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221Current Opinion

Platelet function testing in acute cardiac care – is there a role for prediction or prevention of stent thrombosis and bleeding?Dániel Aradi1,5; Jean-Philippe Collet2; Johannes Mair3; Mario Plebani4; Béla Merkely5; Allan S. Jaffe6; Martin Möckel7; Evangelos Giannitsis8; Kristian Thygesen9; Jurrien M. ten Berg10; Christian Mueller11; Robert F. Storey12; Bertil Lindahl13; Kurt Huber14; on behalf of the Study Group on Biomarkers in Cardiology of the Acute Cardiovascular Care Association of the European Society of Cardiology and the Working Group on Thrombosis of the European Society of Cardiology1Department of Cardiology, Heart Center Balatonfüred, Hungary; 2ACTION Study Group-Institut de Cardiologie – INSERM UMRS-ICAN 1166 Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 3Department of Internal Medicine III – Cardiology and Angiology, Innsbruck Medical University, Innsbruck, Austria; 4Department of Laboratory Medicine, University_Hospital, Padova, Italy; 5Heart and Vascular Center, Semmelweis University, Budapest, Hungary; 6Division of Cardiovascular Diseases, Department of Medicine and Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA; 7Emergency Medicine and Chest Pain Units, Campus Virchow-Klinikum und Campus Mitte Charité – Universitätsmedizin Berlin, Germany; 8Department of Internal Medicine III – University of Heidelberg, Germany; 9Department of Cardiology, Aarhus University Hospital, Aarhus, Denmark; 10Department of Cardiology and Center of Platelet Function Research, St Antonius Hospital, Nieuwegein, the Netherlands; 11Department of Cardiology and Cardiovascular Research Institute Basel, University Hospital Basel, Switzerland; 12Department of Cardiovascular Science, University of Sheffield, Sheffield, UK; 13Department of Medical Sciences and Uppsala Clinical Research Center, Uppsala University, Sweden; 143rd Department of Medicine, Cardiology and Intensive Care, Wilhelminenhospital, Vienna, Austria

SummaryThe role of platelet function testing in acute coronary syndrome pa-tients undergoing percutaneous coronary intervention remains con-troversial despite the fact that high platelet reactivity is an indepen-dent predictor of stent thrombosis and emerging evidence suggests also a link between low platelet reactivity and bleeding. In this expert opinion paper, the Study Group on Biomarkers in Cardiology of the

Acute Cardiovascular Care Association and the Working Group on Thrombosis of the European Society of Cardiology aim to provide an overview of current evidence in this area and recommendations for practicing clinicians.

KeywordsAntiplatelet agents, platelet pharmacology, acute myocardial infarction

Correspondence to:Daniel Aradi, MD, PhDHeart and Vascular CenterSemmelweis University, Hungary2 Gyogy Sq. Balatonfüred, 8230 HungaryTel.: +36 302355639E-mail: daniel_aradi@yahoo.com

Financial support:This work was supported by the Association for the Promotion of Research on Arterio-sclerosis, Thrombosis, and Vascular Biology (ATVB), Viennna, Austria to KH and by a research grant from the Hungarian Academy of Sciences to DA.

Received: May 20, 2014Accepted after major revision: September 5, 2014Epub ahead of print: November 20, 2014

http://dx.doi.org/10.1160/TH14-05-0449Thromb Haemost 2015; 113: 221–230

Introduction

Antiplatelet agents are essential to prevent recurrent coronary events in patients undergoing percutaneous coronary interven-tions (PCI) (1–3). While increasing the level of platelet inhibition significantly decreases the risk of recurrent thrombotic events (4), the price to pay is an increased risk of bleeding (5, 6). Although the delicate balance between thrombosis and bleeding was already documented for clopidogrel administration (7), it has become cen-tral with novel, more potent P2Y12-inhibitors such as prasugrel, ti-cagrelor and cangrelor (5, 6, 8). Several assays have been devel-oped to measure platelet reactivity with the aim of a better predic-tion of ischaemic and/or bleeding complications (3, 9). However, due to lack of evidence that platelet function-guided P2Y12-in-hibitior treatment may improve outcomes, current guidelines rec-ommend platelet function testing (PFT) only in selected patients and not as part of clinical routine (1, 2, 10). Although there are major gaps in knowledge regarding when and how to use PFT, re-

cent publications have substantially widened our understanding. Therefore, the Study Group on Biomarkers in Cardiology of the Acute Cardiovascular Care Association and the Working Group on Thrombosis of the European Society of Cardiology aimed to re-view the available evidence of the usefulness of PFT in acute car-diac care for predicting adverse events and guidance of antiplatelet therapy. Additionally, pre-analytical and analytical issues and limi-tations of different methods are presented, because their under-standing is essential for proper use of these assays.

