Laboratory Monitoring of Antiplatelet Therapy

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Laboratory Monitoring of Antiplatelet Therapy Thomas Gremmel*,†, Deepak L. Bhatt‡ and Alan D. Michelson§

* Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria, †Department of Internal Medicine, Cardiology and Nephrology, Landesklinikum Wiener Neustadt, Wiener Neustadt, Austria, ‡Department of Cardiovascular Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, United States, §Center for Platelet Research Studies, Dana-Farber/Boston Children´s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, United States

INTRODUCTION 653 METHODS 655 Measurement of Thromboxane B2 (TXB2) 655 Light Transmission Aggregometry (LTA) 656 VerifyNow Assay 657 Impedance Aggregometry 657 Thromboelastography (TEG) 657 Platelet Function Analyzer (PFA)-100 657 INNOVANCE Platelet Function Analyzer (PFA)-200 Cone and Plate(let) Analyzer (Impact-R) 657 Flow Cytometry 657 Genetic Testing 658 CORRELATIONS BETWEEN DIFFERENT TEST SYSTEMS 658 Aspirin 658 Clopidogrel 659 ASSOCIATION OF TEST RESULTS WITH CLINICAL OUTCOMES 659 Aspirin 659 P2Y12 Receptor Antagonists 661 GPIIb-IIIa Antagonists 663 Vorapaxar 663 POTENTIAL MECHANISMS OF AN INADEQUATE RESPONSE TO ANTIPLATELET THERAPY 663 Aspirin 663 P2Y12 Receptor Antagonists 663 GPIIb-IIIa Antagonist 664 Vorapaxar 664 PREDICTORS OF AN INADEQUATE RESPONSE TO ANTIPLATELET THERAPY 665 Demographics 665 Comorbidities 665 Medications 666 Genetics 667 THERAPEUTIC ADJUSTMENTS BASED ON TEST RESULTS 668 Dose of Antiplatelet Agent 668 Type of Antiplatelet Agent 669 Dose and Type of Antiplatelet Agent 670 CURRENT GUIDELINES 671 CONCLUSIONS 671 REFERENCES 671

Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00036-9 Copyright © 2019 Elsevier Inc. All rights reserved.

INTRODUCTION

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Platelets are the smallest blood cells, numbering 150–350
109/L in healthy individuals.1 In their resting state, platelets circulate in a discoid form maintained via inhibition of platelet activation by (A) endothelial-derived nitric oxide, (B) endothelial-derived prostaglandin I2 (prostacyclin), and (C) scavenging of adenosine diphosphate (ADP) by endothelial surface CD39 (Chapter 17).1,2 The ability of platelets to adhere to a damaged vessel wall and form aggregates was first described in the 19th century by Bizzozero (see Foreword to this book).3–5 Platelet adhesion is initiated by binding of exposed collagen to platelet surface glycoprotein (GP) VI and integrin α2β1,6,7 and binding of von Willebrand factor (VWF) to the GPIb-IX-V complex.1,8,9 Following the activation of the coagulation cascade, thrombin, one of the strongest platelet agonists, is generated and activates further platelets via protease-activated receptor (PAR)-1 and PAR-4 (Chapter 13).10,11 Important positive feedback loops for platelet activation are provided by (A) the platelet ADP receptors P2Y1 and P2Y12 (Chapter 14),12–14 (B) the serotonin 5-HT2A receptors and (C) the thromboxane prostanoid receptor.1,2,15 While ADP and serotonin are released from platelet dense granules,16 thromboxane A2 (TXA2) is generated by the platelet cyclooxygenase (COX)-1-dependent signaling pathway.15 Platelet aggregation is mediated by binding of fibrinogen and, at high shear stress, VWF to the activated molecular conformation of integrin αIIbβ3 (GPIIb-IIIa) (Chapter 12).1,2,17 Monocyte-platelet aggregates are formed via the interaction of P-selectin on the surface of activated platelets with its counterreceptor P-selectin glycoprotein ligand-1 on leukocytes (Chapter 16).18 Besides the long-established physiological function of platelets in hemostasis, undesirable intravascular platelet activation at the site of atherosclerotic plaque rupture plays a key role in the processes ultimately resulting in vessel occlusion and end organ damage.19,20 The resultant ischemic events such as myocardial infarction (MI) or stroke are the major causes of death in the industrialized world. Consequently, antiplatelet therapy became a cornerstone in the secondary prevention of adverse cardiovascular outcomes.15,21 The molecular targets of currently approved antiplatelet agents are shown in Fig. 36.1. Aspirin was the first widely prescribed antiplatelet drug, and remains the most frequently used platelet inhibitor in acute and long-term secondary prophylaxis of ischemic events (Chapter 50).22 By acetylation of serine residue 529 on COX-1 and -2, aspirin irreversibly blocks the generation of prostaglandin G2 and H2, and the biosynthesis of TXA2 (Fig. 36.1 and Table 36.1).15,23 Its beneficial effects in the secondary prevention of cardiovascular events are welldocumented by many clinical studies and metaanalyses

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PART III Clinical Tests of Platelet Function

Fig. 36.1 Molecular targets of antiplatelet agents. FDA-approved antiplatelet agents are shown in blue boxes. Novel antiplatelet agents in development are shown by red bars. Abbreviations: ADP, adenosine diphosphate; COX1, cyclooxygenase 1; GP, glycoprotein; 5-HT; 5hydroxytryptamine (serotonin); LMWH, low molecular weight heparin; NO, nitric oxide; PAR, protease-activated receptor; PI3Kβ, β isoform of phosphoinositide 3-kinase; PG, prostaglandin; PSGL-1, P-selectin glycoprotein ligand 1; TX, thromboxane; UFH, unfractionated heparin; VWF, von Willebrand factor. (Modified with permission from Michelson AD. Nat Rev Drug Discov 2010.15)

indicating a 20% reduction in ischemic outcomes by low-dose aspirin in high-risk patients.24,25 The group of Food and Drug Administration (FDA)-approved ADP P2Y12 receptor antagonists comprises five agents: ticlopidine, clopidogrel, prasugrel, ticagrelor, and cangrelor (Fig. 36.1) (Chapter 51). The first three of these are thienopyridines which need to be metabolized by the cytochrome (CYP) P-450 family of enzymes in the liver in order to become pharmacologically active and exert their antiplatelet effect (Table 36.1).26,27 While ticlopidine is not recommended by current guidelines due to its numerous side effects, clopidogrel together with aspirin is the treatment of choice following elective percutaneous coronary intervention

(PCI) or peripheral angioplasty with stent implantation,28 and prasugrel plus aspirin is a therapeutic option in acute coronary syndrome (ACS) patients undergoing PCI and stenting.29–31 Ticagrelor is a triazolopyrimidine acting as a direct and reversible inhibitor at the ADP P2Y12 receptor,27,32 and is approved for use in combination with aspirin in ACS patients without and with PCI (Table 36.1).30,31,33 Cangrelor, a modified adenosine triphosphate derivative, is a direct and reversible ADP P2Y12 receptor antagonist with a short half-life of 3– 5 min, and can only be given intravenously (Table 36.1).21,34 Its administration together with aspirin is approved for P2Y12 inhibitor-naïve patients undergoing PCI.30,31,35 The

Laboratory Monitoring of Antiplatelet Therapy

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TABLE 36.1 Available Antiplatelet Therapies Mechanism

Agent

Structure

Route/Dosing

Clinical Use

COX-1 inhibition

Aspirin

Acetylsalicylic acid

Oral/Daily

Irreversible P2Y12 antagonism

Ticlopidine

Thienopyridine

Oral/Twice daily

Clopidogrel

Thienopyridine

Oral/Daily

Prasugrel

Thienopyridine

Oral/Daily

Ticagrelor

Cyclopentyl-triazolopyrimidine ATP analog Murine human chimeric Fab fragment KGD-containing cyclic heptapeptide Nonpeptide mimetic based on RGD Tricyclic himbacine derivative 2-Oxoquinoline derivative Pyrimidopyridine derivative Acetylsalicylic acid/ pyrimidopyridine derivative

Oral/Twice daily IV IV

Coronary artery disease, cerebrovascular disease, peripheral artery disease, stents, CABG, CEA Cerebrovascular disease, coronary stents (now rarely used) Prior myocardial infarction, ischemic stroke, or symptomatic peripheral artery disease, as monotherapy; ACS or coronary stenting, as part of dual antiplatelet therapy with aspirin Patients with ACS treated with stents, as part of dual antiplatelet therapy with aspirin Patients with ACS, as part of dual antiplatelet therapy with aspirin PCI when not pretreated with oral P2Y12 antagonist PCI

IV

ACS, PCI

IV

ACS, PCI

Oral/Daily

Prior myocardial infarction, peripheral artery disease

Oral/Twice Daily Oral/Twice Daily

Peripheral artery disease Stroke or TIA when used with aspirin

Oral/Twice Daily

Stroke, TIA

Reversible P2Y12 antagonism GPIIb-IIIa inhibition

Cangrelor Abciximab Eptifibatide Tirofiban

PAR-1 inhibition

Vorapaxar

PDE inhibition

Cilostazol Dipyridamole

Combination

Aspirin/Dipyridamole (Aggrenox)

Abbreviations: ACS, acute coronary syndrome; CABG, coronary artery bypass graft; CEA, carotid endarterectomy; COX, cyclooxygenase; GPIIb-IIIa, glycoprotein IIb-IIIa; IV, intravenous; KGD, Lys-Gly-Asp; PAR-1, protease-activated receptor-1; PCI, percutaneous coronary intervention; PDE, phosphodiesterase; RGD, Arg-Gly-Asp; TIA, transient ischemic attack.

GPIIb-IIIa (αIIbβ3) receptor antagonists abciximab, tirofiban, and eptifibatide are intravenous antiplatelet agents (Chapter 52).15,21 They immediately block the fibrinogen receptor, i.e., integrin αIIbβ3 (GPIIb-IIIa), on the surface of activated platelets thereby inhibiting platelet-to-platelet aggregation (Fig. 36.1, Table 36.1), and have a rather short biological half-life ranging from 10 min for abciximab to 2 h for tirofiban and 2.5 h for eptifibatide.36 GPIIb-IIIa receptor antagonists are predominantly administered in special circumstances in the periinterventional setting, in particular in ACS patients with a high thrombotic burden within the coronary arteries and no-reflow syndrome following PCI and stenting.37 Dipyridamole and cilostazol are phosphodiesterase inhibitors with antiplatelet (Fig. 36.1) and vasodilatory effects that are prescribed in some countries in the secondary prevention of ischemic stroke and peripheral artery disease (PAD; Table 36.1), respectively (Chapter 54).38 Moreover, several studies evaluated cilostazol in addition to aspirin and clopidogrel therapy in patients undergoing PCI and suggested favorable outcomes compared with conventional dual antiplatelet therapy (DAPT).39–41 Finally, with the PAR-1 antagonist vorapaxar, a third pathway of platelet activation can be selectively inhibited (Fig. 36.1 and Table 36.1) (Chapter 53).15,21 Based on the results of two large Phase III clinical trials in patients with stable cardiovascular disease and ACS,42,43 respectively, vorapaxar was approved for use in addition to standard antiplatelet therapy in patients with a history of MI or symptomatic PAD to prevent future ischemic events. Unfractionated and low-molecular weight heparin, as well as lepirudin, argatroban, bivalirudin, and dabigatran, are anticoagulants which inhibit thrombin, thereby allowing less platelet activation (Fig. 36.1), although unfractionated heparin was also shown to activate platelets in some studies.15 Despite the introduction of the above-described novel agents and major advances in antiplatelet treatment,

atherothrombotic events still impair the prognosis of many patients with cardiovascular disease (Chapter 26). The occurrence of ischemic outcomes in patients receiving state-of-theart antiplatelet therapy led to the concept of an interindividual response variability to antiplatelet drugs.44 In order to measure the extent of platelet inhibition by different antiplatelet agents, a number of test systems were developed and subsequently evaluated in numerous clinical studies over the last two decades—as will be discussed in this chapter.

METHODS The various methods for the assessment of the response to antiplatelet therapy are based on different underlying principles, and can be divided into the measurement thromboxane B2 (TXB2, a soluble marker of platelet activation), test systems capturing surrogate parameters of platelet aggregation, flow cytometric assays and genetic testing (Figs. 36.2 and 36.3).45,46

Measurement of Thromboxane B2 (TXB2) TXB2 is a stable degradation product of TXA2 which is excreted by the kidneys as 11-dehydro TXB2 (d-TXB2).47 Its serum and urinary concentrations can be measured by enzyme-linked immunosorbent assays, and are indicative of the extent of TXA2 generation. Because aspirin exerts its antiplatelet effect by blocking the biosynthesis of TXA2 (Fig. 36.1),23 the measurement of serum TXB2 is considered the most direct and optimal way to assess aspirinmediated platelet inhibition.46,48 High levels of serum TXB2 and—to a lesser extent—urinary d-TXB2 during aspirin therapy may reflect the inability of aspirin to adequately block TXA2 generation, a phenomenon often referred to as “aspirin resistance.”49 Indeed, several studies found a wide variability of serum TXB2 and urinary d-TXB2 levels in aspirin-treated patients.50–52

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PART III Clinical Tests of Platelet Function

Figure 36.2 Laboratory tests to monitor inhibition of arachidonic acid-stimulated platelets by aspirin.

Figure 36.3 Laboratory tests to monitor inhibition of ADP-stimulated platelets by clopidogrel.

