial, which demonstrated that patients receiving dual antiplatelet therapy within 5 days of CABG displayed increased rates of bleeding compared to patients who discontinued therapy 5 days prior to CABG.73 The latter evidence explains the current ACC/AHA (American College of Cardiology/American Heart Association) clinical practice guideline recommendation to withhold clopidogrel for at least 5 days before CABG, provided that the clinical situation permits withdrawal.76 However, despite these recommendations, data from the CRUSADE trial demonstrated that the overwhelming majority of patients with ACS requiring CABG have their surgery within

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