Methodological considerations for platelet function testing

During in vivo conditions of arterial injury, the triad of platelet ad-hesion-activation-aggregation leads to formation of platelet-rich aggregates that are stabilised via the simultaneous initiation of the coagulation cascade (11). The main pathways where antiplatelet

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222 Aradi et al. Platelet function testing in ACS

drugs interfere with these processes include a) thromboxane-A2 generation inhibition by acetylic salicylic acid (ASA), b) P2Y12 ADP-receptor inhibition, c) PAR-1 thrombin receptor inhibition, and finally d) glycoprotein (GP)IIbIIIa receptor blockage. Al-though several different platelet function assays were developed to monitor the effect of antiplatelet agents, the most reliable, clinically best validated and most widely used assays reflect the antiplatelet effect of P2Y12-inhibitors (3, 9). In contrast, measuring ASA re-sponse is usually not specific and may identify artefactually high rate of patients deemed “ASA resistants” (12). Having established a new reference method for aspirin response testing, Kovács et al. (13, 14) recently demonstrated that despite heterogeneous results regarding aspirin responsiveness in platelet assays, all compliant patients and healthy individuals showed fully acetylated cyclo-oxygenase-1 izo-enzymes after seven days of low-dose aspirin treatment. In addition, the largest available clinical trials have neither supported the clinical value of aspirin response screening (15) nor suggested any benefit from using an increased dose of as-pirin in patients after PCI (16). No data exist on the clinical role of measuring PAR-1 receptor inhibition or the effect of glycoprotein IIbIIIa inhibitors. Therefore, the current manuscript will focus on platelet function measurements related to treatment with P2Y12-inhibitors in patients with acute coronary syndrome (ACS) undergoing PCI.

Pre-analytical considerations

As in all laboratory procedures, errors of sample collection, prep-aration and handling may largely influence the obtained results and therefore the manufacturers’ instructions should be followed

carefully. Blood samples should ideally be drawn by direct veni-puncture with a needle of at least 21 gauge without or only mini-mal tourniquet to avoid platelet activation. The first ~5 ml of blood should be discarded to avoid spontaneous platelet aggre-gation. Whether sampling from indwelling venous or arterial lines affects the measurement results due to in-vitro platelet activation has to be evaluated for each assay, separately. Ideally, platelet func-tion should be measured at the point of care (POC) or soon after sample arrival in the laboratory. Some assays mandate an initial waiting period (such as 30 minutes [min] in case of Multiplate® ADP test and 10 min in case of VerifyNow® P2Y12 assay) after blood collection in order to let the blood equilibrate with antico-agulant at room temperature. Recent studies suggest that this in-itial waiting period is important when blood is drawn into citrated tubes; however, the use of hirudin may allow immediate sample processing (17, 18). Blood tubes should never be stored in the refrigerator, they should be kept at room temperatures.

Importantly, samples should be preferably processed within 2 hours of collection and not stored for longer durations due to the initiation of platelet damage within the sample that may contribute to false low platelet reactivity values. Therefore, if not measured as POC, samples for platelet function testing must be transported rapidly by a messenger. Although data is not robust, some results suggest that pneumatic tube delivery may initiate in vitro platelet activation dur-ing transport (19); therefore, if possible, manual transport should be preferred over pneumatic delivery. The type of anticoagulation (e. g. citrate, heparin or hirudin) has to be strictly followed depending on the methodology used (such as hirudin for Multiplate® and citrate for VerifyNow® and VASP®), because the type of anticoagulant may sig-nificantly interfere with the obtained results (20).

Table 1: Comparison between the most widely applied platelet function assays.

Assessment principle

Medium for assessment

Anticoagulant in collection tube*

Agonist

Addition of PGE1

Timeframe for sample processing*

Total measurement time

Reported value

Value range

Point-of-care

Standard process

P2Y12-specificity

Manufacturer

*: Recommended by the manufacturer.

VerifyNow P2Y12

Turbidimetric

Whole blood

Sodium citrate 3.2 %

ADP 20 µM

+

10 min – 4 hours

< 10 min

Platelet reactivity unit (PRU)

0–400 PRU

+++

+

++

Accriva Diagnostics, San Diego, CA, USA

Multiplate ADP test

Impedance

Whole blood

Hirudin > 15 µg/ml

ADP 6.4 µM

-(only in case of ADP-test HS)

30 min – 4 hours

< 10 min

Unit (U)

0–200 U

+

+

+

Roche Diagnostics, Rotkreuz, Switzer-land

VASP P2Y12 kit

Fluorescent intensity

Whole blood

Sodium citrate 0.109 M or 0.129 M

ADP 20 µM

+

Within 48 hours

2–3 h

Platelet reactivity index (PRI, %)

0–100 %

-

+

+++

Diagnostica Stago, Asnieres Sur Seine, France

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223Aradi et al. Platelet function testing in ACS

Are platelet function assays equal?