However, if “resistance” is defined in pharmacological terms as the failure of aspirin to completely inactivate its molecular target platelet COX-1, then aspirin resistance is either a very rare phenomenon or does not exist at all.2,53–56 Based on this pharmacological definition, a study of 400 healthy volunteers could not identify a single case of aspirin resistance.53

Light Transmission Aggregometry (LTA) LTA was independently developed in 1962 by Born and O´ Brien and is considered the historical gold standard of platelet function testing (Chapter 34).45,46,57 It requires two centrifugations steps in order to obtain platelet-rich and platelet-poor plasma.58 The latter is used to set a baseline optical density. Thereafter, LTA captures the increase in light transmittance through platelet-rich plasma when an agonist is added to the sample and platelets start to aggregate, and displays it as surrogate marker of platelet aggregation in aggregation % (Figs. 36.2 and 36.3). The major advantage of LTA is the extensive experimental and clinical data gathered with this method over decades. Moreover, since LTA can be performed with

different agonists, it enables the selective assessment of the inhibition of multiple pathways of platelet activation in a sample. However, although LTA is the most frequently used method to assess platelet function and recommendations of the International Society on Thrombosis and Haemostasis (ISTH) on how to perform LTA were published in 2013,58 its validity and clinical applicability in the monitoring of antiplatelet therapy are still limited by the lack of standardization. In particular, differences in preanalytical variables such as blood sampling, variations in the required centrifugation steps and different concentrations of the agonists used to initiate platelet aggregation impair the comparability of its results between different laboratories. Other limitations of LTA are that the procedure is rather time-consuming, expensive and highly operator-dependent. Finally, due to the use of plasma instead of whole blood, the interaction of platelets with other blood cells is lost in LTA.45,46 For these reasons, the ISTH consensus document on LTA concludes that analytical and preanalytical factors must be carefully controlled for by expert personnel and LTA should only be performed in specialized institutions.58

Laboratory Monitoring of Antiplatelet Therapy

VerifyNow Assay The VerifyNow system (Accriva Diagnostics, San Diego, CA, USA) is a turbidimetric optical detection system, which measures platelet aggregation as an increase in light transmittance in whole blood (Chapter 33).45,46,59 The assay device contains reagents based on microbead agglutination technology, namely a lyophilized preparation of human fibrinogen-coated beads, platelet agonists, preservative and buffer. Citrateanticoagulated whole blood is dispensed automatically from the blood collection tube into the assay device by the instrument. Thereafter, arachidonic acid (AA) or ADP is incorporated into the assay channel to induce platelet activation and light transmittance increases as activated platelets bind and aggregate the fibrinogen-coated beads (Figs. 36.2 and 36.3). The instrument measures this change in optical signal and reports results in Aspirin (ARU) or P2Y12 Reaction Units (PRU). Higher ARU and PRU reflect greater AA- and ADPmediated platelet reactivity, respectively. The VerifyNow system offers a fast and highly standardized way to evaluate platelet response to aspirin and P2Y12 inhibitors. Furthermore, due to the automated procedure it is less operatordependent than other platelet function tests and fulfils the criteria of a point-of-care assay.

Impedance Aggregometry Impedance aggregometry is a whole blood platelet function test measuring the increase in electrical impedance between two electrodes when platelets start to adhere to the electrodes and aggregate following their activation by various agonists used device for imped(Chapter 34).45,46 The most commonly ® ance aggregometry is the Multiplate analyzer (Roche Diagnostics, Rotkreuz, Switzerland), which performs multiple electrode platelet aggregometry (MEA).60 The impedance rise is detected in two separate sensor units with two electrodes each, and translated into aggregation units (AU) or area under the curve of AU (AU
min), which corresponds to AU
10 (Figs. 36.2 and 36.3). As with LTA, MEA allows the assessment of the response to different antiplatelet agents at the same time. However, no prior sample processing is required for MEA and the test follows a standardized protocol offering a better comparability of the obtained results between different laboratories than LTA. Nevertheless, MEA is not a true point-of-care test since pipetting is required, and preanalytical variables, in particular the anticoagulant used for blood sampling, may affect its results.

Thromboelastography (TEG) TEG was invented >50 years ago and updated to a more plateletspecific method in the form of the TEG Platelet Mapping system (Haemonetics, Braintree, Massachusetts, USA) (Chapter 33).45,46 In addition to platelet function, the TEG Platelet Mapping system measures the platelet contribution to clot strength (Figs. 36.2 and 36.3). The test works with whole blood but requires pipetting and cannot therefore be considered a true point-of-care assay. Moreover, clinical data on the monitoring of antiplatelet therapy with this method are limited to very few studies.61

Platelet Function Analyzer (PFA)-100 The PFA-100 (Siemens, Munich, Germany) is a point-of-care test and was one of the first methods used for the monitoring of aspirin therapy (Chapter 33).45,46 In this device, citrateanticoagulated whole blood is aspirated through a stainless steel capillary under vacuum. Platelets are then passed through a membrane with a central aperture under high shear

657

conditions. The membrane is coated with collagen + epinephrine or collagen + ADP resulting in platelet adhesion and aggregation. The system measures the time platelets require to occlude the aperture, reported as closure time. Of note, collagen/epinephrine and collagen/ADP closure time is censored at a maximum of 300 s. The PFA-100 has been widely used to study aspirin response, but is not sensitive to antiplatelet therapy with P2Y12 receptor antagonists.46,62

INNOVANCE Platelet Function Analyzer (PFA)-200 The INNOVANCE PFA-200 system (Siemens, Munich, Germany) is a further development of the PFA-100 with a higher sensitivity to the effects of P2Y12 inhibitors than its predecessor.63 Its major limitation is that to date only a few studies on this relatively new method have been published.64

Cone and Plate(let) Analyzer (Impact-R) The Impact-R (Matis Medical, Beersel, Belgium) is a whole blood test system which applies high shear stress through an acrylnitril-butadien-styrene cone to initiate platelet activation and adhesion to a polystyrene well (Chapter 33).45,46,65 The well is then washed with tap water and stained with MayGr€ unwald solution. Thereafter, the samples are analyzed with an inverted light microscope connected to an image analyzer (Galai, Migdal Haemek, Israel), and platelet adhesion is determined by examination of the percentage of total area covered with platelets (surface coverage %).66 Another read-out of this method is the aggregate size as determined by the image analyzer. If the system is used to assess the response to antiplatelet therapy, blood samples are preincubated with the respective agonist before high shear stress is applied. This leads to platelet activation and microaggregate formation of uninhibited platelets in the tube resulting in reduced platelet adhesion to the well as reflected by reduced surface coverage. The lower the surface coverage, the lower is the inhibitory response to the respective antiplatelet agent. Accordingly, the Impact-R is often referred to as “inverse aggregometry.” Since the method is laborious, time-consuming, and highly operatordependent, it has not become a routine method for monitoring antiplatelet therapy and only few data are available on this assay.64,67,68

Flow Cytometry Vasodilator-Stimulated Phosphoprotein (VASP) Phosphorylation assay The VASP assay (Diagnostica Stago, Marseille, France) is a standardized flow cytometric test,45,46,69 and considered to be among the most specific methods to monitor P2Y12 inhibition because it does not rely on co-activation of the P2Y1 receptor by ADP (Chapter 35). Samples of citrate-anticoagulated whole blood are incubated in vitro with prostaglandin E1 (PGE1), with or without ADP, before fixation. After 10 min, platelets are permeabilized, labeled with a primary monoclonal antibody against serine 239-phosphorylated VASP (clone 16C2) or its isotype, followed by a secondary fluorescein isothiocyanateconjugated polyclonal goat anti-mouse antibody. All procedures are performed at room temperature and the samples are analyzed in a flow cytometer. The platelet population is identified by its forward and side scatter distribution, and 10,000 platelet events are gated (Fig. 36.3). The extent of VASP phosphorylation is measured by geometric mean fluorescence intensity (MFI) values in the presence of PGE1 without (T1) or with ADP (T2). After subtraction of the negative isotypic control

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PART III Clinical Tests of Platelet Function

values from the corresponding fluorescence values, the platelet reactivity index (PRI) is calculated according to the following formula: PRI% ¼ ½T1 ðPGE1 Þ  T2 ðPGE1 + ADPÞ=T1 ðPGE1 Þ
100 The PRI represents the mean percentage platelet reactivity and is inversely correlated with platelet inhibition by P2Y12 receptor blockers. An advantage of this method is that samples can be mailed at room temperature to a core laboratory. A disadvantage of this method is that it requires expensive flow cytometry equipment and highly trained technical staff. An ELISA method is also available.

Platelet Surface P-selectin Expression Upon platelet activation, P-selectin is translocated from the αgranule membrane to the platelet surface where it serves as counterreceptor of P-selectin glycoprotein ligand-1 on leukocytes and mediates the formation of leukocyte-platelet aggregates (Fig. 36.1).18,70 Since P-selectin is exclusively expressed on the surface of activated platelets, it is a marker of platelet activation. By measuring agonist-induced P-selectin expression by flow cytometry (Chapter 35), platelet surface P-selectin can be used to assess the response to antiplatelet therapy.

Activation of Integrin αIIbβ3 (GPIIb-IIIa) Platelet activation leads to the transformation of integrin αIIbβ3 on the platelet surface to its activated molecular conformation,1 which enables fibrinogen- and VWF-mediated platelet-toplatelet aggregation (Chapter 12).71 As for the assessment of platelet surface P-selectin expression, the measurement of agonist-induced integrin αIIbβ3 activation by flow cytometry allows for the selective investigation of the inhibition of different pathways of platelet activation in the same sample.70

Monocyte-Platelet Aggregate Formation Circulating monocyte-platelet aggregates can be assessed by flow cytometry (Chapter 35),70 and were shown to be a more sensitive marker of platelet activation than platelet surface Pselectin expression in several pathophysiological circumstances including MI.18,72,73 By measuring monocyte-platelet aggregate levels after stimulation of a sample with platelet agonists, the inhibitory response to antiplatelet agents can be determined.

Genetic Testing

of 377 miRs in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum.80 They reported markedly higher levels of miRs in platelet-rich plasma compared to serum and platelet-poor plasma, and showed that antiplatelet therapy decreases miR levels. In a second step, they assessed the levels of 92 miRs in nine healthy individuals using custom-made quantitative real-time polymerase chain reaction (PCR) plates at four different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. They showed that plasma levels of the platelet miRs miR-126, miR-150, miR-191, and miR223 decreased on further platelet inhibition, and validated these findings in an independent cohort of 33 patients with symptomatic atherosclerosis. Another study measured circulating levels of selected miRs before and after the therapeutic switch from DAPT with aspirin and clopidogrel to aspirin and ticagrelor, and found that levels of miR-126, miR-150, and miR-223 significantly decreased, while levels of miR-96 increased in these patients.81 Kaudewitz et al. assessed miRs in 125 patients with a history of ACS who had undergone detailed assessment of platelet function 30 days after the acute event.82 They reported that miR-223 and other abundant platelet miRs showed significant positive correlations with the VASP assay. Moreover, miR-126 and miR-223 correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in 669 individuals of the population-based Bruneck study, and inhibition of miR-126 in mice reduced platelet aggregation.82 Finally, miR-126 was shown to affect P2Y12 receptor expression in mice. Consequently, the quantification of miRs using real-time PCR may become useful in the monitoring of antiplatelet therapy. However, further studies are needed to identify the most sensitive miRs for the various antiplatelet agents.

CORRELATIONS BETWEEN DIFFERENT TEST SYSTEMS Since most of the methods used in the laboratory monitoring of antiplatelet agents evaluate on-treatment platelet reactivity or surrogate markers thereof, a number of studies investigated the correlations between different test systems to determine if these are interchangeable in the assessment of the response to antiplatelet therapy. The resulting publications predominantly focused on the comparison of methods measuring platelet response to aspirin and clopidogrel treatment, respectively.

Single Nucleotide Polymorphisms

Aspirin

Genotyping of the hepatic CYP P-450 enzyme system allows the identification of loss-of-function and gain-of-function polymorphisms of CYP isoenzymes that were associated with an impaired and enhanced response to clopidogrel therapy, respectively.74–78 Furthermore, single nucleotide polymorphisms of the ABCB1 gene,76,79 which encodes for the intestinal efflux transporter pump P-glycoprotein, may influence clopidogrel-mediated platelet inhibition and can be determined by genotyping. The detailed effects of these genetic variants on on-treatment residual platelet reactivity and clinical outcomes are described in section “Predictors of an Inadequate Response to Antiplatelet Therapy.”

