Testing antiplatelet therapy

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1 European Heart Journal Supplements (2008) 10 (Supplement A), A28 A34 doi: /eurheartj/sum081 Testing antiplatelet therapy Christian Gachet* and Boris Aleil INSERM U311 Unit, Etablissement Français du Sang Alsace (EFS Alsace), 10, rue Spielmann, PO Box 36, Strasbourg Cédex 67065, France KEYWORDS Platelet function tests; Antiplatelet therapy; Light transmission aggregometry; Flow cytometry; Cardiovascular disease Patient response to antiplatelet agents varies, and poor response may be associated with increased risk of ischaemic events. Monitoring antiplatelet therapy with platelet function tests may identify poor responders who would benefit from a change in therapy, such as a dosage increase. The relevant tests for monitoring aspirin therapy are light transmission aggregometry (LTA) using arachidonic acid as the agonist or the VerifyNow ASA cartridge. The response is all-or-none, and no pre-treatment measurement is required. However, the very low number of aspirin non-responders makes mass monitoring unnecessary. In contrast, there is a wide variability in response to clopidogrel treatment. Clopidogrel monitoring may be justified if it can be demonstrated that poor responders benefit from dose increases. The gold standard to monitor clopidogrel is the vasodilator-stimulated phosphoprotein (VASP) assay due to its P2Y 12 selectivity and the fact that it does not require pre-treatment measurement. However, the relevant cut-off to distinguish poor responders from good responders has not been defined. In contrast to the VASP assay, LTA requires pre-treatment measurement, while the question of threshold is no better defined. The VerifyNow P2Y 12 assay is a good alternative; however, as with the other tests, the relevant thresholds are not clearly defined. The platelet function analyzer PFA-100 can be used to identify patients with severe platelet defects or von Willebrand disease. However, due to its lack of sensitivity, it is not suitable for monitoring antiplatelet therapy. In the context of antiplatelet therapy monitoring, some evidence suggests that the results of platelet function testing can be used to identify patients at risk of cardiac events, but no studies have shown that platelet function testing can be used to guide therapy. It is thus unclear whether the results of platelet function tests can be used in clinical decision-making. Introduction The goal of antiplatelet therapy is to prevent ischaemic events, but in some patients events occur despite chronic treatment with standard doses of one or more antiplatelet agents. 1 Variability has been found in patient responses to aspirin and clopidogrel and lower-than-expected responses may be associated with the occurrence of major adverse cardiac events The availability of laboratory tests to measure platelet function has led to interest in whether such tests can be Boris Aleil did not attend the panel discussion but was invited to coauthor the article Testing Antiplatelet Therapy * Corresponding author. Tel: þ ; fax: þ address: christian.gachet@efs.sante.fr used to guide antiplatelet therapy in patients with coronary heart disease. 19,20 Ideally, such a test could be used to determine the agent and dose that would provide the greatest reduction in risk of ischaemic events without increasing the risk of bleeding. Desirable attributes of platelet function tests are shown in Table Some of the more commonly used tests are shown in Table 2, each of which will be discussed in turn. 21 Methods of platelet function testing Light transmission aggregometry Invented in the 1960s, 22 light transmission aggregometry (LTA) is used to diagnose a variety of acquired and Published on behalf of the European Society of Cardiology. All rights reserved. & The Author For permissions please journals.permissions@oxfordjournals.org.