Available platelet function assays vary significantly regarding the method used to assess platelet reactivity during P2Y12-inhibition: one assay analyses signal transduction changes related to P2Y12-re-ceptor signalling with flow cytometry as a surrogate marker of pla-telet activity (vasodilator stimulated phosphoprotein [VASP] phos-phorylation, VASP-P assay®), others measure the process of pla-telet aggregation itself on metal surfaces (Multiplate®, impedance aggregometry), to fibrinogen-coated beads (VerifyNow®) or in pla-telet-enriched plasma (Light transmission aggregometry, LTA) (3, 9). Even others estimate platelet aggregation in capillaries under flow conditions (PFA-100®, PFA-200®, Innovance P2Y12®) or measure platelet count changes before and after agonist stimu-lation (Plateletworks®) (3). More detailed methodological descrip-tion of the analytical principles and limitations of the different de-vices have been published previously (3, 9). However, due to the heterogeneous way of measuring platelet activation, clinicians should be aware that the correlation between current platelet func-tion assays is only poor-to-fair and classification of patients into groups of response (such as patients with low/high on-treatment platelet reactivity) may not be the same when using different pla-telet devices (21, 22). These results highlight that current assays cannot substitute for each other and results of a specific assay may not be generalised to the whole group of platelet function meth-ods.

Unfortunately, except for a few (23–26), there are no head-to-head comparison studies between available assays. Having pointed out these limitations and based on the available evidence, the Ver-ifyNow®, Multiplate®, VASP-P assay®, and LTA are validated in suf-ficiently large sample size for prediction of stent thrombosis and bleeding in patients with ACS (3, 9). Results with other assays are either controversial (PFA-100®) or limited (Innovance®, Platele-tworks®) (15, 23). Assay standardisation and harmonisation are also important issues for platelet function tests to be used in clini-cal practice. In an analytical sense, no assays are standardised so far, but commercially available methods (e. g. VASP-P®, Multiplate® and VerifyNow®) are harmonised in contrast to LTA and should give comparable results in different laboratories. Lack of standard-isation regarding sample preparation, centrifugation speed, agon-ist concentration, sample storage and the need for expertise in per-forming the method are huge drawbacks for LTA (3). Finally, it may be highlighted that from the three validated, harmonised as-says, the only real point-of-care assay is the VerifyNow® device (▶ Table 1).

How should we interpret platelet function test results?It is important to differentiate between diagnostic and prognostic performance of a marker. Diagnostic biomarkers identify (diag-nose) an underlying disease or condition that would be unknown/equivocal lacking the biomarker test available (27). Diagnostic per-formance is assessed by receiver-operating characteristics (ROC) analysis including the calculation of sensitivity, specificity, positive

(PPV) and negative (NPV) predictive values. Importantly, the last two are largely influenced by the prevalence of the event; therefore, PPV of a diagnostic marker will always be low if the event is rare, such as in case of stent thrombosis (15). In contrast, prognostic biomarkers are used to predict the probability of a future outcome of interest in order to stratify patients into different categories of risk (28). To judge the utility of a prognostic biomarker, the strength of associations (odds ratio [OR] or hazard ratio [HR]) and the value of the marker in reclassifying individuals into new risk categories (net reclassification index, [NRI]) should be used (29). Therefore, platelet reactivity should be evaluated as a prog-nostic biomarker when estimating the risk of recurrent thrombotic or bleeding events after PCI (3, 29).

Essentials for clinicians:

• i) The most reliable, clinically best validated and most widely used assays measure the effect of P2Y12-inhibitors; assessment of response to aspirin, GPI-s or PAR-inhibitors is clinically not established in ACS patients undergoing PCI.

• ii) Available platelet function assays measure different aspects of platelet activation-aggregation, leading to only poor-to-fair correlation between them and possible discordances in patient classification. Therefore, platelet assays cannot substitute for each other.

• iii) Based on the clinical validity and standardisation, recom-mended assays to measure platelet reactivity during P2Y12-in-hibitors include the VASP-P® assay, the VerifyNow® device and the Multiplate® analyser. LTA is not recommended for routine use due to lack of standardisation.

• iv) When assessing the value of platelet function assays in pre-dicting the risk of thrombotic or bleeding complications, the strength of association between the estimate and the outcome should be reported (i. e. OR or HR). Predictive values are not recommended because they are largely influenced by the preva-lence of outcomes that may be misleading regarding clinical utility.

• v) Platelet function testing may be considered to track patient compliance and diagnose premature P2Y12-inhibitor cessation when discontinuation is clinically equivocal.

Role of platelet function testing to predict and prevent adverse events in ACSPlatelet reactivity and stent thrombosis

Stent thrombosis is a rare but serious complication of PCI with multiple determinants including the clinical characteristics of the patients (ACS, diabetes, shock), complexity of the treated lesion, type of stent used and adherence to antiplatelet medication (30–32). High on-treatment platelet reactivity (HPR, also referred as poor responsiveness) during treatment with P2Y12-inhibitors has emerged as a clinically important risk marker for stent throm-bosis (3, 9). A large number of observational studies have corre-lated platelet function estimates and stent thrombosis (33–36)

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224 Aradi et al. Platelet function testing in ACS

(▶ Table 2 and ▶ Table 3). In addition, the ADAPT-DES multi-national, multicentre, prospective registry (15), designed to look at the prognostic utility of PFT in 8,500 patients, demonstrated that HPR to ADP (defined as a PRU greater than 208) was a strong and independent predictor (HR: 2.49, 95 % confidence interval [CI] 1.43–4.31, p< 0.001) of definite or probable stent thrombosis at one year.

