Laboratory Monitoring of Platelet P2Y12 Receptor Inhibitors and Reversal of Antiplatelet Agents

An ACLPS Critical Review

Lisa Senzel, MD, PhD; Tahmeena Ahmed, MBBS; Eric D. Spitzer, MD, PhD


Am J Clin Pathol. 2019;152(1):1-6. 

In This Article

How P2Y12 Inhibitors Work

Activation of the platelet P2Y12 purinergic receptor inhibits adenylyl cyclase, causing an increase in intracellular Ca2+ levels and a decrease in both cAMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) levels. These changes promote platelet aggregation by altering the ligand-binding properties of the glycoprotein IIb/IIIa receptor. Inhibition of the P2Y12 receptor therefore suppresses platelet activation.

Clopidogrel is a prodrug that requires a two-step oxidation process with multiple hepatic cytochrome P-450 (CYP) isoenzymes, mainly CYP2C19, to generate the active thiol metabolite that irreversibly blocks the adenosine diphosphate (ADP) binding site on the P2Y12 receptor. Prasugrel is also a prodrug but is more efficiently activated compared with clopidogrel because only a single-step hepatic CYP oxidation is required. The active metabolite of prasugrel achieves a higher plasma concentration, which translates into more prompt, potent, and predictable platelet inhibitory effects compared with clopidogrel. Ticagrelor is a direct-acting (no metabolism required) oral agent that reversibly inhibits the P2Y12 receptor.

Prasugrel and ticagrelor are characterized by enhanced pharmacodynamic effects compared with clopidogrel. Therefore, switching from a less intensive to a more intensive agent (ie, clopidogrel to prasugrel or ticagrelor) is defined as escalation, and the reverse is defined as de-escalation.[1]

Platelet Function Testing in Patients With Cardiovascular Disease

Platelet P2Y12 receptor inhibitors are routinely prescribed for patients after acute coronary syndromes and percutaneous coronary interventions (eg, placement of stents). POC tests for antiplatelet agents are intended to determine whether the patient shows impaired drug response (resistance), known as high on-treatment platelet reactivity (HPR), which has been associated with ischemic complications in some studies but not others and with variable results among the different methods. Strategies to address HPR include increasing clopidogrel dose or escalating to more potent P2Y12 inhibitors such as ticagrelor or prasugrel. While earlier nonrandomized studies suggested a benefit to personalized antiplatelet therapy based on POC assessment of platelet function, three large randomized prospective trials have failed to demonstrate that such a strategy can reduce cardiac ischemic events.[2] Current evidence does not support changing antiplatelet therapy based on the results of platelet function monitoring tests.[3] High on-treatment platelet reactivity may be a nonmodifiable clinical risk factor in clopidogrel-treated patients. POC testing may be used to allow earlier cardiac operation for a patient stopping a P2Y12 inhibitor before the number of days listed in the drug labeling. Platelet function testing may be useful in helping determine the timing of cardiac surgery following recent discontinuation of a P2Y12 inhibitor in patients with urgent indications for surgery, based on small studies[4,5] involving patients undergoing coronary artery bypass grafting (reviewed in Michelson and Bhatt[2]).

Platelet Function Testing for Neurointerventional Procedures

In the neurointerventional setting, P2Y12 receptor inhibitors are used to reduce the risk of platelet activation when intracranial stents are placed, since they can act as a nidus for thrombosis and subsequent thromboembolic stroke. One retrospective study looked at the pipeline embolization device, a flow-diverting stent to treat intracranial aneurysms.[6] This device provides a scaffold for endothelialization to remodel the parent artery and ultimately excludes the aneurysm from circulation. Due to the high surface coverage area compared with other stents designed for intracranial use, adequate platelet inhibition is particularly important. In the study, clopidogrel nonresponders experienced a significantly higher rate of thromboembolic complications compared with clopidogrel responders. Notably, this risk seemed to be mitigated in nonresponders who were switched to ticagrelor or received a clopidogrel boost within 24 hours preprocedure. Since neurointerventional patients are few compared with coronary disease populations, this study may not be feasible to perform as a large, prospective randomized trial.

Antiplatelet Drug Reversal in Emergencies

Current neurocritical care guidelines[7] suggest platelet transfusion for patients with aspirin- or ADP inhibitor–associated intracranial hemorrhage who will undergo a neurosurgical procedure. In contrast, they suggest against platelet transfusion for patients with antiplatelet-associated intracranial hemorrhage who will not undergo a neurosurgical procedure. In nonsteroidal anti-inflammatory drug or GP IIb/IIIa inhibitor–related ICH, they suggest against platelet transfusion even in the context of neurosurgical intervention. When platelet transfusion is recommended, the suggested initial dose is one single-donor apheresis unit of platelets. Platelet testing is suggested prior to repeat platelet transfusion, if available. Repeat transfusion should be used only for those with persistently abnormal platelet function tests and/or ongoing bleeding, but the utility of these tests in making transfusion decisions has been marginally shown only for aspirin[8] and not for P2Y12 inhibitors. Typical platelet doses for patients on antiplatelet agents requiring urgent surgery or presenting with bleeding would be one apheresis unit or two units for those patients requiring urgent neurosurgery or eye surgery or having ICH.[9]