2 Testing antiplatelet therapy A29 Table 1 Desirable characteristics of laboratory tests to measure platelet function Setting Characteristic Additional comments General Convenient Simple (no operator expertise required), rapid, inexpensive Accurate The test measures what it is supposed to measure Precise Reproducible, different observers agree on interpretation Standardized Test procedure is well described, standards are available, reference ranges are established, and quality control programmes are available; results are comparable between different centres Monitoring of haemostatic or antiplatelet therapy Specific Clinically relevant Modifiable Cost-effective Measures the effect of the drug on its target Results independently correlated with clinical outcome Altering treatment based on the results of the test improves clinical outcome Benefits of testing outweigh the direct and indirect costs of testing and follow-up Adapted from Harrison et al. 21, Copyright 2007, with permission from Elsevier. inherited platelet defects and remains the gold standard for testing platelet aggregation. 21 Platelet aggregation can be quantitatively measured by recording the transmission of a light beam across a suspension of constantly agitated platelets in an aggregometer. In clinical investigation, platelet aggregation is studied using platelet-rich citrated plasma (cprp). The blood is collected in 3.8% sodium dihydrate citrate (one part per nine parts of blood) and centrifuged at 190 g for 15 min at ambient temperature. The cprp is stored at ambient temperature in stoppered tubes under CO 2 to avoid ph variations. The maximal delay between the collection of the blood and the end of the study of aggregation is 3 h. 23 To better define the platelet defect and eliminate the influence of plasma proteins, clotting factors, and anticoagulants, a suspension of washed platelets may be used. Blood collected on acid-citrate-dextrose is Table 2 Test Light transmission aggregometry VerifyNow TM Flow cytometry centrifuged at þ378c to obtain the cprp. The platelet pellet is washed twice, according to Mustard s method. 24 Platelet aggregation or agglutination is induced by an endogenous agent such as adenosine diphosphate (ADP), adrenaline, collagen, thrombin, arachidonic acid, or platelet-activating factor-acether; or laboratory tools such as ristocetin (which allows activation by von Willebrand factor), convulxin (which is a strong activator of the collagen receptor GPVI), or the ionophore A23187, to name a few. 23 The quantitative study of the aggregation curves (maximum amplitude of aggregation, amplitude at defined time points, slope of the initial increase, shape change) identifies the physiological and biochemical mechanisms that control platelet aggregation; allows recognition and classification of the hereditary or acquired platelet abnormalities that lead to clinical haemorrhagic or thrombotic manifestations; and determines the effect of drugs that inhibit platelet aggregation and their mechanism of action. 19,21,23 The clinical usefulness of LTA in monitoring antiplatelet therapy is limited by the need for a large sample volume, complexity of sample preparation, high level of expertise required, timeconsuming protocols, high cost, and variability of results from different laboratories. 20,21 Furthermore, there is no consensus on the concentrations of agonists that should be used in LTA platelet function testing. VerifyNow TM Platelet function tests Flow cytometry using the VASP assay PFA-100 w Method Measures low-shear, platelet-to-platelet aggregation in response to agonists Fully automated platelet aggregometer used to measure levels of antiplatelet therapy Uses whole-blood samples to measure platelet glycoproteins and activation markers by light-emitting fluorescence and detects platelet activation in vivo or in response to agonists Used to monitor P2Y 12 platelet receptor inhibition Measures high-shear platelet adhesion and aggregation VASP, vasodilator-stimulated phosphoprotein. Adapted from Harrison et al. 21, Copyright 2007, with permission from Elsevier. VerifyNow (Accumetrics, Inc., San Diego, CA; formerly known as Ultegra Rapid Platelet Function Assay) was developed as a point-of-care, whole-blood, semiautomated, cartridge-based platelet function test to determine the response to antiplatelet agents. 25 It measures the agglutination of fibrinogen-coated beads by platelets stimulated by an agonist in citrated whole