Platelet reactivity to ADP can be considered as a valuable prog-nostic biomarker to improve the prediction of stent thrombosis beyond well-known clinical risk factors in patients after PCI (3, 9). However, the risk associated with HPR is modulated by the clinical characteristics and procedural results of the studied individual. As for example, the predictive accuracy of HPR to ADP was higher in patients with ACS (adjusted HR: 3.91 (95 % CI: 1.51–10.11),

p=0.005) than in stable coronary artery disease (adjusted HR: 1.49 (95 % CI: 0.35–6.36), p=0.59) during 30 days of follow-up in the ADAPT-DES registry (37).

Compared to the acute PCI setting, HPR to ADP appears less relevant in ACS patients managed without revascularisation. In the platelet function substudy of the TRILOGY-ACS trial (38), in which medically managed ACS patients were enrolled, platelet reactivity was not an independent predictor of death, myocardial infarction or stroke in the adjusted analysis.

Finally, despite the robust evidence on HPR during clopidogrel treatment, the relevance of HPR in patients exposed to prasugrel or ticagrelor is unclear. HPR is much less prevalent in prasugrel or ticagrelor-treated patients than in patients on clopidogrel (39, 40), and its correlation with outcome is still not established. Only one

Table 2: Association of platelet reactivity, stent thrombosis and bleeding in studies including acute coronary syndrome patients and using commercially available platelet function assays*.

First author(ref)

Stone (15)(ADAPT-DES)

Park (55)

Cuisset (52)

Cuisset (26)

Bonello (53)

Sibbing (50) (ISAR)

Siller-Matula (24)

*only studies above 300 included patients are shown. BARC: Bleeding Academic Research Consortium; Def/prob: definite or probable; NR: not reported; ST: stent thrombosis.

N

8,448

2,849

1,542

598

301

2,533

402

Device

VerifyNow

VerifyNow

VASP

VASP

VASP

Multiplate

Multiplate

Year

2013

2011

2013

2009

2012

2010

2012

ACS

52 %

44 %

100 %

100 %

100 %

12 %

34 %

Follow-up

1 year

2.2 years

6 months

30 days

1 year

Bleeding: in-hospitalST: 30 days

1 year

P2Y12-inhibitor

clopidogrel

clopidogrel

25 %: prasugrel75 %: clopidogrel

clopidogrel

prasugrel

clopidogrel

clopidogrel

Bleeding assessed

ADAPT major

TIMI major

BARC 2,3,4

-

TIMI major

TIMI major

TIMI major

Stent thrombosis assessed

def/prob

def/prob

NR

def/prob

def/prob

def

def/prob

Table 3: Prognostic value of platelet reactivity for prediction of stent thrombosis and bleeding.

First author(ref)

Stone (15, 57)(ADAPT-DES)

Park (55)

Cuisset (52)

Cuisset (26)

Bonello (53)

Sibbing (50)(ISAR)

Siller-Matula (24)

Pooled*

*Pooled odds ratio was calculated by pooling events with the Mantel-Heanszel method into a fixed-effect meta-analysis from studies reporting events for stent thrombosis and/or bleeding (see Figure 1). NR: not reported; OR: odds ratio, ST: stent thrombosis.

Device

VerifyNow

VerifyNow

VASP

VASP

VASP

Multiplate

Multiplate

Cut-off for ST

> 208 PRU

> 235 PRU

-

NR

50 % PRI

> 46 U

> 48 U

n=14 533

OR for ST

HR: 2.49 (1.43–4.31)

HR: 1.45 (0.27–7.92)

-

NR

OR: 7.45 (1.49–41.36)

OR: 6.44 (2.38–17.38)

OR: 36.9 (4.3–319)

OR: 3.27 (2.21–4.85)

P

0.001

0.67

-

0.10

0.02

< 0.001

< 0.001

< 0.00001

Cut-off for bleeding

< 95 PRU

< 235 PRU

< 10 % PRI

-

< 16 % PRI

< 19U

< 20U

n=15 673

OR for bleeding

OR: 1.48 (1.21–1.81)

HR: 0.78 (0.35–1.69)

OR: 4.7 (2.7–8.3)

-

OR: 2.64 (0.52–13.36)

OR: 2.6 (1.3–5.2)

NR

OR: 1.68 (1.41–2.00)

P

< 0.001

0.52

< 0.001

-

0.24

0.005

0.32

< 0.0001

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225Aradi et al. Platelet function testing in ACS

small study suggests that prasugrel-treated patients with HPR may have elevated risk for thrombotic events (41), but this needs to be confirmed independently.

