In keeping with the rising incidence of atherothrombotic disease, reliable platelet function testing has become more important. In acute coronary syndromes (ACS), platelets often play a starring role in the pathogenesis of disease and are a critical target for pharmacotherapy (1,2). In particular, percutaneous coronary intervention (PCI) with coronary artery stents—a widely used intervention to manage ischemic heart disease—requires dual antiplatelet therapy with aspirin and platelet P2Y12 inhibitors to prevent stent thrombosis (3).

Dual antiplatelet therapy has also significantly reduced major cardiac events in patients with ACS. In these cases, platelet function testing helps determine drug efficacy and/or sensitivity, patients’ compliance with medication regimens, and optimal timing of urgent surgery after oral antiplatelet therapy ends (1).

Platelet function assays also have become more important owing to their role in minimizing the side effects of over- or under-treatment. For example, some patients being treated with the P2Y12 inhibitor clopidogrel have the status of “high on-treatment platelet reactivity” (HPR) and are at increased risk for thrombotic events despite being compliant with their treatment regimen (3,5). More potent P2Y12 receptor inhibitors, like prasugrel and ticagrelor, are available, but they come with an increased risk of bleeding (3). Notably, aspirin typically produces more reliable and predictable effects because it delivers a high level of COX-1 inhibition even at low doses, so monitoring response is usually not essential (1).

To balance the risks and benefits of these medications, several platelet function tests are aimed at establishing a therapeutic window for platelet inhibition with the intent of tailoring antiplatelet therapy in the treatment of ACS (3).

Several unique technologies have been devised to assess platelet function by measuring platelet activation and aggregation in response to a variety of agonists (1). This review summarizes different methods of platelet function testing in the setting of antiplatelet therapy.

Methods for Assessing Platelet Function

VerifyNow

The VerifyNow P2Y12 assay is the simplest and most reliable method of evaluating response to P2Y12 inhibitors as well as sensitivity to aspirin. However, evidence is lacking that demonstrates this assay supports improved clinical outcomes when it is used for therapy guidance (1).

VerifyNow uses anticoagulated whole blood for turbidometric detection of platelet aggregation. A single-use cartridge with separate wells contains a chrome-plated mixing ball, fibrinogen-coated beads, and a platelet agonist. Activated platelets bind to nearby platelets via the fibrinogen-coated beads, thereby aggregating both platelets and beads with subsequent reduction in turbidity (and increase in light transmittance). VerifyNow assays are available for aspirin, P2Y12 inhibitor, and GIIb/IIIa inhibitors (1,3,6).

Several studies have assessed the prognostic and clinical uses of VerifyNow on clopidogrel, with the specific intent of establishing numeric thresholds for adequate and inadequate platelet inhibition (1). Marcucci et al. defined HPR while on clopidogrel as P2Y12 reaction units (PRU) ≥240 for patients (n=683) undergoing PCI with bare metal stents and found that value to be a significant and independent predictor of cardiovascular death and nonfatal myocardial infarction (MI) (1).

Similarly, Price et al. used PRU ≥235 as a cutoff for HPR on clopidogrel and found that value to be predictive of stent thrombosis. The ARMYDA-PRO group looked at major adverse cardiovascular events for 30 days in each quartile distribution for PRU and found that adverse events occurred more frequently in patients with PRU levels in the upper quartile than compared with those in the lower quartile. The GRAVITAS study, which involved standard versus high-dose clopidogrel based on platelet function testing after PCI, assessed the efficacy of tailoring clopidogrel dose in a large group of patients (n=5429) with HPR determined by the VerifyNow P2Y12 assay with a PRU >230 cutoff (1). This study found a PRU <208 was associated with significantly lower risk of reaching adverse events at 60 days.

Finally, the ADAPT-DES study exploring platelet reactivity and clinical outcomes after coronary artery implantation of drug-eluting stents also investigated a large cohort (n=8665) that used PRU cutoffs of >208 and >230 with the endpoint of clinically relevant bleeding, stent thrombosis, and MI. The investigators found HPR an independent predictor of stent thrombosis and/or MI in the first 12 months but not a predictor of death.