Lordkipanidze et al. compared the results obtained by LTA after stimulation with AA (LTA AA), LTA after stimulation with ADP (LTA ADP), the VerifyNow aspirin assay, impedance aggregometry after stimulation with AA, the PFA-100, and urinary dTXB2 in the assessment of aspirin response in 201 patients with stable coronary artery disease (CAD).83 A maximal aggregation 20% by LTA AA was defined as aspirin resistance based on previous studies showing an increased risk of adverse cardiac events above this threshold.84–86 Cut-off values for the identification of aspirin-resistant patients by the other methods were defined according to results obtained in healthy individuals, the manufacturer instructions and previous studies linking residual platelet aggregation to adverse outcomes, respectively. With use of these assay-specific thresholds, the prevalence of aspirin resistance ranged from 2.8% to 59.5% in their study population.83 The results obtained by the various test systems showed poor agreement and poor correlations between themselves with the best though still weak correlations between

MicroRNAs (miRs) Recent studies found a substantial platelet contribution to the circulating miR pool and identified miRs that are responsive to antiplatelet therapy.80,81 In detail, Willeit et al. created a profile

Laboratory Monitoring of Antiplatelet Therapy

LTA and impedance aggregometry. The authors therefore concluded that the different platelet function tests may not be equally suitable for measuring aspirin’s antiplatelet effect. Gremmel et al. correlated LTA AA, the VerifyNow aspirin assay, MEA after stimulation with AA (MEA AA), the PFA-100 and the Impact-R after stimulation with AA (Impact-R AA) with urinary d-TXB2 in 225 patients with atherosclerotic cardiovascular disease.68 All patients were on DAPT with aspirin and clopidogrel and had undergone angioplasty with stent implantation 1 day before blood sampling. The highest quartile of the results obtained by LTA AA, the VerifyNow aspirin assay, MEA AA, the PFA-100 and d-TXB2, and the lowest quartile of surface coverage % by the Impact-R AA were defined as high on-treatment residual platelet reactivity to AA (HRPR AA). In line with the previous study, all platelet function tests correlated poorly with d-TXB2, and only MEA AA showed a weak but significant correlation with d-TXB2 (r ¼ 0.14).68 The results from the five platelet function tests also correlated poorly with each other, and only results by LTA AA and the Impact-R AA correlated significantly (r ¼ 0.19). Further, all platelet function assays showed a poor agreement with d-TXB2 regarding the determination of HRPR AA. Sensitivities and specificities of the five platelet function tests for HRPR AA as defined by d-TXB2 ranged from 17.5% to 44.6%, and from 70.8% to 77.9%, respectively. This is in line with a study by Santilli et al. who found that serum TXB2 as marker for cyclooxygenase activity was persistently suppressed by low-dose aspirin, while the antiplatelet effect of aspirin was variably detected by LTA AA and the VerifyNow aspirin assay.55 Likewise, Frelinger et al. revealed no significant correlations of serum TXB2 concentrations with LTA AA, LTA ADP, the VerifyNow aspirin assay and TEG with AA as an agonist in 165 normal subjects following the intake of 81 mg aspirin for seven consecutive days.52 Moreover, even serum TXB2 and urinary dTXB2 did not correlate significantly after the initiation of aspirin in their study population. One study correlated residual platelet aggregation by LTA AA, the VerifyNow aspirin assay and MEA AA with platelet surface expression of P-selectin and activated GPIIb-IIIa in response to AA in 316 patients on DAPT with aspirin and clopidogrel.87 The authors reported statistically significant, but poor correlations of all aggregation tests with AA-induced P-selectin expression, while only LTA AA correlated significantly with the expression of activated GPIIb-IIIa in response to AA. In summary, the measurement of serum TXB2 is the most direct way to assess COX-1 activity and may be the preferred method to identify poor responders to aspirin therapy,48 while platelet function tests may help to identify patients with high platelet reactivity, be it due to an inadequate response to antiplatelet treatment or due to other stimuli, e.g., platelet activation by different agonists or high shear stress. Platelet function assays do not mirror serum TXB2 and urinary d-TXB2 results, suggesting that thromboxane inhibition can be circumvented in assays that determine platelet function. Furthermore, platelet function tests used for the assessment of aspirin response correlate poorly with each other, and the results obtained with one of these methods cannot be extrapolated to those obtained with the others.

Clopidogrel Paniccia et al. compared the results obtained by the VerifyNow P2Y12 assay and the PFA-100 collagen/ADP cartridge with LTA ADP in 1267 CAD patients on DAPT with aspirin and clopidogrel following PCI.88 Moreover, the VASP assay was performed in a subgroup of 115 patients of their study population. They found significant, but only moderate correlations of the

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VerifyNow P2Y12 assay and the VASP assay with LTA ADP, whereas the PFA-100 collagen/ADP closure time did not correlate with LTA ADP. Lordkipanidze et al. measured on-treatment platelet reactivity to ADP by LTA, the VerifyNow P2Y12 assay, impedance aggregometry and the PFA-100 collagen/ADP cartridge in 116 patients with stable CAD before and after the initiation of clopidogrel therapy.89 Although all methods were sensitive to clopidogrel intake, none could distinguish categorically between patients who had or had not ingested clopidogrel. Furthermore, the agreement of the different platelet function tests regarding the identification of patients with an insufficient response to clopidogrel was low, and the assays correlated only poorly to moderately with each other with the best correlation between LTA ADP and the VerifyNow P2Y12 assay.89 Gremmel et al. correlated on-treatment platelet reactivity by the VerifyNow P2Y12 assay, the VASP assay, MEA after stimulation with ADP (MEA ADP) and the Impact-R after stimulation with ADP (Impact-R ADP) with LTA ADP in 80 patients receiving DAPT with aspirin and clopidogrel 1 day after percutaneous angioplasty with stent implantation.67 The highest quartile of the results obtained by LTA ADP, the VerifyNow P2Y12 assay, the VASP assay, and MEA ADP, and the lowest quartile of surface coverage % by the Impact-R ADP were defined as HRPR ADP. Sensitivities and specificities of the different methods were based on the results from LTA ADP. The results from all four assays correlated significantly, but at best moderately with those from LTA ADP, and the VerifyNow P2Y12 assay revealed the strongest correlation with LTA ADP (r ¼ 0.61). The respective correlation coefficient was close to that reported by the two above-mentioned studies88,89 and another publication,90 whereas a study including data obtained before and after the start of clopidogrel treatment revealed an even higher correlation coefficient between the VerifyNow P2Y12 assay and LTA ADP.91 Sensitivities and specificities of the four platelet function tests for HRPR by LTA ADP ranged from 35% to 55%, and from 78.3% to 85%, respectively.67 In another study, the same group correlated residual platelet aggregation by LTA ADP, the VerifyNow P2Y12 assay and MEA ADP with platelet surface expression of P-selectin and activated GPIIb-IIIa in response to ADP in 316 patients on DAPT with aspirin and clopidogrel.87 They found significant correlations of all aggregation tests with ADP-induced expression of P-selectin and activated GPIIbIIIa. The best, but still only moderate correlation was observed between on-treatment platelet reactivity by the VerifyNow P2Y12 assay and the expression of activated GPIIb-IIIa in response to ADP (r ¼ 0.68). In summary, the various test systems used for monitoring clopidogrel response correlate at best moderately with each other, and the different methods are therefore not interchangeable. It can be assumed that this also applies to the measurement of the response to other P2Y12 inhibitors by the described platelet function tests. However, studies comparing multiple test systems in the assessment of platelet response to prasugrel or ticagrelor in the same patient population have not been reported.

ASSOCIATION OF TEST RESULTS WITH CLINICAL OUTCOMES Aspirin A number of studies have reported associations of the extent of aspirin-mediated platelet inhibition with the occurrence of major adverse cardiovascular events (MACE) in different manifestations of atherosclerosis.50,51,84,92–96 Gum et al. performed LTA in 326 patients with stable CAD who were

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receiving 325 mg aspirin daily for 7 days and no other antiplatelet agent.84 HRPR was defined as the combination of mean aggregation 20% by LTA AA and mean aggregation 70% by LTA ADP, and seen in 5.2% of the patients. During the follow up of 679  185 days, HRPR was independently associated with a higher risk of death, MI or cerebrovascular accident compared to an adequate aspirin response. Chen et al. investigated the impact of aspirin response on the incidence of myonecrosis after nonurgent PCI.92 Applying a threshold of 550 ARU by the VerifyNow aspirin assay they identified 29 patients (19.2%) with HRPR AA in a cohort of 151 patients, and reported that poor response to aspirin was an independent predictor of CK-MB and troponin I elevation following PCI. In another study, the same group of authors found HRPR by the VerifyNow aspirin assay in 128 aspirintreated patients (27.4%) with stable CAD, and observed that these patients suffered the primary endpoint of ACS, stroke, cardiovascular death, and transient ischemic attack more frequently within 1 year than patients who were classified as aspirin-sensitive (n ¼ 340, 72.6%; 15.6% vs. 5.3%).93 In line with the results of the above-mentioned studies, Breet et al. showed that LTA AA and the VerifyNow aspirin assay were able to identify patients at risk of atherothrombotic events in a population of 951 aspirin-treated patients with elective PCI.94 Eikelboom et al. obtained urinary samples from 5529 Canadian patients enrolled in the HOPE (Heart Outcomes Prevention Evaluation) study.50 Using a nested case-control design, they measured urinary d-TXB2 levels in 488 aspirintreated patients with MI, stroke or cardiovascular death during 5 years of follow-up and in 488 sex- and age-matched control subjects also receiving aspirin who did not have an event. After adjustment for baseline differences, the odds for the composite endpoint increased with each increasing quartile of urinary d-TXB2, with patients in the upper quartile having a 1.8-times higher risk than those in the lower quartile. Furthermore, patients in the upper quartile had a 2-fold higher risk of MI and a 3.5-fold higher risk of cardiovascular death than those in the lower quartile.50 In a subsequent study, Eikelboom et al. were able to confirm these findings by measuring urinary d-TXB2 in 3261 aspirin-treated patients from the CHARISMA (Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization,

Management, and Avoidance) trial and showing that d-TXB2 concentrations were a determinant of MI, stroke, or cardiovascular death in these patients.51 Specifically, d-TXB2 levels in the highest quartile were associated with a significantly increased risk of the primary endpoint compared to the lowest quartile. Of note, randomization to clopidogrel versus placebo (which was performed in the CHARISMA trial) did not reduce the hazard of cardiovascular events in patients in the highest quartile of urinary d-TXB2.51 Frelinger et al. assessed serum TXB2 and PFA-100 collagen/epinephrine and collagen/ADP closure times as well as AAstimulated platelet surface expression of P-selectin and activated GPIIb-IIIa, and leukocyte-platelet aggregates in 700 consecutive patients before coronary angiography.95 All patients had received 81 or 325 mg of aspirin daily for 3 days prior to study enrollment. After adjustment for covariables including sex, aspirin dose, clopidogrel use, and TIMI (thrombolysis in myocardial infarction) risk score, both serum TXB2 and PFA-100 collagen/ADP closure time were independently associated with MACE at 2 years, whereas AA-stimulated platelet markers and PFA-100 collagen/epinephrine closure time were not linked to the occurrence of adverse outcomes. The authors concluded that residual platelet COX-1 function as determined by serum TXB2 and COX-1independent platelet function measured by PFA-100 collagen/ ADP closure time, but not indirect COX-1- dependent assays, correlate with subsequent clinical events in aspirin-treated patients. Thus, their results suggest that multiple mechanisms including but not confined to inadequate inhibition of COX1, are responsible for poor outcomes in aspirin-treated patients.95 Mayer et al. performed MEA AA in 7090 consecutive patients on daily aspirin therapy directly before PCI.96 The upper quintile of patients (n ¼ 1414) according to MEA AA measurements was defined as the HRPR cohort. The primary endpoint of death or stent thrombosis over 1 year occurred significantly more often in patients with HRPR AA than in those with adequate aspirin-mediated platelet inhibition (6.2% vs. 3.7%), and HRPR AA was found to be an independent predictor of the primary endpoint. Cut-offs of on-treatment residual platelet reactivity to AA that have been associated with ischemic outcomes are given in Table 36.2 for the various platelet function tests.

TABLE 36.2 Cut-Offs of On-Treatment Residual Platelet Reactivity to Adenosine Diphosphate and Arachidonic Acid Associated With Ischemic Outcomes and Bleeding Complications for the Various Platelet Function Tests Platelet Function Test

Ischemic Outcomes

LTA, maximal aggregation

ADP 43%64, 97 >46%99 >59%99 65%64, 97, 98 >67%100 70%84, 101, 102 >208 PRU103, 104 235 PRU64, 97, 106–108 240 PRU110, 111 >468 AU
min112, 113 48 AU115 >47 mm61 50%98, 101, 117–122 >53.5%124

VerifyNow assays

Impedance aggregometry TEG, platelet-fibrin clot strength VASP assay, PRI

Bleeding Complications AA 20%84, 94, 97, 98

ADP <40%122.124

AA Not available

454 ARU94, 97 550 ARU92, 93

85 PRU105 189 PRU109

Not available

>203 AU
min96 14 AU115 Not available Not applicable

<188 AU
min114 23 AU116 31 mm61 20%123 16%124 10%125

Not available Not available Not applicable

Abbreviations: AA, arachidonic acid; ADP, adenosine diphosphate; ARU, Aspirin Reaction Units; AU, aggregation units; AU
min; area under the curve of AU; LTA, light transmission aggregometry; PRI, platelet reactivity index; PRU, P2Y12 Reaction Units; TEG, thromboelastography; VASP, vasodilatorstimulated phosphoprotein. Superscripts indicate reference numbers.

Laboratory Monitoring of Antiplatelet Therapy

In summary, HRPR during aspirin treatment has repeatedly been linked to ischemic events in cardiovascular disease.50,51,84,92,93,95,96 However, no widely accepted thresholds to identify patients with HRPR AA by the various test systems have been defined to date. Definition of these thresholds is a prerequisite for HRPR AA to become a useful prognostic biomarker in aspirin-treated patients.