Optimal cut-offs to predict stent thrombosis

Universal cut-offs to predict stent thrombosis can only be recom-mended for platelet function assays that are adequately harmon-ised. In line with the recommendations of two recent consensus documents (3, 9), the VerifyNow®, the Multiplate® and the VASP-P® assays fulfill such criteria. Although it might be hypo-thesized that the optimal cut-off values may differ according to the clinical presentation and timing from PCI, there is lack of evidence to separately define thresholds for distinct cohorts. Based on cur-rent data, recommendations can only be given for general patient populations for each standardized device.

The largest dataset correlating platelet reactivity and stent thrombosis was obtained for the VerifyNow® P2Y12 assay and early studies suggested an optimal threshold between 230–240 pla-telet reaction units (PRU) (23, 42). Post-hoc analysis of GRAVI-TAS suggested a somewhat lower cut-off, 208 PRU (43), which was further confirmed to be more accurate in ADAPT-DES (15).

In the case of the Multiplate® analyzer, the largest study per-formed by Sibbing et al., including 1,608 patients, suggested the optimal cut-off of 46 units (U; equals to 468 AU*min) for predict-ing stent thrombosis, with a HR of 5.4 (95 % CI: 1.9–15.6, p = 0.002) at one-year follow-up. (44) Two studies later confirmed the higher risk for stent thrombosis using this cut-off as compared to patients below 46 U (24, 45).

Regarding VASP phosphorylation, most of the early studies suggested 50 % platelet reactivity index (PRI) to predict throm-botic complications (46, 47). Although some more recent studies proposed a slightly higher cut-off (60 %) (21, 48), this has not been confirmed in a sufficiently large cohort of patients so far.

Platelet function testing to predict bleeding

Major bleeding events have also highly heterogeneous background and are difficult to predict (2, 49). Beyond well-known risk factors, low on-treatment platelet reactivity (LPR) has been suggested to be of interest by several studies (50, 51). In particular, increase in the risk of bleeding with the use of more potent P2Y12-inhibitors is getting closer to the magnitude of reduction in stent thrombosis or myocardial infarction (5, 6, 8). Therefore, there is a considerable need to better understand and prevent such complications.

However, the value and accuracy of LPR to predict bleeding in patients treated with P2Y12-inhibitors remains debated with both positive (15, 50–54) and negative studies published so far (23, 55) (▶ Tables 2–3). One important limitation is the lack of standard-isation in the definition of bleeding events, duration of follow-up and adjudication of bleeding events in most studies. Notably, the largest study so far, the ADAPT-DES registry, demonstrated that platelet reactivity to ADP independently predicts major bleeding events one year after PCI (HR for PRU< 208: 1.37, 95 %CI: 1.12–1.64, p=0.002) (15).

In patients treated with prasugrel, two studies have shown a sig-nificant association between the level of platelet inhibition and bleeding (52, 53). In the study of Bonello et al., 301 ACS patients after PCI were followed for one year. The risk of bleeding was higher in patients below 16 % PRI (15.6 % vs 3.3 %; p< 0.001) (53). In the study of Cuisset et al., 1,542 ACS patients were followed for six months after PCI, and those with very low on-treatment pla-telet reactivity (PRI< 10 %) had significantly higher risk for bleed-ing, according to Bleeding Academic Research Consortium (BARC) class 2–4 (OR: 4.7, 95 %CI: 2.7–8.3; p< 0.001) (52). No-tably, most of the bleeding events were minor (class 2) and a large proportion took place during in-hospital period; therefore, the rel-evance of this cut-off regarding major spontaneous bleeding is not clear. No studies were published regarding the association between platelet reactivity and bleeding in ticagrelor-treated patients to date; however, pharmacodynamic studies suggest that ticagrelor is at least as potent as prasugrel in ACS patients (56).

Based on the discussed evidence, the suggested preliminary cut-off s to predict bleeding in ACS patients undergoing PCI include 95 PRU with VerifyNow® (57), 19 U with the Multiplate® (50) and 10 % with the VASP® assay. (52) The suggested cut-off for VASP® is differ-ent from that suggested by the last consensus document (9); how-ever, the cut-point of 10 % PRI was validated in a large study that was not available at the time of the prior consensus document. It needs to be highlighted that these cut-offs were mostly driven from studies of clopidogrel treatment; the relevance of these thresholds in the prasugrel and ticagrelor era needs to be verified.

Essentials for clinicians:

• i) HPR during clopidogrel therapy independently predicts the occurrence of stent thrombosis in ACS patients undergoing acute PCI (▶ Figure 1). However, clinical and procedural find-ings are also influential, and therefore, platelet reactivity should be evaluated in the clinical context of the patient.

Table 4: Best validated cut-offs of platelet reactivity to predict stent thrombosis and bleeding.

VerifyNow

Multiplate

VASP-P

PRU: platelet reactivity unit, U: aggregation unit, PRI: platelet reactivity index.