3 A30 C. Gachet and B. Aleil blood. It was initially developed to measure the antiplatelet effects of glycoprotein (GP) IIb/IIIa inhibitors. 25,26 Samples for VerifyNow are collected in vacuum tubes containing sodium citrate or another anticoagulant. The specimen tube is inserted into a staging well for temperature control and subsequently into a sample well that contains a needle that inserts directly into the closed vacuum tube. 25 To determine the level of platelet inhibition in patients receiving GP IIb/IIIa inhibitor therapy, VerifyNow uses a thrombin-receptor-activating peptide (iso-trap) as the agonist. Light transmittance changes as the TRAP-activated platelets bind to the fibrinogen-coated beads and fall out of solution; the rate of change in light transmittance is reported as platelet-aggregation units (PAU). Results are reported as raw PAU and as a percentage of the baseline (pre-treatment) PAU. 25 New VerifyNow cartridges have been introduced with more sensitivity and specificity forthetwomostwidelyusedantiplatelettreatments: VerifyNow Aspirin, for monitoring aspirin treatment, 21 and VerifyNow P2Y 12, for monitoring clopidogrel treatment. 27 Results from all three VerifyNow tests correlate well with those of LTA, 25,28,29 which is not surprising since the VerifyNow assay is based on platelet aggregation.itsgreatadvantageisthatitissemi-automated, calibrated, and standardized. A minor problem is the size of the tubes, which often are not well filled, and the sample may clot before the test has been performed (B. Aleil, unpublished). It should be kept in mind that, as an aggregation test, VerifyNow is sensitive to associated treatments such as GP IIb/IIIa inhibitors, regardless of the cartridge type. 25 Becauseitisanaggregationtest, VerifyNow cannot provide more information than LTA, and assessment of clear thresholds to evaluate the inhibitory effects of a particular drug is still a matter of debate, except for aspirin (see below). Flow cytometry Flow cytometry rapidly measures specific fluorescent characteristics of a large number of individual cells. In the flow cytometer, the cell suspension passes through a flow chamber where each individual cell is illuminated by a focused laser beam. Most frequently, cells have been labelled with one or more fluorescent antibodies. The laser light activates the fluorophore at the excitation wavelength and detectors process the emitted fluorescence and light-scattering properties of each cell. The intensity of the emitted light is directly proportional to the antigen density or the characteristics of the cell being tested. Flow cytometry assays can measure circulating activated platelets, in vitro platelet activation, and platelet leucocyte interactions, to diagnose inherited or acquired platelet disorders, monitor antiplatelet therapy, and so on. 30 Platelet activation in vitro can be measured using whole blood or platelet suspensions of either cprp or washed platelets. Platelets are activated by an agonist and activation-dependent markers are measured. These can be P-selectin exposure or activated GP IIb/IIIa. Alternatively, binding of fibrinogen can also be measured. Advantages of flow cytometry include the use of whole blood in minute quantities requiring minimal handling and its wide range of applications. It has a high degree of accuracy, providing results that correlate well with those of LTA. However, although useful for research, flow cytometry is not practical for measuring the response to antiplatelet agents in a clinical setting. Its limitations include the expense of the unit and complex sample preparation by specially trained personnel. 30 Several years ago, a very important development in platelet function analysis by flow cytometry came from the use of fixed and permeabilized platelets to specifically label the intra-cellular vasodilator-stimulated phosphoprotein (VASP). 31 VASP is an intra-cellular regulatory protein, that is non-phosphorylated in the basal state and phosphorylated through the cyclic adenosine monophosphate (camp) biochemical cascade. 32 This cascade is activated by prostaglandin E1 and inhibited by ADP via the P2Y 12 receptors. Results show either VASP phosphorylation, indicating P2Y 12 receptor inhibition, or dephosphorylation, indicating P2Y 12 receptor activity. Therefore, the VASP assay can be used to monitor thienopyridine therapy. Results are shown as an index of platelet reactivity to ADP ranging from 0 100%, where lower percentages indicate greater inhibition of the P2Y 12 receptor. Results of the VASP assay correlate well with those of platelet aggregometry and tests of activation by flow cytometry. The use of the commercially available kit reduces inter-laboratory variability of results. The test can be performed in,1 h, and the blood specimen can be stored or shipped to a core laboratory at room temperature for up to 48 h without affecting the results. 