In contrast to patients with HPR, patients with low platelet reactivity (LPR) have a high risk of significant bleeding events, especially in the setting of PCI. To delineate these risks, clinicians use platelet function testing to target a therapeutic window of platelet reactivity (5). This has been referred to as the optimal level of platelet reactivity (OPR) which is flanked by LPR and HPR cutoffs to prevent bleeding and thrombotic events, respectively. The ARMYDA-PROVE study, which assessed platelet reactivity in response to clopidogrel before elective PCI (n=732), identified more adverse bleeding events at an LPR cutoff of <178 and significantly more ischemic events at an HPR cutoff of >239 (3).

VerifyNow has been used similarly for aspirin therapy, where again, patients with HPR, despite being on standard aspirin treatment, have been identified. These patients have significantly more frequent nonfatal acute MI and ischemic stroke compared with those showing optimal platelet inhibition (5).

Platelet Function Analyzer-100

The Platelet Function Analyzer-100 (PFA-100) is a sensitive screening tool for qualitative platelet defects but is not recommended for monitoring antiplatelet therapy due to the lack of sensitivity and specificity for the effects of aspirin and P2Y12 inhibitors (1).

PFA-100 is a rapid point-of-care test that uses small quantities of whole blood and is reproducible and standardized (1,5). This cartridge-based system includes a capillary, sample reservoir, and aperture containing a membrane coated with either collagen and epinephrine or collagen and adenosine diphosphate (ADP). Citrated blood is aspirated at high shear rates through a disposable cartridge. Functional platelets in whole blood that meet the membrane are activated and aggregate at the aperture.

The PFA-100 endpoint, known as the closure time, is the duration from the start of the test to occlusion of the aperture (1,6). Gianetti et al. found increased platelet activation to be an independent predictor of recurrent ischemic events.

Light Transmittance Aggregometry

Light transmittance aggregometry for many years has been the gold standard in platelet function testing. However, it is no longer used in daily clinical practice for monitoring antiplatelet therapy due to lack of standardization, problems with spurious platelet activation secondary to centrifugation, and the high complexity of the test precluding its use as a point-of-care system (1,3).

This method uses turbidometric optical detection to assess pharmacodynamic response to various agonists. As platelet aggregation occurs in response to the addition of the agonist, the sample becomes more translucent and light transmittance increases. Common agonists include ADP, epinephrine, collagen, thrombin receptor-activating peptide, arachidonic acid, and ristocetin.

Light transmittance aggregometry allows quantification of the final common pathway of platelet aggregation via GPIIb/IIIa-dependent bridging (1,3,6). Several studies have shown a relationship between HPR and risk of future atherothrombotic events. Despite its limitations, the method may still provide prognostic information, especially in patients taking clopidogrel.

Vasodilator-Stimulated Phosphoprotein Phosphorylation Assay

The vasodilator-stimulated phosphoprotein (VASP) assay is a flow cytometric test that indirectly measures P2Y12 receptor inhibition. The assay uses intracellular fluorescently labeled antibodies against phosphorylated vasodilator-stimulated phosphoprotein. VASP is an intracellular actin regulatory protein that is normally unphosphorylated in resting conditions, and its phosphorylation is regulated by the cyclic adenosine monophosphate (cAMP) cascade. Stimulation of the P2Y12 receptor by ADP leads to inhibition of adenylate cyclase and reduction in cAMP levels via prostaglandin E1 (PGE1) activation. Persistence of PGE1-stimulated VASP phosphorylation in the presence of ADP correlates with P2Y12 receptor inhibition (1).

The VASP assay uses whole blood mixed with PGE1 or PGE1 and ADP incubated at room temperature. The specimen is then fixed with a paraformaldehyde solution and the cells permeabilized. Following incubation with fluorescently labeled antibodies, the amount of phosphorylated VASP is measured by flow cytometric detection of the bound fluorophore.