Stent thrombosis

Cut-off

> 208 PRU

> 46 U

> 50 % PRI

n

11,245

1,608

640

references

(15,43)

(70)

(47,53,71)

Bleeding

Cut-off

< 95 PRU

< 19 U

< 10 % PRI

n

8,449

2533

1,542

references

(57)

(50)

(52)

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226 Aradi et al. Platelet function testing in ACS

• ii) The prognostic value of HPR is unclear in ACS patients managed conservatively or in patients treated with prasugrel or ticagrelor.

• iii) Although the cut-offs for predicting higher risk of stent thrombosis may depend on the clinical presentation and are currently still under validation, the proposed thresholds for commercially available assays are 208 PRU in case of Ver-ifyNow®, 46 U in case of Multiplate® and 50 % in case of VASP-P® assay (▶ Table 4).

• iv) LPR appears to be associated with bleeding complications (▶ Figure 1). Preliminary cut-offs for higher bleeding risk are 95 PRU with VerifyNow®, 19 U with Multiplate® and 10 % with the VASP-P® assay, but further studies are needed to verify such thresholds (▶ Table 4).

Modifying treatment based on platelet function testing: is there a role to prevent bleeding or stent thrombosis?

Although platelet reactivity to ADP might be a valuable risk marker to predict the risk of stent thrombosis and bleeding in ACS patients after PCI, it remains uncertain whether it can be used for guidance to improve clinical outcomes. The rationale behind this is that platelet reactivity is a modifiable trait potentially behaving as a risk factor: potent P2Y12-inhibitors effectively decrease platelet

reactivity (39, 40) while it increases with reduced doses or by switching to less potent agents (58). Platelet reactivity to ADP is not only a marker of the drug effect but incorporates many pa-tient-related co-morbidities affecting baseline platelet activity, such as age, diabetes and renal insufficiency (3). The impact of these unmodifiable risk markers on clinical outcomes cannot be overcome by adjusting the level of P2Y12-inhibition.

The first randomised, controlled clinical trial to study platelet reactivity-guided treatment modification was the GRAVITAS study (59) that included 2,214 low-to-intermediate risk patients undergoing PCI. Only 10 % of the patients had acute myocardial infarction (AMI) during enrollment. Using a cut-off value of 230 PRU (Verify Now® assay) to separate patients with HPR, the clini-cal effect of 150 vs 75 mg clopidogrel was compared on the six-month incidence of death, myocardial infarction or stent throm-bosis. As a result, the primary endpoint was identical in both ran-domised arms (2.3 % vs 2.3 %). (59) The ARCTIC multicentre ran-domised trial (60) aimed at comparing the potential clinical bene-fits of a platelet function-guided approach and conventional clini-cal-guided strategy in intermediate-risk patients undergoing PCI including 27 % with AMI. In the study, not only platelet reactivity to ADP was used to define HPR with the cut-off of 235 PRU (Ver-ify Now® assay), also percent inhibition compared to baseline and platelet reactivity to arachidonic acid were used (60). The one-year primary endpoint of death from any cause, myocardial infarction, stent thrombosis, stroke/transient ischaemic attack or urgent rev-

Figure 1: Relative risk for stent thrombosis (A) and bleeding (B) in patients with high or low platelet reactivity. Data are plotted from studies using commercially available platelet function assays (VerifyNow®, Multiplate®, VASP®) including more than 300 patients. Studies are ordered by increasing weight in the overall results. CI, confidence interval.

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227Aradi et al. Platelet function testing in ACS

ascularisation did not differ between the conventional and the monitoring group (31.1 % vs 34.6 %), the latter receiving mostly dose elevations of clopidogrel and GPIIbIIIa inhibitor infusions during PCI (60).

It is important to re-emphasise that risk reduction may only be achieved in patients at risk (61). Therefore, currently available studies on platelet function testing and tailored antiplatelet ther-apy may have been hampered by routine selection of low-risk indi-viduals and not high-risk patients with ACS (59, 60, 62).

The available randomized studies do not support the role of platelet reactivity biomarkers in improving outcomes after acute PCI. However, our understanding on platelet reactivity has largely changed since the design and initiation of these studies: i) platelet reactivity to ADP is a reliable prognostic marker mostly in ACS, but less valuable in stable angina (37); ii) while platelet reactivity to ADP may be a good prognostic marker, measuring ASA response and percent P2Y12-inhibiton is not recommended (3); iii) high-dose clopidogrel provides modest and variable benefit in ACS pa-

tients with HPR and should not be used (45); iv) mortality, defi-nite/probable stent thrombosis together with major bleeding are the most appropriate endpoints to assess safety and efficacy in such trials (instead of e. g. periprocedural troponin release).