P2Y12 Receptor Antagonists Clopidogrel The clinical implications of an inadequate response to clopidogrel therapy have been evaluated by a large number of studies. HRPR ADP by all established platelet function tests has been associated with the occurrence of adverse ischemic events in different patient populations (Fig. 36.4),126 in particular in patients undergoing PCI.61,64,99–101,103,106,107,110,112,117,127,128 Geisler et al. studied the response to a loading dose of 600mg clopidogrel by LTA ADP in 379 consecutive patients undergoing PCI for symptomatic CAD. Platelet inhibition <30% was defined as poor response to clopidogrel, and shown to be independently associated with the occurrence of ischemic outcomes within 3 months after PCI.127 Gurbel et al. measured postprocedural platelet reactivity by LTA ADP in 297 patients with nonemergent PCI and followed these patients for postdischarge ischemic events for up to 2 years.99 Patients who subsequently suffered ischemic outcomes (n ¼ 81, 27%) had displayed significantly higher on-treatment platelet reactivity to ADP at baseline. Using a receiver-operating characteristic (ROC) curve analysis, cut points of >46% aggregation following 5 μM ADP stimulation and >59% aggregation following 20 μM ADP stimulation were independently associated with MACE, and therefore defined as HRPR ADP.99 Similar cut-off values were found by ROC curve analysis in another study using the same ADP concentrations,64 while other studies using 10 μM ADP as platelet agonist defined residual aggregation >70% and >67%, respectively, as HRPR by LTA ADP.100–102 Price et al. measured on-treatment platelet reactivity by the VerifyNow P2Y12 assay in 380 patients following PCI with sirolimus-eluting stent implantation.106 ROC curve analysis was used to determine the best threshold to predict cardiovascular death, MI, or stent thrombosis at 6 months. The optimal cut-off value for the combined endpoint was on-treatment platelet reactivity 235 PRU,

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which was similar to the upper tertile of obtained platelet aggregation results (231 PRU). Patients with platelet reactivity values equal to or above this threshold suffered significantly higher rates of cardiovascular death (2.8% vs. 0%, P ¼ 0.04), stent thrombosis (4.6% vs. 0%, P ¼ 0.004), and the combined endpoint (6.5% vs. 1.0%, P ¼ 0.008).106 Using the same approach, Marcucci et al. found a cut-off value of 240 PRU by the VerifyNow P2Y12 assay to identify clopidogrel-treated ACS patients at risk of cardiovascular death or nonfatal MI within 12 months after PCI.110 Likewise, Spiliopoulos et al. reported that according to ROC curve analysis a threshold of 234 PRU was the best predictor of MACE in 100 patients with clopidogrel therapy undergoing infrainguinal angioplasty or stenting for PAD.107 Other studies used the same or comparable cut-off values for HRPR by the VerifyNow P2Y12 assay,64,108,111,129 whereas the prospective ADAPT-DES (Assessment of Dual AntiPlatelet Therapy with Drug Eluting Stents) registry found on-treatment platelet reactivity >208 PRU to be associated with stent thrombosis at 1 month as well as with stent thrombosis and MI at 1 year in 8665 patients on DAPT after PCI.103 The latter is consistent with the post hoc analysis of the GRAVITAS (Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety) trial, which showed that residual aggregation <208 PRU was associated with significantly improved clinical outcomes.104 Sibbing et al. assessed on-treatment platelet reactivity by MEA ADP in 1608 patients directly before PCI.112 All patients had received a 600 mg loading dose of clopidogrel prior to blood sampling, and the upper quintile of patients according to MEA measurements (>416 AU
min) was defined as patients with HRPR ADP (n ¼ 323). Of note, calculation of the optimal cutoff value for HRPR ADP by ROC curve analysis after completion of the follow-up resulted in a similar threshold of >468 AU
min. Over the 30-day follow-up, patients with poor response to clopidogrel had a significantly higher incidence of definite stent thrombosis than patients with adequate clopidogrelmediated platelet inhibition (2.2% vs. 0.2%, P < 0.0001). The composite of death or stent thrombosis occurred in 3.1% of patients with HRPR ADP as compared to 0.6% of normal responders (P < 0.001).112 Within 6 months, definite stent thrombosis (2.5% vs. 0.4%, P < 0.001) and the composite of definite and probable stent thrombosis (4.1% vs. 0.7%, P < 0.0001) were seen more frequently in patients with HRPR compared to those with adequate clopidogrel-mediated platelet inhibition.113 Bonello et al. performed the VASP assay to evaluate

Despite being treated with the same dose of clopidogrel, more than 40% of patients had a notable change in on-treatment platelet reactivity when serial samples were measured

Resting platelets

Stronger Platelets harder to activate Risk of ischemia Risk of bleeding

Activated platelets

Platelet inhibition

Weaker Platelets easier to activate Risk of ischemia Risk of bleeding

Figure 36.4 Measurements of platelet function vary over time in a significant proportion of clopidogrel-treated patients. Distribution of platelet inhibition in patients on clopidogrel with the arrows indicating individual changes in platelet inhibition over time. Treatment adjustment according to platelet function testing at a single time point might therefore not be sufficient for guiding antiplatelet therapy. (Reproduced with permission from Hochholzer et al. J Am Coll Cardiol 2014.126)

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the response to a loading dose of 300mg clopidogrel in 144 patients before PCI.117 With use of ROC curve analysis, they identified a PRI >50% to be linked with an increased risk of 6-month postprocedural MACE, a threshold for HRPR ADP equal or similar to the ones determined by other studies.101,118–121 Gurbel et al. investigated the prognostic utility of the strength of ADP- and thrombin-stimulated platelet-fibrin clots measured by TEG in 225 consecutive patients after elective stenting treated with aspirin and clopidogrel.61 By ROC curve analysis an ADP-stimulated platelet-fibrin clot strength >47 mm had the best predictive value for ischemic events over 3 years. Finally, patients on DAPT exhibiting a poor response to both aspirin and clopidogrel may be at an even greater ischemic risk than patients with HRPR AA or HRPR ADP alone.97,98,115 On the other hand, low on-treatment residual platelet reactivity to ADP (LRPR ADP) during clopidogrel therapy has been related to an increased bleeding risk following PCI and in patients undergoing cardiac surgery (Fig. 36.4).61,109,114,126,130–135 Cuisset et al. observed 16 non-CABG (coronary artery bypass grafting)-related TIMI hemorrhagic complications including five major and 11 minor bleeds within 30 days after ACS in 597 patients on DAPT with aspirin and clopidogrel.130 Patients who subsequently developed bleeding complications had significantly lower on-treatment platelet reactivity by LTA ADP (43  14% vs. 56  19%, P ¼ 0.002) and the VASP assay (43  14% vs. 54  23%, P ¼ 0.04). Results in the first quartile according to LTA ADP (on-treatment platelet reactivity <40% by LTA ADP) were defined as LRPR ADP, and patients with LRPR ADP suffered significantly more TIMI major and minor bleedings than those in the others quartiles (6.6% vs. 1.4%, P ¼ 0.001). In line with these findings, Tsukahara et al. reported the first quartile of ADP-stimulated platelet aggregation by LTA to be associated with a higher risk of bleeding events within 16 months in 184 clopidogrel- or ticlopidine-treated PCI patients.131 Moreover, on-treatment platelet reactivity <40% by LTA ADP correlated with 92% of severe coagulopathies that required multiple transfusions in 45 patients receiving clopidogrel within 6 days before on-pump CABG.132 Using ROC curve analysis, Patti et al. identified a cut-off value of 189 PRU by the VerifyNow P2Y12 assay as best predictor of 30-day TIMI major bleedings in 310 patients on aspirin and clopidogrel therapy after PCI.109 In contrast, in another study in 300 clopidogrel-treated PCI patients, on-treatment platelet reactivity 85 PRU at 1 month was an independent risk factor for bleeding complications over 1 year.105 Sibbing et al. used ROC curve analysis to define residual platelet aggregation <188 AU
min by MEA ADP as LRPR ADP, and reported that the risk of an in-hospital TIMI major bleeding was significantly higher in patients with enhanced response to clopidogrel (n ¼ 975) as compared with the remaining patients (n ¼ 1558) following PCI.114 Ranucci et al. associated the results obtained by MEA ADP in 87 patients with clopidogrel- or ticlopidine treatment until at least 1 week before cardiac surgery with postoperative bleeding.133 Some studies also related platelet reactivity by TEG with the occurrence of bleeding events in clopidogrel-treated patients. Thus, Gurbel et al. found an ADP-induced platelet-fibrin clot strength 31 mm by TEG to predict bleeding complications within 3 years after elective PCI.61 Furthermore, two studies suggested that TEG may be used to guide the timing of CABG in patients receiving clopidogrel.134,135 Cut-off values for HRPR ADP and LRPR ADP that were previously associated with ischemic events or bleeding are summarized in two international consensus documents on the definition of on-treatment platelet reactivity to ADP.136,137 Discrepancies of thresholds for HRPR and LRPR despite the application of the same test system may be attributable to the timing of testing, to differences in the studied patient

populations and to the use of different ADP concentrations (Table 36.2).

Prasugrel Data linking on-treatment residual platelet reactivity with ischemic events post PCI are scarce for prasugrel-treated patients compared to patients receiving clopidogrel, most likely because prasugrel exerts a stronger and more consistent antiplatelet effect than clopidogrel,138–141 and HRPR ADP despite prasugrel therapy is an infrequent phenomenon.142,143 While most studies observed HRPR ADP in less than 10% of prasugrel-treated patients,144–152 Bonello et al. found a poor response to prasugrel by the VASP assay in 25.2% of 301 patients who underwent successful PCI for ACS.122 The discrepancy between this study and the other publications may be explained by the timing of testing and the applied method: Bonello et al. assessed on-treatment platelet reactivity within 12 h after prasugrel loading dose,122 whereas other studies predominantly measured prasugrel response at a later time point.145,146,148–152 Moreover, Bonello et al. used the VASP assay,122 while most of the other studies used the VerifyNow P2Y12 assay.145–148,150–152 However, in the study by Bonello et al. HRPR ADP during prasugrel therapy resulted in a significantly higher risk of thrombotic events at 1 month and 1 year after PCI,122,124 and a PRI 16% by the VASP assay was associated with an increase in bleeding complications over 1 year.124 Likewise, Cuisset et al. reported that the use of prasugrel was the main predictor of LRPR by the VASP assay (defined as a PRI 10%) in 1542 ACS patients undergoing PCI, and LRPR was the strongest predictor of bleeding complications at 6 months.125 In summary, in line with the findings in clopidogreltreated patients, in patients receiving prasugrel HRPR ADP may be associated with the occurrence of ischemic outcomes and LRPR ADP seems to be linked to an increased bleeding risk (Table 36.2).

Ticagrelor The incidence of HRPR ADP may be even lower with ticagrelor compared to prasugrel.144,150,152–159 A metaanalysis including 14 studies and 1822 patients found HRPR ADP in 1.5% and 9.8% of ticagrelor- (n ¼ 805) and prasugrel-treated patients (n ¼ 1017), respectively.144 Likewise, a metaanalysis by Lhermusier et al., which analyzed data from 29 studies with 5395 patients, concluded that a maintenance dose of 90 mg ticagrelor twice daily is associated with significantly lower on-treatment residual platelet reactivity by LTA ADP, the VerifyNow P2Y12 assay and the VASP assay than 10 mg prasugrel per day.159 Due to the very low rate of HRPR ADP with ticagrelor there are to date no larger studies showing an association of HRPR ADP with ischemic outcomes in ticagrelor-treated patients. Some reports, however, suggest that LRPR ADP during ticagrelor is associated with an increased risk of bleeding complications (Table 36.2).116,123,150

Cangrelor In a pharmacodynamic substudy of 167 patients from the CHAMPION PCI and PLATFORM trials, patients randomized to cangrelor exhibited, as expected, a significantly lower platelet reactivity to ADP, and lower rates of HRPR by LTA ADP and the VerifyNow P2Y12 assay during infusion than patients receiving placebo (LTA with 5 μM ADP: 0% vs. 55% HRPR; LTA with 20 μM ADP: 6% vs. 93% HRPR; VerifyNow P2Y12 assay: 11% vs. 69% HRPR; all P < 0.001).160–162 Due to the low rates of HRPR ADP during cangrelor infusion and the fact

Laboratory Monitoring of Antiplatelet Therapy

that cangrelor is only administered in the peri-interventional period, there are to date no studies associating cangrelor response with the occurrence of MACE.

GPIIb-IIIa Antagonists As with cangrelor, GPIIb-IIIa receptor blockers are only administered in the peri-interventional setting.15,21 Thus, data linking the extent of platelet inhibition by GPIIb-IIIa antagonists with clinical outcomes are scarce. In 2001, Steinhubl et al. sought to determine the optimal level of platelet inhibition by GPIIb-IIIa receptor blockers to minimize thrombotic complications in patients undergoing PCI.163 Platelet aggregation was measured with the Ultegra Rapid Platelet Function Assay, a former version of the VerifyNow test using thrombin receptor activating peptide (TRAP) as the platelet agonist, before and 10 min, 1 h, 8 h, and 24 h after the initiation of a GPIIb-IIIa antagonist in 503 PCI patients. Eighty-four percent of the patients received abciximab, 9% received tirofiban, and 7% were treated with eptifibatide. The primary endpoint was defined as the composite of death, MI and urgent target vessel revascularization within 7 days after PCI. One quarter of all patients had <95% platelet inhibition at 10 min after the start of therapy and suffered significantly more MACE compared to patients with 95% platelet inhibition (14.4% vs. 6.4%; P ¼ 0.006). Patients whose platelet function was <70% inhibited at 8 h had a MACE rate of 25% vs. 8.1% for those 70% inhibited (P ¼ 0.009). Platelet aggregation inhibition 95% at 10 min after the initiation of therapy was independently associated with a significant decrease in the incidence of the primary endpoint (odds ratio 0.46, 95% CI 0.22 to 0.96, P ¼ 0.04).163 However, due to changes in antiplatelet therapy over the last two decades and the advent of new antiplatelet agents, it remains unclear if these results are applicable to current patients undergoing PCI.

663

activation despite conventional DAPT could be candidates for additional antiplatelet therapy with vorapaxar.

POTENTIAL MECHANISMS OF AN INADEQUATE RESPONSE TO ANTIPLATELET THERAPY Aspirin Nonadherence has been identified as a main reason for HRPR and adverse outcomes in patients on antiplatelet therapy.55,168– 170 Besides this “pseudo-resistance,” delayed and reduced drug absorption due to the prescription of enteric-coated instead of immediate-release aspirin formulations was associated with HRPR AA in several studies.53,56,171,172 Moreover, the proximity of the binding sites for aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) within the core of COX-1173 carries a risk of competitive drug-drug interactions,174–177 and is probably responsible for many literature reports of aspirin resistance.56 Specifically, some NSAIDs may prevent acetylation of serine 529 by aspirin and thereby the irreversible inhibition of platelet COX-1.178 High platelet turnover leading to faster recovery of platelet COX-1 is considered another important mechanism for increased platelet reactivity to AA despite aspirin therapy, particularly in patients with diabetes mellitus,179,180 obesity,181 essential thrombocythemia,182,183 myeloproliferative neoplasms, and cardiac surgery.56,184 In diabetic patients, higher aspirin esterase activity with increased blood plasma hydrolysis of acetylsalicylic acid may further contribute to impaired aspirin-mediated platelet inhibition.185 In addition, HRPR AA may be the result of underlying intrinsic platelet hyperreactivity prior to the institution of aspirin therapy.52 Strategies to optimize low-dose aspirin therapy are given in Table 36.3.186–192 Variables affecting aspirin response are discussed in section “predictors of an inadequate response to antiplatelet therapy.”