5,33,34 PFA-100 w The PFA-100 (Dade-Behring, Deerfield, IL) was developed for first-line screening for platelet dysfunction as an alternative to the bleeding time. 35 Citrated whole blood (0.8 ml) is placed in a disposable cartridge in the reservoir of the PFA-100. A vacuum passes the sample under high shear forces (.4000 s 21 ) through a collagen and ADP- or collagen and epinephrine-coated membrane. When the membrane aperture is occluded by a platelet plug (within 110 s) and system flow terminates, closure time (CT) is reached. Results are reported in seconds to CT. 35,36 As with other platelet function tests, each laboratory using the PFA-100 must establish baselines and normal ranges in healthy volunteers. The system is very easy to use, automated, and quick. It is sensitive to von Willebrand disease and to severe platelet disorders such as Glanzmann thrombasthenia or Bernard Soulier syndrome. However, the PFA-100 system lacks sensitivity to mild inherited or drug-induced defects. 20,36 The PFA-100 does not adequately measure inhibition of platelet aggregation in response to clopidogrel. Patients receiving aspirin treatment may have prolonged or normal CT. 36 Many variables can affect the results, including platelet count, red blood cells, platelet reactivity to collagen, and plasma von Willebrand factor. Therefore, the PFA-100 system is not suitable for

4 Testing antiplatelet therapy A31 antiplatelet therapy monitoring. 20 Its best use may be in determining an in vitro bleeding time to identify patients who require further testing. 20,35 Monitoring of antiplatelet drugs Testing platelet function to monitor antiplatelet effects of defined drugs has two related but distinct goals: to determine if the drugs efficiently affect their molecular targets and to try to predict secondary ischaemic events. Concerning the latter, although several studies have shown correlation between high on-treatment residual platelet reactivity and ischaemic events, no prospective clinical trial has been conducted to determine whether changing therapy in response to test results leads to better clinical outcomes. Monitoring the pharmacological effectiveness of antiplatelet drugs is important in the context of antiplatelet drug resistance or variability of the response to antiplatelet drugs. 20,37 Monitoring antiplatelet therapy should ensure that, in case of secondary ischaemic events, the biological effectiveness of the drug is not responsible for treatment failure. Variability of response has been reported with GP IIb/IIIa inhibitors, 38 aspirin, 2 4,17 and clopidogrel. 5 10,37 The mechanisms of action of these drugs are very different, and the mechanisms underlying variability or resistance are also very different. The issue of so-called aspirin resistance and the issue of variability of response to clopidogrel will be discussed separately. Concerning GP IIb/IIIa inhibitors, variability was reported early in their use. 38 However, these compounds are no longer used as chronic treatments, so the issue of their monitoring is not within the scope of this paper. Monitoring of aspirin Low-dose (75 mg/day) aspirin irreversibly acetylates Ser 529 of cyclo-oxygenase (COX)-1 (while higher and repeated doses are required to acetylate COX-2). This results in the complete inhibition of the ability of platelets and mature megakaryocytes to synthesize thromboxane (Tx) A This effect is reached 1 h after ingestion, while the plasma concentration peaks min after aspirin ingestion and the half-life of aspirin is min in plasma. 40 It takes 5 6 days to recover 50% of normal platelet function. It is important to understand that, depending on the agonist used, aspirin is a potent or a weak inhibitor of platelet aggregation. Indeed, when arachidonic acid is used to trigger platelet activation and aggregation, the response to aspirin treatment is null, while at the same time aggregation in response to any other agonist is only mildly reduced. This is because the involvement of TxA 2 synthesis in the overall mechanism of platelet aggregation is modest, although physiologically important, and varies from weak agonists such as ADP to strong agonists such as thrombin or collagen. It also depends on the concentration of the agonist used. Thus, the best agonist to use to monitor aspirin treatment is arachidonic acid, whatever the test, including LTA and flow cytometry. 20,40 Since the response is of an all-or-none type, and provided the right agonist is used, there is no need for pre-treatment measurement. Under these conditions, the rate of poor responders to aspirin is reported to be,5%, including a number of non-compliant patients. 20 A small number of poor responders to aspirin may benefit from an increase in the aspirin dosage up to 325 mg/day. 2,41 Some studies report a higher percentage of aspirin non-responders because they are using an imprecise definition of aspirin resistance and inappropriate platelet function tests. 