Of note, this assay is insensitive to low levels of P2Y12 receptor inhibition and can lead to an inappropriately high number of patients classified as HPR (1). Bonello et al. used VASP phosphorylation analysis to demonstrate the association between clopidogrel resistance and increased major adverse cardiac events. This method requires skilled personnel and specialized equipment and has variability in its results (1).

Whole Blood Impedance Aggregometry

Whole blood impedance aggregometry using the Multiplate analyzer with ADP is one of the recommended near-patient devices for monitoring platelet inhibition during P2Y12 inhibitor therapy (5). The Multiplate device uses citrated whole blood and measures increases in electrical impedance in aggregation units as activated platelets attach and coat electrodes (1,3,4,6). This rapid and comprehensive platelet function test involves adding platelet agonists manually and is prognostically useful in clopidogrel-treated patients undergoing PCI. However, evidence is lacking that shows it improves clinical outcomes when used for guiding therapy (1).

Thromboelastography/Thromboelastometry

Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) technologies use whole blood to measure dynamic hemostatic changes in clot formation. Whole blood is placed in a cup with a suspended pin that is connected to a computer (7). ROTEM uses an oscillating pin to measure resistance during clot formation. This resistance is interpreted as a curve that describes the viscoelastic properties during clot initiation to clot termination. In TEG, the cup oscillates during clot formation and this movement detects increased resistance. TEG and ROTEM may be used as rapid tests to assess patients’ bleeding due to antiplatelet therapy. Both tests also assess platelet inhibition in the context of evaluating drug efficacy or sensitivity (7).

PlateletMapping is an additional technology in the TEG system that measures platelet function in the presence of antiplatelet therapy. This assay uses arachidonic acid and ADP as agonists. TEG PlateletMapping compares standard TEG results in fully activated blood—where thrombin causes full platelet activation—with TEG results from blood activated with a combination of snake venom and a weak platelet agonist such as ADP or arachidonic acid (the venom converts fibrinogen to fibrin) (8). Studies evaluating TEG in the setting of anti-platelet therapy have shown varying ability to predict bleeding tendency (8).

Conclusion

With the standard use of dual anti-platelet therapy in atherothrombotic disease and specifically ACS, laboratories have developed numerous methods for assessing platelet function (see Table 1). While the effects of aspirin are typically reliable and the need to monitor its response less significant, platelet function testing may help assess adherence to therapy. Conversely, patients’ variable responses to clopidogrel is one of this therapy’s limitations. HPR while on this drug is associated with major adverse cardiovascular events.

The best assays to monitor clopidogrel therapy include VerifyNow and Multiplate ADP test. However, due to the lack of support by clinical trials, these tests are not currently recommended for routine use in certain situations, such as in low-risk PCI patients and in higher risk patients transitioning from clopidogrel to potent drugs such as ticagrelor and prasugrel. These studies detected no differences regarding hard clinical end points.

Furthermore, randomized studies on individualized antiplatelet therapy based on platelet function testing have many limitations, including enrollment of only low- to intermediate-risk cohorts and limited use of potent antiplatelet agents.

Tailoring antiplatelet therapy in stable (low-risk) PCI patients is difficult as this cohort already has a low frequency of atherothrombotic events. However, there is a great need for studies that focus on high-risk populations, including patients who may need to replace clopidogrel with more potent drugs.

Overall, the prognostic value of platelet function testing for risk prediction of ischemic and bleeding events when using P2Y12 receptor inhibitors is well established. Determining a therapeutic window of platelet inhibition should be the primary goal of these assays to help guide the choice of antiplatelet therapy and prevent complications.

Maggie Yell, MD, is the hematopathology fellow at West Virginia University in Morgantown.+Email: myell@hsc.wvu.edu

Jeffrey A. Vos, MD, is the program director for the hematopathology fellowship and pathology residency programs at West Virginia University in Morgantown.+Email: jvos@hsc.wvu.edu

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