Vorapaxar In the TRACER (Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome) pharmacodynamic substudy vorapaxar caused a potent inhibition of PAR1 mediated platelet aggregation.164 Two hours after administration of a loading dose of 40 mg vorapaxar, maximal aggregation by LTA in response to TRAP was 68% (IQR 53%–75%) in patients receiving placebo (n ¼ 41) vs. 3% (IQR 2%–6%) in those receiving vorapaxar (n ¼ 44; P < 0.0001). Thus, 89% and 100% of vorapaxar-treated patients had 80% inhibition of TRAP-stimulated platelet aggregation at 2 and 4 h, respectively, post loading. High levels of inhibition of TRAP-stimulated platelet aggregation were maintained throughout vorapaxar therapy. On-treatment platelet reactivity to ADP at 4 h and 1 month post loading, and PAR-1 receptor number at 1 month were significantly lower in patients receiving vorapaxar.164 Studies linking an inadequate response to vorapaxar with clinical outcomes have not been reported to date. However, it has been shown that many patients receiving and responding well to DAPT with aspirin plus clopidogrel or aspirin plus prasugrel are still susceptible to platelet activation via PAR-1 and PAR4.165,166 Gremmel et al. assessed clinical outcomes and PAR-1 mediated platelet activation by measuring platelet surface expression of P-selectin and activated GPIIb-IIIa in response to TRAP in 108 patients with DAPT undergoing infrainguinal angioplasty and stenting for symptomatic PAD.167 They found high levels of TRAP-stimulated platelet surface Pselectin and activated GPIIb-IIIa to be independent predictors of the primary endpoint of atherothrombotic events and target vessel restenosis at 2 years. It may therefore be speculated that patients with high PAR-1-mediated platelet

P2Y12 Receptor Antagonists Clopidogrel Esterases degrade approximately 85% of absorbed clopidogrel, leaving only 15% to be converted by the hepatic CYP P-450 enzyme system to the active metabolite required for inhibition of the platelet ADP P2Y12 receptor.193 The CYP P-450 isoenzymes involved in this 2-step process are CYP1A2, CYP3A4, CYP3A5, CYP2B6, CYP2C9 and CYP2C19.193 Consequently, factors influencing intestinal absorption and—most importantly— those interfering with metabolic activation of the prodrug, affect clopidogrel response and account for the wide intra- and interindividual variability of on-clopidogrel platelet reactivity (Fig. 36.4).126,178 Other reasons for insufficient platelet inhibition during clopidogrel therapy comprise nonadherence,169 an increased platelet turnover and high intrinsic platelet reactivity to ADP prior to thienopyridine exposure.178,194 High intrinsic platelet reactivity before clopidogrel intake was shown to predict 6-h, 18- to 24-h and 15-day ADP-stimulated monocyte-platelet aggregate formation, P-selectin expression and platelet aggregation by LTA in patients undergoing planned PCI.194 Since clopidogrel targets only the P2Y12 receptor on human platelets,15,26 the second ADP receptor P2Y1 may still allow platelet activation by ADP in clopidogrel-treated patients. P2Y1 activation initiates ADP-induced platelet aggregation, and is responsible for platelet shape change,13 while P2Y12 activation results in amplification and stabilization of the aggregation response. There is a complex interplay between P2Y1 and P2Y12, and co-activation of both is necessary for full platelet aggregation.14 Recently, a study investigating a modified diadenosine tetraphosphate derivative as

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PART III Clinical Tests of Platelet Function

TABLE 36.3 Optimizing Low-Dose Aspirin Therapy

Suggested Action

Evidence (Reference Number)

1. Use the lowest effective dose (i.e., 75–100 mg daily)

186–188

2. Consider BID dosing in patients with type 2 diabetes mellitus and essential thrombocythemia

179, 180, 183

3. a. Prefer nonentericcoated formulations

53, 171, 172

b. Improve adherence c. Avoid concomitant administration of ibuprofen and naproxen 4. a. Avoid concomitant administration of gastrotoxic medications (nonsteroidal antiinflammatory drugs and high-dose corticosteroids) b. Consider proton pump inhibitors in high-risk patients. Consider eradication in H. pylori-positive patients

55

174–177, 189

Clinical Implication Maximize clinical efficacy; minimize gastrointestinal toxicity and drug-drug interactions Ensure persistent inhibition of platelet function throughout the dosing interval; clinical benefit remains untested Improve extent and duration of platelet inhibition Avoid misclassification of “resistance” Avoid interference with the antiplatelet effect of low-dose aspirin

190

Improve gastrointestinal safety

191, 192

Improve gastrointestinal safety

Modified with permission from Patrono C. J Am Coll Cardiol 2015.56

synergistic inhibitor of both P2Y1 and P2Y12 yielded promising results in two animal models and samples from healthy individuals.195 Finally, other as-yet unidentified mechanisms may contribute to HRPR ADP despite clopidogrel therapy.196 Factors that have previously been associated with inadequate clopidogrelmediated platelet inhibition are discussed in section “predictors of an inadequate response to antiplatelet therapy.”

Prasugrel Since prasugrel is also a thienopyridine requiring biotransformation to become pharmacologically active, the mechanisms influencing prasugrel response are potentially the same as with clopidogrel.178,197 In contrast to clopidogrel, however, prasugrel does not have an inactivation pathway consuming most of the absorbed dose.193,197 Instead, prasugrel is hydrolyzed to an intermediate metabolite by the same intestinal esterases that are responsible for the inactivation of clopidogrel. The intermediate metabolite is then oxidized by CYP3A4, CYP3A5, CYP2B6, CYP2C9, and CYP2C19 to the active form.193,198 A proportion of the active metabolite of prasugrel may even be produced within the small intestine which expresses CYP isoenzymes, in particular CYP3A4 and CYP2C9.199 Due to metabolic activation of the majority of the administered dose and only one step of hepatic conversion,193 prasugrel acts faster and is more potent than clopidogrel, and is less susceptible to influencing factors.138–143

Ticagrelor Ticagrelor is an adenosine triphosphate analog belonging to the triazolopyrimidine class.193 It binds reversibly and in a noncompetitive manner to the P2Y12 receptor at a distinct site to the thienopyridines.200 Unlike clopidogrel and prasugrel, it directly inhibits ADP-induced platelet activation following intestinal absorption. Although ticagrelor is also metabolized by CYP P-450 (in particular by CYP3A4) to generate an active metabolite,193,200,201 its antiplatelet effect may not be altered by factors influencing biotransformation because both the parent drug and the active metabolite exhibit an equal antiplatelet potency.200,202 Thus, nonadherence as well as differences in ticagrelor absorption, intrinsic platelet reactivity and platelet turnover may be mainly responsible for variations in ticagrelor-mediated platelet inhibition.178,194 Furthermore, as in patients receiving thienopyridines, the P2Y1 receptor may still allow ADP-induced platelet activation in ticagrelor-treated patients.13,14,195 P2Y12 activation counteracts the antiplatelet effects of prostacyclin, which inhibits platelet function by increasing levels of cyclic adenosine monophosphate through activation of adenylyl cyclase.203,204 Consequently, P2Y12 receptor inhibitors exert their antithrombotic effects in part by fostering the antiplatelet potency of prostacyclin.204 By inhibiting prostacyclin synthesis, concomitant aspirin therapy may attenuate the antiplatelet effects of P2Y12 inhibition. Indeed, it has been speculated that the combination of ticagrelor with high-dose aspirin at many study sites in North America in the PLATO (Platelet Inhibition and Patient Outcomes) trial may have led to the less pronounced reduction of ischemic outcomes by ticagrelor compared to study sites in the rest of the world. However, this was not noted in the TRITON-TIMI (Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel-Thrombolysis in Myocardial Infarction) 38 trial with prasugrel.205,206

Cangrelor Cangrelor is a modified adenosine triphosphate derivative acting as direct and reversible P2Y12 inhibitor with a short half-life of 3–5 min.21,34 It is administered intravenously thereby avoiding nonadherence and malabsorption as potential influencing factors. Variations in cangrelor response may be explained by differences in intrinsic platelet reactivity and ADP-induced platelet activation via P2Y1.13,194,195

GPIIb-IIIa Antagonists As with cangrelor, the three currently available GPIIb-IIIa receptor antagonists abciximab, tirofiban, and eptifibatide are intravenous antiplatelet agents.15,21 On-treatment platelet reactivity in patients receiving GPIIb-IIIa inhibitors may depend on intrinsic platelet reactivity prior to antiplatelet therapy.194 However, data on potential influencing factors for the response to GPIIb-IIIa antagonists are scarce.163

Vorapaxar Vorapaxar acts as an antagonist at the thrombin receptor PAR-1 on human platelets.15,21 Besides nonadherence, malabsorption and high intrinsic platelet reactivity,194 platelet activation via the second thrombin receptor PAR-4 may account for an inadequate response to vorapaxar.10,11 However, studies on impaired vorapaxar-mediated platelet inhibition and potential clinical consequences have not yet been reported.

Laboratory Monitoring of Antiplatelet Therapy

PREDICTORS OF AN INADEQUATE RESPONSE TO ANTIPLATELET THERAPY Over the last two decades, many predictors of an inadequate response to different antiplatelet agents have been identified.207 It should be noted, however, that most of these influencing factors affect the results of some, but not all, platelet function tests,208 and their clinical relevance in the context of antiplatelet treatment is often uncertain.

Demographics Age Gremmel et al. found a pronounced age dependency of ADPstimulated platelet reactivity by LTA and the VerifyNow P2Y12 assay in the initial phase of clopidogrel therapy in 191 patients after angioplasty and stenting.209 In their study, on-treatment platelet reactivity to ADP independently increased with the patients´ age, and HRPR ADP was seen more frequently in patients aged 75 years or older than in younger patients. They speculated that similar to other CYP P-450 substrates,210–212 hepatic biotransformation of clopidogrel may decrease with aging resulting in lower levels of the active metabolite and less clopidogrel-mediated platelet inhibition.209 In addition, the higher incidence of comorbidities such as diabetes and chronic kidney disease (CKD) and pharmacological interactions may in part be responsible for attenuated antiplatelet effects of clopidogrel in the elderly.207 The significant correlation of age with ADPstimulated platelet reactivity during clopidogrel therapy was confirmed in subsequent studies.213–215 Silvain et al. reported that the use of a higher maintenance dose of clopidogrel (150 mg/day) or 10 mg prasugrel daily could blunt, but not completely eliminate the differences in antiplatelet response between patients 75 years and younger patients.213 Another investigation observed a higher rate of HRPR ADP also in ticagrelor-treated patients aged 70 years or older, suggesting that impaired metabolism of thienopyridines may not be the only reason for increased on-treatment platelet reactivity to ADP in the elderly.215

Sex Female sex was associated with HRPR ADP during DAPT with aspirin and clopidogrel.110,216,217 This could at least partially account for the increased in-hospital mortality in women suffering an ACS noted in some studies.218 The underlying mechanism for decreased clopidogrel-mediated platelet inhibition in female patients remains unclear, but high intrinsic platelet reactivity might play a role.219

Body Mass Index (BMI) Several studies identified BMI and obesity as independent predictors of poor response to clopidogrel, respectively.220–224 The most likely reason for inadequate platelet inhibition by clopidogrel in obese patients is underdosing of clopidogrel in relation to body weight. However, even the administration of up to three additional loading doses of 600 mg clopidogrel did not achieve adequate P2Y12 inhibition in many obese patients.223 Thus, additional factors might promote HRPR ADP in patients with a high BMI. Specifically, an impaired hepatic metabolism of clopidogrel possibly due to fatty liver disease could account for the attenuation of clopidogrel-mediated platelet inhibition in obese patients. Moreover, the increased inflammatory status in obesity may contribute to poor clopidogrel response.225–227 In contrast to clopidogrel, the antiplatelet activity of prasugrel and ticagrelor did not correlate with body weight in a recent analysis.228

665

Comorbidities Diabetes Insufficient platelet inhibition by aspirin and clopidogrel is frequently seen in patients with diabetes (Chapter 27).229,230 The main reasons are an increased platelet turnover and impaired hepatic metabolism of clopidogrel resulting in lower levels of its active metabolite.179,229,231 Accordingly, in comparison with clopidogrel, administration of the less CYP P-450dependent prasugrel and the CYP P-450-independent ticagrelor resulted in stronger inhibition of ADP-stimulated platelet reactivity in patients with diabetes.231 Ticagrelor seems to be even superior to prasugrel at reducing platelet reactivity in patients with diabetes,232 and yielded a consistently high level of platelet inhibition regardless of diabetes status.233

Chronic Kidney Disease Chronic renal insufficiency is associated with a worse prognosis in cardiovascular disease,234 which may be attributable to inadequate platelet inhibition by standard antiplatelet therapy.235,236 Furthermore, poor response to aspirin and clopidogrel was observed more frequently in patients with more advanced stages of kidney failure,236–240 and may be explained by high intrinsic platelet reactivity and the presence of comorbidities such as diabetes in CKD.52,194,236,237 The addition of cilostazol to standard treatment with clopidogrel resulted in a lower rate of HRPR ADP in CKD,240 suggesting that patients with renal insufficiency could benefit from more intense antiplatelet therapy. Likewise, ticagrelor achieved faster and greater platelet inhibition than clopidogrel in patients undergoing hemodialysis.241 Moreover, ticagrelor significantly reduced ischemic endpoints and mortality compared with clopidogrel in ACS patients with CKD.242 On the other hand, antiplatelet treatment in CKD is complicated by a pronounced bleeding risk.243 Therefore, clinical outcome trials are important prior to the introduction of alternative antiplatelet regimens in advanced CKD.