20,40 The VerifyNow Aspirin assay also uses arachidonic acid (1 mm). Upon its conversion into TxA 2, platelet activation and agglutination with the fibrinogen-coated beads occur. The change in light transmittance is reported as aspirin reaction units (ARU). A pre-treatment measurement is not required, and the patient s response to aspirin is reported simply in ARU. When patients not receiving aspirin were tested with VerifyNow, all had.550 ARU, which became the threshold for aspirin response. 25 There is good correlation between arachidonic-induced LTA and VerifyNow Aspirin. 29 Investigators have used this device in numerous studies. 2,3,29,41,42 Of course, since it is a platelet aggregation-based test, the results of the aspirin assay are influenced by GP IIb/IIIa inhibitors, phosphodiesterase inhibitors, thienopyridines, and other COX inhibitors such as non-steroidal anti-inflammatory drugs. 25 These assays (arachidonic acid-induced platelet activation and/or aggregation, VerifyNow Aspirin) are the only relevant tests to monitor aspirin. In contrast, any other agonist-induced platelet response, as well as PFA-100, should be rejected. Since extra-platelet sources of TxA 2 also exist, measurement of urinary TxB 2 is not recommended. 40 Monitoring of clopidogrel The mechanism of action of the thienopyridine compounds such as ticlopidine and clopidogrel is completely different from that of aspirin and, in contrast to aspirin, the platelet response to the agonist (which is ADP in this case) and the response to the treatment are not of an all-or-none type. Clopidogrel is a prodrug, which has to be metabolized in the liver to produce an active metabolite. 43 The latter covalently binds the P2Y 12 platelet ADP receptor and inhibits the binding of ADP and the subsequent activation of the receptor. 8,43 The variability of the response to a loading dose ( mg) or to the chronic dose of 75 mg/day has been documented for years and reported again in more recent studies. 6,10,18,44,45 Thus, the rationale exists to ask whether low responders are at risk of secondary events, 9,12,14,16 while high responders could in theory be at risk for bleeding. So far, no clear prospective clinical trial has been performed to establish the clinical outcome and relevance of modifying the treatment regimen on the basis of biological markers, although in clinical practice cardiologists tend to increase the dose of clopidogrel in the so-called low responders. 46 An important issue is choosing the relevant test to perform in an appropriate fashion. As already mentioned,

5 A32 C. Gachet and B. Aleil PFA-100 is not sensitive enough to monitor clopidogrel. Concerning LTA, the only appropriate agonist is ADP, since clopidogrel inhibits one of the two platelet ADP receptors. 8 However, there is a large distribution of response to ADP, in addition to the above-mentioned inter-individual variability in the response to clopidogrel. Indeed, the overlap between the results of ADP-induced platelet aggregation of non-treated and treated individuals is 80% (Aleil et al., unpublished data). Moreover, interpretation of data obtained from LTA varies from institution to institution and there is no consensus regarding thresholds to define good or poor response. For these reasons, it is necessary to measure platelet aggregation before and after clopidogrel treatment to determine the absolute change in aggregation (the baseline aggregation minus the post-treatment aggregation) and to establish a percentage of inhibition. Nonresponsiveness has been defined as,10% reduction in platelet aggregation. 6 These values are arbitrary. Needless to say, in routine clinical practice, it is not often possible to test patients before and after treatment. Therefore, more selective and standardized alternative tests are required to monitor clopidogrel. 20 The VerifyNow P2Y 12 test uses ADP (20 mm) with prostaglandin E1 (PGE1) (22 nm) as agonists. 25 PGE1 is added to increase intra-platelet camp activity, which increases the test s sensitivity to ADP in relation to P2Y 12 receptors and suppresses P2Y 1 -activated increases in intracellular calcium levels. Each assay records two results: P2Y 12 reaction units (PRU) and percentage of inhibition. 25 The PRU result indicates the amount of P2Y 12 - specific ADP aggregation and is determined by calculating the rate and extent of platelet aggregation in the ADP detection well. Percentage of inhibition is the percentage change from baseline aggregation, and is calculated from the PRU result and the BASE result, which is an independent measurement based on the rate and extent of platelet aggregation in the TRAP channel. The BASE serves as an estimate of the patient s baseline platelet function independent of P2Y 12 receptor inhibition (% inhibition ¼ [BASE2PRU/BASE]100). 47 A good correlation has been established between the VerifyNow P2Y 12 test and several other methods including LTA and the VASP assay. 