A recently published prospective, single-centre registry (45) well illustrates the importance of patient characteristics and anti-platelet intervention for future randomised platelet function studies. In this registry, authors non-randomly compared the clini-cal safety and efficacy of prasugrel vs high-dose clopidogrel in a very high-risk cohort of ACS patients with HPR, including almost 50 % with ST-segment elevation myocardial infarction (STEMI), 5 % with cardiogenic shock and up to 85 % with AMI (▶ Table 5). According to the findings, switching patients with HPR to prasu-grel showed a significant reduction in the rate of death, myocardial infarction, stent thrombosis or stroke compared with high-dose clopidogrel, without increasing the risk of bleeding (45). In addi-tion to this registry, there are two other non-randomised studies (ISAR-HPR and MADONNA) (63, 64), using the Multiplate® ana-

Table 5: Comparison between randomized trials and registries aiming to test the clinical benefit of treatment modification based on platelet function testing.

Patient risk profile

Study design

Patient number

ACS (%)

AMI (%)

STEMI (%)

Shock (%)

Mortality

Antiplatelet intervention, if HPR

High-dose clopidogrel (guided or HPR group)

High-dose ASA

Prasugrel

PFT assay

Results

Comparison

1° Endpoint

1° Results

*Comparison between high-dose clopidogrel and prasugrel. Abbreviations: ACS: acute coronary syndrome; AMI: acute myocardial infarction; ASA: acetylic salicylic acid; CV: cardiovascular, HPR: high platelet reactivity; PFT: platelet function test, STEMI: ST-segment elevation myocardial infarction; ST: stent thrombosis.

GRAVITAS(59)

RCT

2,214

40 %

10 %

0.4 %

0 %

0.8 %

100 %

-

-

VerifyNow

150 vs 75 mg clopi-dogrel in HPR

CV death, MI or ST at 6 months

2.3 % vs 2.3 %p=0.97

ARCTIC(60)

RCT

2,440

27 %

NA

0 %

0 %

2 %

80 %

45 %

12 %

VerifyNow

Monitoring vs con-ventional therapy

CV death, MI, urgent revasc., stroke at 1 year

31.1 % vs 34.6 %p=0.10

RECLOSE-2 ACS (69)

Prospective registry

1789

100 %

NA

46 %

6 %

5 %

100 %

-

-

LTA

HPR + high-dose clopidogrel vs no HPR

CV death, MI, urgent revasc., stroke at 2 years

14.6 % vs 8.7 %

PÉCS REGISTRY(45)

Prospective registry

741

100 %

84 %

48 %

4.5 %

8.2 %

58 %

-

42 %

Multiplate

High-dose clopido-grel vs prasugrel in HPR

All-cause death, MI, ST or stroke at 1 year

16.5 % vs 7.1 %*p< 0.0001

ISAR-HPR(64)

Historical control study

999

50 %

33 %

15 %

N/A

1.2 % (30-day)

15 %

-

20 %

Multiplate

Monitoring vs con-ventional therapy

Death or ST at 30 days

1.2 % vs 3.7 % p=0.009

MADONNA(63)

Prospective registry

798

37 %

37 %

12 %

N/A

1.6 % (30-day)

100 %

-

14 %

Multiplate

Monitoring vs con-ventional therapy

ST at 30 days

0.2 % vs 1.9 % p=0.027

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228 Aradi et al. Platelet function testing in ACS

lyser and suggesting a benefit for platelet function testing in reduc-ing the risk of stent thrombosis in all-comers undergoing PCI. However, the premature termination of the TRIGGER-PCI (62) trial, which aimed to evaluate the benefit of switching low-risk elective patients after successful drug-eluting stent (DES) implan-tation to prasugrel, highlights the importance of targeting high-risk patients undergoing PCI in future clinical trials with platelet function testing. With this respect, one of the most important on-going trial is the TROPICAL-ACS (NCT01959451) study that aims to test the non-inferiority of Multiplate-guided P2Y12-in-hibitor selection compared to one-year use of prasugrel in high-risk ACS patients after PCI.

Although some studies are on their way to test the hypothesis (ANTARCTIC: NCT0153844), there is currently no proof for the utility of platelet function assays in preventing bleeding events in elderly patients undergoing PCI. Therefore, although reducing the dose of prasugrel/ticagrelor or switching back to clopidogrel to reach a potential therapeutic window for ADP-dependent platelet reactivity may be appealing, there is no data that patients titrated into an optimal range of platelet reactivity might benefit compared to regular antiplatelet therapy with prasugrel or ticagrelor.

Platelet function testing may be helpful according to a study to define the optimal time of cardiac surgery in patients pretreated with clopidogrel. In the TARGET-CABG trial (65), patients pre-treated with clopidogrel underwent cardiac surgery either after waiting sufficient antiplatelet washout time or guided by thrombe-lastography. As a result, there were no difference between the two groups in bleeding complications, however, the guided group had approximately 50 % shorter waiting time before operation (65). This is the reason why platelet function testing has obtained a class IIa indication in thoracic and cardiac surgery guidelines, to define the optimal time window before cardiac operation is performed in clopidogrel-treated patients (66, 67).