Smoking Cigarette smoking may foster the conversion of clopidogrel to its active metabolite by inducing CYP1A2 and CYP2B6,244–246 and thereby enhance clopidogrel-mediated platelet inhibition. Indeed, several studies using LTA ADP, the VerifyNow P2Y12 assay and the VASP assay observed significantly lower platelet reactivity and a lower rate of HRPR ADP in clopidogrel-treated patients smoking 10 cigarettes per day,216,246–249 whereas smoking status did not influence prasugrel response.246 These findings may provide an explanation for the reduced clinical benefit of clopidogrel in nonsmokers in major randomized clinical trials.250,251 However, other large analyses yielded no significant association of smoking with the extent of clopidogrelmediated platelet inhibition,252,253 and no significant interaction of smoking status with the clinical efficacy of prasugrel vs. clopidogrel.253 Accordingly, the effect of smoking on clopidogrel response remains controversial and may at least in part depend on the test system used for monitoring on-treatment platelet reactivity.208

Dyslipidemia Wadowski et al. reported a significant inverse correlation of platelet surface P-selectin expression in response to ADP and platelet reactivity by the VerifyNow P2Y12 assay and the Impact-R ADP with high-density lipoprotein cholesterol

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PART III Clinical Tests of Platelet Function

(HDL-C) in 314 patients on DAPT with aspirin and clopidogrel.254 Patients with low levels of HDL-C (HDL-C 35 mg/ dL) exhibited significantly higher ADP-stimulated P-selectin expression and platelet aggregation by both platelet function tests compared to those with normal HDL-C levels. Furthermore, HRPR by the VerifyNow P2Y12 assay was seen more frequently in patients with low HDL-C.254 These data are in line with a previous study by Tselepis et al. who found an inverse association of HDL-C with platelet activation parameters,255 and are possibly due to a modulation of platelet function by HDL-C.256–258 Thus, antiplatelet properties may contribute to the beneficial effects of HDL-C on the occurrence of cardiovascular events.259,260

Anemia Anemia is frequently associated with a hypercoagulable state,261,262 and a higher incidence of thrombotic events was described especially in patients with hemolytic anemia.261,263 The latter may in part be due to detrimental platelet activation by ADP released from red blood cells.264 Moreover, iron deficiency anemia often leads to thrombocytosis.265,266 Giustino et al. reported an independent correlation of anemia with HRPR by the VerifyNow P2Y12 assay in 8413 patients on DAPT with aspirin and clopidogrel.267 Further, in their study, anemic patients had higher platelet reactivity by the VerifyNow aspirin assay. Likewise, Wadowski et al. found increased ADPstimulated monocyte-platelet aggregate formation and platelet reactivity in anemic patients receiving clopidogrel (n ¼ 306) or prasugrel/ticagrelor (n ¼ 109) therapy,268 suggesting a direct influence of anemia on the response to P2Y12 inhibitors. In contrast, a recent cohort level metaanalysis attributed the inverse association between hemoglobin concentrations and platelet reactivity by the VerifyNow P2Y12 assay to a laboratory error.269

Inflammation Increased platelet turnover and high intrinsic platelet activation in inflammation may result in poor response to antiplatelet therapy.270 Gremmel et al. observed an independent association of elevated interleukin-6 (IL-6) and high-sensitivity Creactive protein (hsCRP) levels with AA-stimulated platelet reactivity in 288 patients on aspirin therapy.271 In addition, HRPR AA as defined by various methods was seen more frequently in patients with high concentrations of both inflammatory markers.271 Likewise, other studies reported inverse correlations of aspirin and clopidogrel responsiveness with IL-6 and hsCRP, respectively, in patients undergoing PCI.115,226,272 Bernlochner et al. described a significant association of heightened levels of CRP, white blood cell count and fibrinogen with platelet reactivity by MEA ADP in 1223 stable patients under chronic aspirin and clopidogrel therapy.227 Inadequate platelet inhibition may be one reason for the pronounced risk of ischemic events in chronic inflammatory states.115,226,272

Left Ventricular Ejection Fraction Reduced left ventricular ejection fraction has repeatedly been linked with HRPR ADP during clopidogrel therapy,102,113,273 possibly due to diminished hepatic metabolism of clopidogrel in heart failure.

Vitamin D Deficiency One study reported that the rate of HRPR ADP increased with decreasing vitamin D quartiles in 249 clopidogrel- and 254

ticagrelor-treated patients, suggesting an attenuated response to ADP receptor antagonists in patients with vitamin D deficiency.274 Aspirin-mediated platelet inhibition was not affected by vitamin D levels. The underlying mechanisms as well as potential clinical implications of these findings remain unknown.

Medications Enteric-Coated Aspirin In contrast to immediate-release aspirin, enteric-coated formulations delay and reduce aspirin absorption resulting in HRPR AA despite patient adherence and adequate dosing.53,56,171,172

Nonsteroidal Antiinflammatory Drugs A pharmacokinetic interaction with aspirin has been shown for ibuprofen174,175,189 and naproxen175–177, and is most likely a consequence of the proximity of their binding sites within the core of COX-1.173 Strategies to avoid an attenuation of aspirin-mediated platelet inhibition by NSAIDs comprise stopping ibuprofen and naproxen or switching to an NSAID that does not interfere with the antiplatelet action of aspirin, e.g., diclofenac or celecoxib.174,189 Alternatively, the interaction with ibuprofen can be bypassed by administering aspirin 2 h before a single daily dose of ibuprofen.174 The latter is ineffective, however, if multiple daily doses of ibuprofen are given.174

High-Dose Aspirin High-dose aspirin therapy appeared to neutralize the additional benefit of ticagrelor compared with clopidogrel in the PLATO trial.205 The potential underlying mechanism is discussed in section “potential mechanisms of an inadequate response to antiplatelet therapy.” However, no studies have yet proven an interaction between high-dose aspirin and ticagrelor.

Proton Pump Inhibitors (PPIs) PPIs are metabolized to a variable degree by CYP3A4 and particularly CYP2C19, which are also involved in the bioactivation of clopidogrel.275,276 The potential interaction between clopidogrel and PPIs was first investigated by Gilard et al. who randomized 124 patients on DAPT with aspirin and clopidogrel to 20 mg omeprazole daily versus placebo for 1 week following PCI.277 Clopidogrel response was tested by the VASP assay on days 1 and 7 in both treatment groups. At day 7, patients receiving omeprazole exhibited a significantly higher PRI compared to the placebo group, suggesting an attenuation of clopidogrel-mediated platelet inhibition by omeprazole.277 Subsequent studies were able to confirm the pharmacokinetic interaction between clopidogrel and omeprazole,278,279 but found no significant influence of pantoprazole,278–280 dexlansoprazole281 or lansoprazole280,281 on the antiplatelet effect of clopidogrel. Studies investigating the association between clopidogrel response and the use of esomeprazole yielded conflicting results.278,281 A retrospective analysis in 8205 clopidogreltreated ACS patients reported a higher incidence of ischemic events in those concomitantly taking a PPI.282 In order to evaluate the clinical relevance of the clopidogrel-omeprazole interaction, the COGENT (Clopidogrel and the Optimization of Gastrointestinal Events) trial randomly assigned 3873 patients with aspirin and clopidogrel therapy to 20 mg omeprazole per day versus placebo.283 Over a median follow-up of 106 days, omeprazole significantly reduced upper gastrointestinal bleeding compared with placebo without increasing the primary

Laboratory Monitoring of Antiplatelet Therapy

cardiovascular endpoint of MI, revascularization, stroke and cardiovascular death.283 Moreover, a subanalysis of the PLATO trial revealed a higher risk of adverse events in both clopidogreland ticagrelor-treated patients receiving PPIs.284 Consequently, the association of PPI use with MACE in some studies may be due to confounding factors,285 with PPI use being a marker for, rather than a cause of, higher rates of ischemic outcomes.284

Calcium Channel Blockers (CCBs) Dihydropyridine CCBs inhibit isoenzyme CYP3A4 and could therefore interfere with hepatic conversion of clopidogrel to its active metabolite.286,287 Gremmel et al. assessed ontreatment platelet reactivity to ADP by LTA and the VerifyNow P2Y12 assay in 162 patients on DAPT with aspirin and clopidogrel 24 h after angioplasty with stenting.288 They observed significantly higher residual platelet aggregation, and a higher rate of HRPR ADP by both methods in patients with concomitant CCB therapy indicating impaired clopidogrel-mediated platelet inhibition. In a subsequent study, they were able to confirm these findings in a larger cohort with flow cytometric assays capturing platelet activation in response to ADP.289 Similar results were obtained in other studies using LTA ADP or the VASP assay,290,291 whereas one large analysis found no significant association of CCB therapy with ADP-stimulated platelet reactivity by MEA ADP.292 This discrepancy may be explained by the fact that the various test systems capture different aspects of platelet activation,67,68,87 and that the identification of influencing factors for clopidogrel-mediated platelet inhibition is to some extent assay-dependent.208 While some studies reported increased MACE rates in clopidogrel-treated patients receiving CCBs,290,293 others found no such association.292,294,295 Large prospective randomized trials on the clinical impact of a potential interaction between clopidogrel and CCBs have not been reported.

Statins

667

concentrations of ticagrelor and delays its antiplatelet effects.314 The administration of intravenous antiplatelet agents such as cangrelor or GPIIb-IIIa antagonists may achieve immediate comprehensive platelet inhibition despite the concomitant use of morphine or fentanyl.315 However, these strategies should only be considered if the coadministration of morphine or fentanyl and oral P2Y12 inhibitors results in more MACE in future clinical studies.

Vitamin K Antagonists Since coumarin derivatives and thienopyridines are both metabolized by the CYP P-450 enzyme system,316,317 their concomitant prescription carries the risk of a drug-drug interaction. Indeed, a study in 1223 clopidogrel-treated PCI patients revealed significantly higher platelet reactivity by MEA ADP in those with simultaneous phenprocoumon therapy.318 The clinical implications of this pharmacokinetic interaction remain unknown, but may be of particular interest because these two types of antithrombotic agents also interact at a pharmacodynamic level.

β-Blockers Different β-blockers exert antiplatelet effects by stimulating nitric oxide production,319,320 reducing in vivo catecholamine levels,321 or through interaction with platelet membrane macromolecules.322,323 Recently, Lee et al. found significantly lower levels of leukocyte-platelet aggregates and a better response to clopidogrel as assessed by MEA ADP in patients with concomitant β-blocker therapy following angioplasty with stenting.324 It remains to be established to what extent the beneficial effects of β-blockers in cardiovascular disease are attributable to antiplatelet properties.325

Genetics Loss-of-Function Polymorphisms

In contrast to hydrophilic statins such as pravastatin, lipophilic statins such as atorvastatin, simvastatin, lovastatin or fluvastatin may compete with clopidogrel for isoenzyme CYP3A4 of the hepatic CYP P-450 system,296,297 and could influence clopidogrel response. In line with this hypothesis, Lau et al. reported that atorvastatin but not pravastatin decreased the antiplatelet activity of clopidogrel in a dose-dependent manner in 44 patients following PCI.298 Subsequent studies investigating the potential interaction between lipophilic statins and clopidogrel metabolism yielded controversial results.299–303 The same applies to the hydrophilic high-intensity statin rosuvastatin, which undergoes inactivation via CYP2C9 and CYP2C19,304 and was associated with reduced clopidogrelmediated platelet inhibition in some but not all studies.302,305–308 Since statins are an integral part of cardiovascular pharmacotherapy, a prospective randomized trial evaluating the clinical consequences of a possible clopidogrel-statin interaction does not seem feasible. Observational studies and retrospective analyses, however, do not suggest a significant increase in adverse outcomes in clopidogrel-treated patients with concomitant statin therapy.294,300,301,309

Loss-of-function polymorphisms of the CYP P-450 enzyme system impair hepatic metabolism of clopidogrel to its active form,77,79 resulting in HRPR ADP and an increased risk of MACE during clopidogrel therapy.74–77,326–329 In particular, carriers of the *2 allelic variant of CYP2C19 were more likely to exhibit a poor response to clopidogrel and develop ischemic outcomes post PCI in numerous studies.74,76,77,326,327,329 Furthermore, CYP2C9 loss-of-function genotypes were associated with heightened on-treatment platelet reactivity and the occurrence of stent thrombosis in clopidogrel-treated patients.75,328,329 Single nucleotide polymorphisms of the ABCB1 gene may alter clopidogrel absorption,79 and were linked with a higher risk of cardiovascular events.76,330 In contrast to clopidogrel, the above-mentioned CYP P-450 and ABCB1 genetic variants did not affect inhibition of platelet aggregation or MACE rates in patients receiving prasugrel or ticagrelor in most studies.330–335 However, a recent study suggested that carriage of the C-allele of the rs5751876 adenosine A2a receptor polymorphism predisposes for HRPR ADP during ticagrelor therapy.336

Morphine/Fentanyl

Gain-of-Function Polymorphisms

Numerous studies showed that morphine delays the absorption of clopidogrel, prasugrel and ticagrelor,310,311 and attenuates P2Y12 receptor inhibition.311–313 Recently, the PACIFY (Platelet Aggregation with Ticagrelor Inhibition and Fentanyl) trial revealed that fentanyl, a potent opiate routinely given in US catheterization laboratories, also lowers plasma