42 However, since the test is based on aggregation, although highly standardized and specific for P2Y 12, there is no clear cut-off between low and high responders, and a certain extent of overlap between untreated and treated patients is observed. 42 One specific problem of the VerifyNow P2Y 12 test is that the results are expressed as percentage inhibition and approximately a third of untreated subjects exhibit up to 15% inhibition. 42 How could these patients be distinguished from low responders to clopidogrel treatment? Also, what is the threshold from which a patient should be considered a good responder? Finally, the results of the P2Y 12 assay are influenced by treatment with GP IIb/IIIa inhibitors and phosphodiesterase inhibitors. Thus, the VerifyNow P2Y 12 test, although Food and Drug Administration (FDA) approved, highly standardized, and easy to perform, would benefit from further improvements. 25,42 The gold standard test to monitor P2Y 12 inhibitors is the VASP assay, the assay kit developed by Biocytex and Stago, which results from the standardization of the method established by Geiger and Schwartz in Ulrich Walter s group. 31 The principle of the test has been explained. Its correlation with other methods including LTA and VerifyNow P2Y 12 has been established in vitro and ex vivo. 5,42 As with LTA, VerifyNow P2Y 12, or flow cytometric measurement of P-selectin exposure or fibrinogen binding, a wide range of responses to clopidogrel treatment was recorded. 5,32,34 However, the great advantages of the VASP assay over the other tests are its selectivity for P2Y 12, its insensitivity towards GP IIb/ IIIa inhibitors, the high stability of the blood samples, and the fact that it is the only test where a single measurement allows interpretation of the data. 33,48 No pre-treatment value is required. The platelet reactivity index (PRI) calculated from the assay value is 80% in untreated individuals. 5 At least two types of threshold have been proposed. One is mean PRI 2 SD (70%), which encompasses the values of non-treated subjects. A third of clopidogrel-treated patients were indistinguishable from untreated patients. 5 This threshold tells us only that the patient had more or less responded to the treatment at a biological level. A second type of threshold has been put forward by others in order to prevent recurrence of ischaemic events during clopidogrel treatment. 49 This threshold has been defined as 50% inhibition of PRI, on the observation that, among those receiving clopidogrel, none of the subjects with PRI,50% presented with ischaemic events, while 100% of subjects with recurrence of thrombosis had PRI.50%. 49 Should we really monitor antiplatelet therapy? It is probably too early to answer this question, and more research is required. However, in practice, more and more patients receive increasing doses of clopidogrel, as well as other antiplatelet drugs. In many cases, the rationale for increasing the dose is not validated, such as, for example, the fact that some physicians systematically double the dose of clopidogrel in patients with a higher body mass index. 50 Such practices should not be allowed, as they do not correspond to any scientific data. Monitoring clopidogrel would be useful if it were demonstrated that the biological response could be modified in a single individual by modifying the dose; in other words, that a dose response relationship exists on an individual basis. This would appear to be the case. 27,45,51 Also to be demonstrated is that the biological modification results in an improved clinical outcome. Large, prospective clinical trials are currently underway to answer these questions. Concerning aspirin, since,5% of patients are poor responders, of which a proportion are non-compliant, it does not seem reasonable to propose the monitoring of every patient on aspirin therapy. Rather, in case of any doubt, increasing the dosage may be sufficient to

6 Testing antiplatelet therapy A33 ensure that the drug is not responsible for the treatment failure. 41 In conclusion, platelet function tests are not able to predict clinical events, although high on-treatment residual platelet reactivity is correlated with increased risk of recurrent ischaemic events. However, testing may ensure that the drugs reach their target and exert the expected pharmacological effect. Thus, in case of ischaemic events despite efficient treatment, the drug could be exonerated. Conflict of interest: C.G. declares he has received lecture and consultancy fees from sanofi-aventis/s.o. and Sankyo-Eli Lilly. His laboratory has received grants from Stago and sanofiaventis/s.o. B.A. declares he has no conflicts of interest. Funding Sponsored by an educational grant from Daiichi Sankyo Europe GmbH and Eli Lilly and Company. References 1. Meadows TA, Bhatt DL. Clinical aspects of platelet inhibitors and thrombus formation. 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