Essentials for clinicians:

• i) There is no evidence that treatment modification based on platelet function testing can improve outcomes in patients undergoing PCI.

• ii) Although further clinical research is needed to evaluate the role of platelet function testing in preventing stent thrombosis and bleeding, platelet function assessment in clinical routine is currently not recommended. Unless contraindications exist, ACS patients should be treated with prasugrel or ticagrelor.

• iii) Treatment modification based on platelet function testing may be justifiable in selected patients suffering thrombotic or bleeding complication while being compliant to P2Y12-inhbitor treatment.

Summary for clinical practice

Platelet reactivity to ADP, assessed by commercially available pla-telet function assays (such as VerifyNow®, Multiplate®, VASP-P®), is a valuable prognostic biomarker to predict the risk of stent throm-

bosis and bleeding after acute PCI (3, 15). The association of pla-telet inhibition with bleeding and thrombosis is statistically inde-pendent and clinically relevant. Adding platelet reactivity on top of known clinical risk factors of stent thrombosis significantly im-proved the predictive value (c-statistic) of scoring systems (23). Based on the currently available evidence, the best preliminary cut-offs for risk stratification include 95 and 208 PRU for Ver-ifyNow®, 19 and 46 U for Multiplate® and 10 and 50 % PRI for VASP-P® for bleeding and stent thrombosis, respectively (see also ▶ Table 4). These suggested cut-offs might be different according to clinical presentation, timing from PCI, procedural success and ethnicity and, therefore, need further validation. In addition, most of the data stem from clopidogrel-treated patients; smaller studies have been published regarding prasugrel and no clinical data is available for patients treated with ticagrelor. Since current Euro-pean Society of Cardiology ACS guidelines (1, 2, 10) recommend the use of prasugrel or ticagrelor as first-line treatments and keep clopidogrel only for patients who cannot be treated with novel P2Y12-receptor inhibitors, there is currently no role for routine pla-telet function testing in ACS patients. In selected ACS patients on clopidogrel, the usefulness of platelet function testing in guiding antiplatelet therapy is still uncertain, although the available rando-mised studies were not including high-risk patients, and generally excluded those with STEMI (59, 60). In addition, the economic impact of platelet function testing-guided antiplatelet approach needs detailed evaluation, since the availability of generic clopido-grel may trigger significant cost savings for the tailored approach (68).

In case of high platelet reactivity is revealed, switching over to novel P2Y12-inhibitors is recommended and dose increase in clopidogrel is discouraged (45). In case of an unanticipated bleed-ing event, platelet function testing might help to determine the level of P2Y12-inhibition that may be useful to manage the compli-cation.

Further randomised studies testing the impact of tailored anti-platelet treatment in ACS patients should evaluate whether reach-ing a therapeutic window of P2Y12-inhibition is beneficial to im-prove net clinical benefits; however, until positive results are ob-tained, such attempts should not be part of routine practice. In ad-dition to platelet function studies with prasugrel or ticagrelor, fu-ture trials should focus on the potential value of platelet function testing to minimize bleeding complications in patients treated with a combination of novel or conventional oral anticoagulants and double antiplatelet therapy where the risk of bleeding in par-ticularly high.

Conflicts of interestDA: Consultant for Verum Diagnostica GmbH, lecture fees from AstraZeneca, DSI/Lilly, Roche, Verum Diagnostica. JPC: Research grants Diagnostica Stago and AstraZeneca. BM: MB lecture fees from AstraZeneca, Lilly, Roche. ASJ: Has or presently consults for most of the major diagnostic companies. EG: Consultant to Astra-Zeneca, lecture fees from AstraZeneca. JMtB: research grants from AstraZeneca; speakers fee AstraZeneca, Merck, DSI/Lilly. Consult-ancy fees from AstraZeneca, DSI/Lilly. RFS: research grants from

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229Aradi et al. Platelet function testing in ACS

AstraZeneca and Merck; research support from Accumetrics; hon-oraria from AstraZeneca, Accumetrics, and Medscape; consultan-cy fees from AstraZeneca, Correvio, Accumetrics, Sanofi-Aventis, Regeneron, PlaqueTec, Roche and Daiichi Sankyo. None of the other authors reorts any conflicts of interest.

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Abbreviations

ACS: acute coronary syndrome; ADP: adenosine di-phosphate; ASA: acetylic salicylic acid; BARC: Bleeding Academic Research Consor-tium classification for bleeding types; GPIIb/IIIa: glycoprotein IIb/IIIa antagonists; HPR: high platelet reactivity; LPR: low platelet reactivity; LTA: light transmission aggregometry; PCI: percutaneous coronary in-tervention; PAR-1: protease activated receptor-1; PFT: platelet func-tion testing; POC: point-of-care; PRI: platelet reactivity index; PRU: platelet reactivity unit; STEMI: ST-segment elevation myocardial in-farction; VASP: vasodilator stimulated phosphoprotein phosphory-lation.

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