The CYP2C19*17 allelic variant was associated with an enhanced response to clopidogrel,337 and an increased bleeding risk in clopidogrel-treated patients.78,338 Homozygous carriers of CYP2C19*17 exhibited the highest rate of bleeding complications in a population of 1524 PCI patients pretreated with a loading dose of 600 mg clopidogrel.78 Similarly, a higher

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PART III Clinical Tests of Platelet Function

incidence of LRPR ADP and more bleedings were observed in prasugrel-treated patients carrying the *17 allelic variant of CYP2C19.338,339 In contrast, carriage of gain-of-function polymorphisms of CYP2C19 was not associated with an increase in bleeding endpoints in the CHARISMA trial.340

MicroRNAs As discussed in section “MicroRNAs (miRs),” miRs may in the future serve as markers to evaluate the response to antiplatelet therapy.80,81 Besides their biomarker potential, however, some miRs such as miR-126 and miR-223 may influence on-treatment platelet reactivity either as regulators of endothelial cell function or by directly modulating platelet activation.82,341,342

THERAPEUTIC ADJUSTMENTS BASED ON TEST RESULTS Dose of Antiplatelet Agent Aspirin The generally accepted chronic aspirin dose is 75, 81, or 100 mg daily, depending on the available commercial formulations of the drug in different countries.56 Because 75 mg of aspirin per day is at least twice as high as the lowest dose necessary and sufficient to fully inhibit platelet COX-1 activity, there are no significant differences in the antiplatelet effects of doses ranging between 75 and 100 mg.56 Increasing aspirin dosage showed no evidence of superiority compared with lower aspirin doses in randomized clinical trials in patients with ACS or cerebrovascular disease.56,186–188 Although 325 mg of aspirin can be administered as a loading dose in the setting of ACS or acute ischemic stroke, prescribing 325 mg aspirin per day for longterm treatment does not produce any additional benefit while exposing the patient to the risk of unnecessary side effects such as gastrointestinal damage, bleeding complications, and possibly negative interactions with ticagrelor.12,33,56,204 Accordingly, the guidelines of the American College of Cardiology/American Heart Association (ACC/AHA) and the European Society of Cardiology (ESC) recommend a daily dose of 75–100 mg aspirin in patients treated with DAPT (Table 36.3).30,31 Although aspirin is usually administered once daily, there is evidence that twice-daily dosing may be beneficial in patients with diabetes mellitus, obesity, essential thrombocythemia, myeloproliferative neoplasms, and cardiac surgery, possibly due to increased platelet turnover (Table 36.3).56,179,180,182– 184, 343–345 Dillinger et al. treated 92 consecutive diabetic patients with 150 mg aspirin once daily or 75 mg aspirin twice daily in a crossover study.343 Residual platelet reactivity to AA was measured by LTA at trough level before morning aspirin intake. These investigators found stronger aspirin-mediated platelet inhibition and a significantly lower rate of HRPR AA (17% vs. 42%) with the twice-daily aspirin regimen compared to 150 mg aspirin once daily. The same group of investigators sequentially treated 32 essential thrombocythemia patients with 100 mg aspirin once daily, then 250 mg once daily, and finally with 100 mg aspirin twice daily.344 In accordance with their previous results, they observed significantly lower ontreatment platelet reactivity by LTA AA with the twice-daily aspirin regimen. Moreover, the rate of HRPR by LTA AA decreased from 97% with 100 mg aspirin per day and 94% with 250 mg aspirin per day to 9% with 100 mg aspirin twice daily. Cavalca et al. randomized 37 patients within 36 h after onpump cardiac surgery to 100 mg aspirin once daily, 100 mg aspirin twice daily or 200 mg aspirin once daily for 90 days.184 At day 7 post surgery, patients on 100 mg aspirin once daily showed a significant increase in serum TXB2 within the 24-h

dosing interval and a higher excretion of urinary d-TXB2. In contrast, the twice-daily aspirin regimen lowered serum TXB2 and prevented a rise in urinary d-TXB2 without affecting prostacyclin metabolite excretion.184 Thus, the administration of two doses of aspirin per day may ensure persistent inhibition of platelet function throughout the dosing interval under the above-mentioned circumstances. However, the clinical benefit of twice-daily aspirin regimens remains untested to date.

Clopidogrel Based on large clinical trials in patients with different manifestations of cardiovascular disease,28,346 the standard dose of clopidogrel is 75 mg daily. Several small studies and one large randomized clinical trial assessed the effects of increasing clopidogrel dosage in the setting of a poor response to clopidogrel.347–349 Trenk et al. measured residual platelet aggregation by LTA ADP in 117 patients on DAPT with aspirin and clopidogrel after elective PCI, and raised the daily dose of clopidogrel to 150 mg in case of HRPR ADP.347 The high maintenance dose significantly reduced on-treatment platelet reactivity from baseline to day 14. In contrast, patients who initially responded well to clopidogrel and therefore received the standard dose of 75 mg clopidogrel per day showed a significant increase in residual platelet aggregation to ADP from baseline to day 14. Gremmel et al. randomly assigned 46 aspirintreated patients with HRPR ADP in at least one of three platelet function tests to 75 versus 150 mg clopidogrel daily for 3 months.348 At baseline, on-treatment platelet reactivity by the VerifyNow P2Y12 assay, the VASP assay and MEA ADP was comparable between both treatment groups, whereas at 3 months, patients receiving the high maintenance dose exhibited a significantly lower platelet reactivity by all three methods than patients in the standard dose group. Furthermore, at 3 months HRPR by at least one of the three test systems was significantly less frequent in patients assigned to 150 mg clopidogrel compared with patients receiving 75 mg clopidogrel per day (33% vs. 87%; P < 0.001).348 In the multicenter, randomized, double-blind, active-control GRAVITAS trial, on-treatment residual platelet reactivity to ADP was measured in 5429 patients following PCI with drug eluting stent implantation for stable CAD or ACS (Table 36.4).349,350 All patients received a dose of 75–162 mg aspirin per day throughout the study period. Those who had not taken a daily dose of 75 mg clopidogrel for at least 7 days prior to the intervention, received a clopidogrel loading dose of 300 mg before PCI or a loading dose of 600 mg clopidogrel within 2 h after PCI. HRPR ADP was defined as PRU 230 by the VerifyNow P2Y12 assay and identified in 2214 patients (40.8%) of the study population. Patients with HRPR ADP were then randomized in a 1:1 fashion to an intensified treatment regimen with a total first-day dose of 600 mg clopidogrel followed by 150 mg clopidogrel daily for 6 months versus a standard regimen with no additional loading dose and 75 mg clopidogrel daily for 6 months. Study visits and platelet function testing by the VerifyNow P2Y12 assay were conducted in both groups at 30 days and 6 months. Compared to standard dose clopidogrel, the high dose regimen provided a 22% absolute reduction in HRPR ADP at 30 days (62% vs. 40%; P < 0.001). However, this laboratory finding did not translate into clinical outcomes: the primary composite endpoint of cardiovascular death, nonfatal MI and stent thrombosis occurred in 2.3% of the patients in both treatment groups over 6 months. Moreover, severe or moderate bleeding according to the GUSTO definition was not significantly increased in the high-dose group (1.4% vs. 2.3%; P ¼ 0.1). Due to the inclusion of a rather low risk population resulting in an overall low MACE

Laboratory Monitoring of Antiplatelet Therapy

669

TABLE 36.4 Comparison of Three Major Randomized Controlled Trials Measuring the Utility of Platelet Function Tests for Guiding Antiplatelet Therapy in Patients With High On-Treatment Residual Platelet Reactivity to Adenosine Diphosphate 349

Trial Feature

GRAVITAS

Patient characteristics

PCI with DES: 58% stable CAD, 27% unstable angina without MI, 15% ACS 2214 VerifyNow P2Y12 assay HRPR cut-off: 230 PRU Clopidogrel 600 mg loading dose, followed by 150 mg maintenance dose

Number of patients Platelet function test Treatment type/ dose in the monitoring group Efficacy outcome (monitoring group vs. conventionaltreatment group) Safety outcome

Limitations

6-Month cardiovascular death. nonfatal MI, or stent thrombosis: 2.3% vs. 2.3%; HR 1.01, P ¼ 0.97 Severe or moderate GUSTO bleeding: 1.4% vs. 2.3%; HR ¼ 0.59, P ¼ 0.1 - Low-risk patient cohort resulted in low event rate, hence underpowered to test the utility of PFM - Suboptimal remedy to overcome HRPR - Randomization done 12 or 24 h after PCI, missing periprocedural events

ARCTIC

351

ANTARCTIC

352

PCI with DES: 27 % NSTE-ACS

Patients aged 75 years undergoing PCI with stenting for ACS

2440 VerifyNow P2Y12 assay HRPR cut-off: 235 PRU or 15% inhibition Clopidogrel 600 mg loading dose plus clopidogrel 150 mg daily (90%) or prasugrel 60 mg loading dose plus prasugrel 10 mg daily (10%) 1-Year death, MI, stent thrombosis, stroke, or urgent revascularization: 34.6% vs. 31.1%; HR 1.13, P ¼ 0.1

877 VerifyNow P2Y12 assay HRPR cut-off: 208 PRU, LRPR cut-off: 85 PRU Prasugrel 5 mg daily (55%); prasugrel 10 mg (4%); clopidogrel 75 mg (39%)

Major STEEPLE bleeding: 2.3% vs. 3.3%; HR ¼ 0.7, P ¼ 0.15 - Low-risk patient cohort - Suboptimal remedy to overcome HRPR in majority of patients - Underpowered for postdischarge event occurrence

1-Year cardiovascular death, MI, stroke, stent thrombosis, urgent revascularization, and BARC-defined bleeding (types 2,3, or 5): 28% vs. 28%; HR: 1.0, P ¼ 0.98 BARC-defined bleeding (types 2,3, or 5): 21% vs. 20%; HR ¼ 1.04, P ¼ 0.77 - Uniform strategy for first 14 days: utility of PFM not tested in early period - Therapeutic approach in the PFM group is primarily to reduce bleeding - Approximately 7.8% of the PFM group did not undergo PFM on either day 14 or day 28

Abbreviations: BARC, Bleeding Academic Research Consortium; CAD, coronary artery disease; DES, drug-eluting stent; GUSTO, Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries; HR, hazard ratio; HRPR, high on-treatment residual platelet reactivity to adenosine diphosphate (ADP); LRPR, low on-treatment residual platelet reactivity to ADP; MI, myocardial infarction; NSTE-ACS, non-ST-segment elevation acute coronary syndrome; PFM; platelet function monitoring; PRU, P2Y12 Reaction Units; STEEPLE, Safety and Efficacy of Enoxaparin in Percutaneous Coronary Intervention Patients, an International Randomized Evaluation. Modified with permission from Gurbel PA et al. Nat Rev Cardiol 2016.350

rate, the GRAVITAS trial was underpowered to test the utility of platelet function monitoring.2 Other study limitations comprised the choice of a suboptimal remedy to overcome HRPR ADP, and the timing of randomization (12–24 h after PCI) which excluded periprocedural events from statistical analysis. Bonello et al. identified 429 patients with HRPR by the VASP assay after a loading dose of 600 mg clopidogrel.119 These patients were randomized to a VASP-guided group (n ¼ 215) who received up to three additional loading doses of 600 mg clopidogrel to achieve a PRI <50% before PCI and a control group (n ¼ 214) who underwent PCI without additional clopidogrel loading. Using the VASP-guided approach the rate of HRPR ADP could be reduced to 8%, and the risk of stent thrombosis and MACE within 30 days after PCI was significantly lower compared to the control group.119 Major bleeding events occurred in 0.9% of the patients in both treatment groups. The same group of authors confirmed the potential of a clopidogrel reloading strategy to decrease the rate of HRPR by the VASP assay in another publication,353 and showed that a VASPguided loading regimen may even be able to overcome HRPR ADP in carriers of the CYP2C19*2 LOF polymorphism.354,355 However, in another study 128 patients, who underwent the above-mentioned reloading with up to three additional boluses of 600 mg clopidogrel due to HRPR by MEA ADP, still suffered significantly more ischemic events within 1 year after PCI than patients without HRPR.356 In conclusion, increasing clopidogrel loading and/or maintenance dose decreases the rate of HRPR ADP.119,347–349,353,354,356 However, the clinical benefit of this strategy is questionable since the only large

randomized controlled trial yielded negative results,349 and smaller studies reported heterogeneous effects of intensified clopidogrel therapy on patient outcomes.119,356,357

Type of Antiplatelet Agent Valgimigli et al. screened 1277 patients with elective PCI at 10 European centers to enroll 93 aspirin, 147 clopidogrel, and 23 dual poor responders based on the results of the VerifyNow aspirin and P2Y12 assays.129 Patients with HRPR were then randomized in a double-blind manner to receive either the GPIIbIIIa antagonist tirofiban (n ¼ 132; bolus followed by an infusion for 14–24 h) or placebo (n ¼ 131) on top of standard aspirin and clopidogrel therapy. The primary endpoint of periprocedural MI defined as troponin I/T elevation 3 times the upper limit of normal within 48 h after PCI was seen less frequently in the tirofiban group than in the placebo group (20.4% vs. 35.1%; P ¼ 0.009). Furthermore, the 30-day MACE rate was reduced in patients receiving tirofiban (3.8% vs. 10.7%; P ¼ 0.031), whereas the incidence of bleeding did not differ between both treatment groups. In the TRIGGER-PCI (Testing Platelet Reactivity in Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy with Prasugrel) trial, platelet response to a loading dose of 600 mg clopidogrel was determined by the VerifyNow P2Y12 assay in stable CAD patients who had undergone successful elective PCI with stent implantation.358 Among 3283 patients with valid PRU measurements, HRPR ADP was found in 625 patients (19%). Of these, 423 patients (67.7%) participated in the randomized study and were assigned in a

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1:1 fashion to 75 mg clopidogrel per day versus a loading dose of 60 mg prasugrel followed by 10 mg prasugrel daily. From baseline to 3 months, prasugrel significantly decreased ontreatment platelet reactivity compared to clopidogrel. However, the primary efficacy endpoint of cardiac death or MI at 6 months occurred in no patient on prasugrel versus one patient on clopidogrel, and the safety endpoint of non-CABG related TIMI major bleeding occurred in three patients on prasugrel versus one patient on clopidogrel. Accordingly, the study failed to demonstrate the clinical utility of switching from clopidogrel to prasugrel based on the results of the VerifyNow P2Y12 assay. Due to the inclusion of a low-risk cohort with a low rate of MACE, the study was underpowered. Moreover, as in the GRAVITAS trial, the authors chose a fixed drug regimen for the intensified treatment group instead of individually tailoring antiplatelet therapy in each patient based on repeated platelet function testing. In contrast to the TRIGGER-PCI trial, smaller studies and registries suggested beneficial effects of switching from clopidogrel to prasugrel or ticagrelor in patients with HRPR ADP without increasing the risk of bleeding.356,357,359–361 Bassez et al. switched 16 prasugrel-treated patients with HRPR by the VASP assay 1 month after an ACS to 90 mg ticagrelor twice daily.362 One month after the initiation of ticagrelor they observed a significant decrease of on-treatment platelet reactivity and none of the patients still exhibited HRPR ADP. Four study participants were even classified as patients with LRPR ADP due to a PRI 10%. These data are in line with another study showing a stronger antiplatelet effect of ticagrelor compared to prasugrel in clopidogrel-treated patients with HRPR ADP,145 while Bernlochner et al. found similar levels of platelet inhibition by both agents in poor responders to clopidogrel.363 The randomized, open-label TROPICAL-ACS (Testing Responsiveness to Platelet Inhibition on Chronic Antiplatelet Treatment for Acute Coronary Syndromes) trial investigated a monitoring-guided de-escalation strategy of thienopyridine therapy in 2610 ACS patients undergoing PCI.364 Patients assigned to the guided de-escalation group (n ¼ 1304) received 10 or 5 mg prasugrel per day (according to the label and the current guideline recommendation) for 1 week followed by 1 week of 75 mg clopidogrel daily. At day 14, on-treatment platelet reactivity was determined by MEA ADP. Patients with HRPR ADP (defined as 46 AU by MEA ADP) were immediately switched back to prasugrel, while those without HRPR ADP continued on clopidogrel. In contrast, patients in the control group (n ¼ 1306) received standard treatment with prasugrel for 12 months. The primary endpoint was a composite of death from cardiovascular causes, MI, stroke and bleeding grade 2 or higher according to BARC criteria at 12 months after randomization, and occurred in 7% and 9% of the patients in the guided de-escalation group and control group, respectively. The authors concluded that guided de-escalation of antiplatelet therapy based on the results of MEA ADP was noninferior to standard treatment with prasugrel at 1 year after PCI.364 Another study, randomized 645 ACS patients with DAPT consisting of aspirin plus prasugrel or ticagrelor 1 month after PCI to unchanged DAPT (n ¼ 323) versus switched DAPT with aspirin plus 75 mg clopidogrel (n ¼ 322).365 Over 1 year, they observed a significant reduction of BARC 2 bleeding complications in the switched DAPT group compared to patients receiving unchanged DAPT (4% vs. 14.9%), whereas no significant differences were reported on the occurrence of ischemic endpoints. In a subsequent analysis, the same group of authors showed that the main benefit regarding the reduction of bleeding events by the switched DAPT regimen was achieved in patients with LRPR by the VASP assay (defined as a PRI 20%) at 1 month.123

In summary, prasugrel and ticagrelor significantly reduce on-treatment platelet reactivity in patients with HRPR ADP despite clopidogrel therapy.145,358,363,366–368 However, it remains unclear if switching to these newer P2Y12 receptor antagonists leads to a better prognosis in patients with poor response to clopidogrel and, if so, which one of these two drugs should be prescribed. Guided early de-escalation of P2Y12 inhibition may become an alternative option in ACS patients managed with PCI, in particular in those at an increased risk of bleeding, though further randomized data are necessary before using this strategy more broadly.

Dose and Type of Antiplatelet Agent 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, 2440 patients scheduled for coronary stenting at 38 sites in France were randomized to a strategy of platelet function monitoring with therapeutic adjustments in case of HRPR by the VerifyNow aspirin or P2Y12 assays versus a conventional strategy without testing and drug adjustment (Table 36.4).350,351 In the monitoring group, response to antiplatelet therapy was assessed before stenting and 2–4 weeks later. HRPR AA during treatment with aspirin called for the administration of intravenous aspirin, and HRPR ADP during treatment with clopidogrel called for the administration of a GPIIb-IIIa inhibitor and an additional bolus of 600 mg clopidogrel or a loading dose of 60 mg prasugrel before stent implantation, followed by a daily dose of 150 mg clopidogrel or 10 mg prasugrel thereafter. Furthermore, patients with HRPR ADP during treatment with clopidogrel at 14–30 days after PCI were switched to 10 mg prasugrel per day or received a 75 mg increase in their daily maintenance dose of clopidogrel. Patients with LRPR during thienopyridine therapy were switched to 75 mg clopidogrel if they were treated with 10 mg prasugrel or 150 mg clopidogrel per day. In the conventional-treatment group the use of all antiplatelet agents was left to the physician´s discretion with the recommendation to follow the current practice and the most recent international guidelines. The primary composite endpoint of death, MI, stent thrombosis, stroke or urgent revascularization within 1 year after stent implantation occurred in 34.6% of the patients in the monitoring group and in 31.1% of those in the conventional-treatment group suggesting no significant improvements in prognosis with treatment modifications based on platelet function testing compared to standard antiplatelet therapy without monitoring.351 Of note, the rate of major STEEPLE bleeding was also similar in both groups (2.3% vs. 3.3%; P ¼ 0.12). As in the previous studies on the clinical utility of platelet function monitoring, the limitations of the ARCTIC trial comprised the inclusion of a lowrisk population and the use of a suboptimal remedy to overcome HRPR in the majority of patients. In addition, the study was underpowered for postdischarge event occurrence.2 Aradi et al. assigned 219 ACS patients with poor response to clopidogrel by MEA ADP 12 to 36 h after PCI to high-dose clopidogrel (n ¼ 128) or standard-dose prasugrel (n ¼ 91).356 The high-dose clopidogrel group received up to three additional loading doses of 600 mg clopidogrel based on MEA ADP to normalize platelet reactivity below the cut-off for HRPR ADP, followed by a daily maintenance dose of 75 or 150 mg clopidogrel. The prasugrel group was instead switched to a loading dose of 60 mg prasugrel followed by 10 mg prasugrel per day. Both intensified treatment regimens significantly decreased ontreatment platelet reactivity. However, prasugrel provided a more potent platelet inhibition than repeated boluses of 600 mg clopidogrel, and the 1-year MACE rate in the prasugrel

Laboratory Monitoring of Antiplatelet Therapy

group was comparable to patients without HRPR ADP. In contrast, patients in the high-dose clopidogrel group suffered significantly more ischemic events than patients without HRPR ADP. Interestingly, BARC type 3 or 5 major bleeding also occurred more frequently with high-dose clopidogrel than with prasugrel.356 In the open-label, randomized, controlled ANTARCTIC (Assessment of a Normal Versus Tailored Dose of Prasugrel After Stenting in Patients Aged >75 Years to Reduce the Composite of Bleeding, Stent Thrombosis and Ischemic Complications) trial, 877 patients aged 75 years or older who had undergone coronary stenting for ACS were assigned to a monitoring group (n ¼ 442) or a conventional group (n ¼ 435; Table 36.4).350,352 All patients initially received 5 mg prasugrel in addition to low-dose aspirin. In those randomized to the monitoring group platelet function testing by the VerifyNow P2Y12 assay was performed 14 days after starting prasugrel to adjust treatment if needed. Specifically, the prasugrel dose was increased to 10 mg per day in case of HRPR ADP (defined as 208 PRU by the VerifyNow P2Y12 assay) or prasugrel was switched to 75 mg clopidogrel daily in case of LRPR ADP (defined as 85 PRU by the VerifyNow P2Y12 assay). Platelet function testing was repeated at day 28 for patients who required adjustment after the first testing. Patients with LRPR ADP during treatment with 10 mg prasugrel were subsequently prescribed 5 mg prasugrel per day, and patients with HRPR ADP during treatment with 75 mg clopidogrel were switched to 5 mg prasugrel daily. Patients with LRPR ADP already receiving 75 mg clopidogrel and patients with HRPR ADP who were on 10 mg prasugrel per day underwent no further therapeutic adjustment at day 28. In patients randomized to the conventional group no platelet function testing was performed throughout the study. At 1 year, the primary composite endpoint of cardiovascular death, MI, stroke, stent thrombosis, urgent revascularization and BARCdefined bleeding complications (types 2, 3, or 5) had occurred in 28% of the patients in both treatment groups. Moreover, the bleeding rate did not differ significantly between groups.352 A limitation of the trial was the uniform strategy for the first 14 days which did not allow for the testing of the utility of platelet function monitoring in the early period after PCI. Furthermore, the therapeutic approach in the monitoring group addressed the minimization of bleeding complications rather than the reduction of ischemic outcomes. Finally, approximately 8% of the monitoring group did not undergo platelet function testing on either day 14 or day 28.2 Jeong et al. randomly assigned 60 patients with HRPR by LTA ADP after a loading dose of 300 mg clopidogrel to receive either 100 mg cilostazol twice daily on top of standard DAPT (n ¼ 30) or a 150 mg maintenance dose clopidogrel in addition to aspirin (n ¼ 30).369 On-treatment platelet reactivity was measured by LTA ADP and the VerifyNow P2Y12 assay at baseline and after 30 days. At 30 days, they found a significantly lower rate of HRPR by LTA ADP in the cilostazol group compared to the high maintenance dose group (3.3% vs. 26.7%; P ¼ 0.012). In addition, adjunctive cilostazol was associated with significantly greater platelet inhibition as assessed by both methods than high-dose clopidogrel therapy.369 The CREATIVE (Clopidogrel Response Evaluation and Antiplatelet Intervention in High Thrombotic Risk PCI Patients) trial used TEG to identify 1078 PCI patients with poor response to clopidogrel.370 All patients were on 100 mg aspirin daily and randomized in a 1:1:1 fashion to standard DAPT with 75 mg clopidogrel per day, a high-dose group with 150 mg clopidogrel daily or a triple therapy group with 75 mg clopidogrel per day plus 100 mg cilostazol twice daily. The addition of cilostazol to DAPT significantly reduced the primary endpoint of death, MI, stroke, and target vessel revascularization at 18 months compared to conventional DAPT (8.5% vs.

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14.4%). High-dose clopidogrel therapy showed a nonsignificant trend towards less MACE compared to standard DAPT (10.6% vs. 14.4%). While BARC-defined minor bleeding occurred more frequently with 150 mg clopidogrel than with conventional DAPT (27.4% vs. 20.3%), there were no significant differences in BARC-defined major bleeding complications between the three treatment groups.370 Overall, these trials, together with the aforementioned studies on treatment modifications based on test results, illustrate how difficult it is to establish the clinical superiority of a monitoring-guided strategy compared with standard antiplatelet therapy. Thus, further studies on how to implement platelet function testing optimally in tailoring treatment are warranted before laboratory monitoring of antiplatelet therapy can be considered for daily clinical practice.

CURRENT GUIDELINES Therapeutic adjustments based on platelet function testing did not result in improved outcomes in large randomized clinical trials.349,351,352,358 Furthermore, there are no adequately powered studies on tailoring antiplatelet treatment according to the results of genetic testing. Accordingly, routine laboratory monitoring of antiplatelet therapy is currently not recommended by the guidelines on DAPT of the ACC/AHA and the ESC.30,31 However, the guidelines of the ESC state that platelet function testing and genotyping may be considered in specific situations where the results may influence the treatment strategy, e.g., in patients with recurrent adverse events despite state-of-the-art antiplatelet therapy or in patients undergoing CABG who have recently received P2Y12 inhibitors. In the latter, platelet function testing may help to guide decisions on the timing of cardiac surgery because of the wide interindividual variability of the magnitude and duration of the antiplatelet effect of P2Y12 receptor antagonists.371–374

CONCLUSIONS The response to antiplatelet therapy varies from one patient to another. While HRPR as assessed by established methods represents an independent risk factor for the occurrence of ischemic events following PCI, LRPR may be associated with an increased risk of bleeding complications. The test systems used to measure on-treatment platelet reactivity are based on different principles. They correlate at best moderately with each other, and their results are influenced by a wide range of different demographic, clinical, and genetic factors. Consequently, these methods are not interchangeable. Tailoring of antiplatelet therapy based on the results of platelet function testing decreases the rate of HRPR and LRPR, but failed to reduce adverse outcomes in large randomized controlled clinical trials. Therefore, laboratory monitoring of antiplatelet therapy should currently be limited to investigational purposes and specific clinical situations in which the results may influence the treatment strategy. REFERENCES 1. Gremmel T, Frelinger 3rd AL, Michelson AD. Platelet physiology. Semin Thromb Hemost 2016;42(3):191–204. 2. Michelson AD, Bhatt DL. How I use laboratory monitoring of antiplatelet therapy. Blood 2017;130(6):713–21. 3. de Gaetano G, Cerletti C. Platelet adhesion and aggregation and fibrin formation in flowing blood: a historical contribution by Giulio Bizzozero. Platelets 2002;13(2):85–9. 4. Brewer DB. Max Schultze (1865), G. Bizzozero (1882) and the discovery of the platelet. Br J Haematol 2006;133(3):251–8. 5. Coller BS. Bizzozero and the discovery of the blood platelet. Lancet 1984;1(8380):804.

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