PATCH [platelet transfusion vs standard care after acute stroke due to spontaneous cerebral hemorrhage associated with antiplatelet therapy][10] was the first randomized trial investigating the issue of platelet transfusion in spontaneous ICH following use of antiplatelet therapy and was driven by the hypothesis that platelet transfusion would reduce the risk of death and dependence compared with standard care. However, the results showed the opposite: the risk of death and dependence at 3 months was higher in patients randomly assigned to administration of platelets than in those assigned to standard care. The effect was consistent across all predefined subgroups and remained even after adjusting for potential modifying and confounding factors.[11] Serious adverse events, mainly ICH hemorrhage growth or infections, were higher in participants who received platelet transfusion: 40 (42%) transfused patients had serious events compared with 28 (29%) participants who received standard care. There was not a significant difference between groups in thromboembolism, but four people in the transfusion group had an event vs one in the standard therapy group. The study involved mainly patients taking aspirin, with a minor population taking dipyridamole and only a handful of patients taking P2Y12 inhibitors. Possible explanations for why platelet transfusion appeared to worsen outcomes in ICH include the following: platelet concentrates contain cytokines, which may potentiate the proinflammatory response to ICH and may cause prothrombotic tendency; transfused platelets vary in aggregation and thrombin generation capabilities; and transfused platelets can suppress new platelet synthesis, possibly decreasing the fraction of young (and most hemostatically effective) platelets.[11] In addition, platelet transfusions may lead to the volume loading and increased intracranial venous pressure that could worsen bleeding; this may be more relevant when the cranium remains "closed" vs when the cranium is "open" during neurosurgery and there is an exit for increased volume/pressure.

What do Platelet Function Tests Actually Measure?

Platelet function tests (PFTs) for P2Y12 blockers can measure either the degree of ADP-induced aggregation or the phosphorylation status of VASP, an intracellular marker for residual P2Y12 receptor activity. ADP-induced light transmission aggregometry (LTA) and VASP phosphorylation by flow cytometry both require extensive laboratory expertise and resources, and they are time-consuming. The VerifyNow P2Y12 assay uses a semiautomated instrument that does not require manual pipetting or specimen preparation and can be performed at the point of care. Other modalities, including multiplate (MP) impedance aggregometry (Roche Diagnostics) and thromboelastography, are also semiautomated.

VerifyNow P2Y12 is based on the ability of activated platelets to bind fibrinogen. Fibrinogen-coated microparticles aggregate in whole blood in proportion to the number of expressed platelet GP IIb/IIIa receptors. The reagent ADP is used as an agonist. Light transmittance increases as activated platelets bind and aggregate fibrinogen-coated beads. The instrument measures this change in optical signal and reports results in PRUs. Older studies using VerifyNow P2Y12 report additional parameters, including baseline PRUs and percent inhibition, based on a separate channel using the thrombin receptor agonist peptide. VerifyNow P2Y12 changed its reporting format in 2012 to a single PRU value with a normal reference range of 194 to 412 PRUs. Notably, this reference range contains the commonly used cutoff for HPR (clopidogrel resistance)—namely, 208 PRUs.[3] Using this cutoff, since higher PRU values represent higher platelet reactivity, values above 208 PRUs represent clopidogrel resistance. A test result between 194 and 208 PRUs could represent either a patient on no drug or a patient who has achieved target levels of clopidogrel. One explanation for this overlap could be that healthy volunteers can have impaired platelet function due to ingestion of medications, garlic, ginger, fish oil, or other foods or supplements. The manufacturer states that interferences include low hematocrit or platelet count and use of GP IIb/IIIa inhibitor drugs but no other medications.

ADP stimulates platelet aggregation through its interaction with two receptors, P2Y1 and P2Y12. Interaction with P2Y1 stimulates platelet shape change while interaction with P2Y12 is needed for continued activation of the GP IIa/IIIb receptor that binds fibrinogen.[12] Importantly, VerifyNow P2Y12 uses prostaglandin E1 (PGE1) to block P2Y1-mediated aggregation, so that only P2Y12-mediated aggregation is measured. In contrast, standard aggregometry using ADP as an agonist will measure both P2Y1- and P2Y12-mediated aggregation. This may explain why some patients appear sensitive to clopidogrel by VerifyNow P2Y12 but seem resistant to clopidogrel when tested by LTA or whole-blood impedance aggregometry (WBA).[13] The aggregometer registers a higher level of platelet reactivity because ADP is stimulating both its P2Y1 receptor and its P2Y12 receptor; the VerifyNow P2Y12 registers lower platelet reactivity because ADP is prevented (by PGE1) from stimulating its P2Y1 receptor. Higher reactivity is interpreted as drug resistance. Levels of PGE1 can vary between patients due to the effects of COX-1 and/or COX-2 inhibitor medications, as well as complex biological mediators.[14] Figure 1 shows WBA tracings from two patients who appear resistant to clopidogrel because their platelets aggregate in response to ADP, exceeding the 5-ohm cutoff. For the patient in the upper tracing, the same-day VerifyNow P2Y12 result was 119 PRUs, which is well within the drug-sensitive cutoff of less than 208 PRUs. It is possible that P2Y12 receptor signaling was inhibited, while P2Y1 receptor signaling was active and affected the WBA result but not the VerifyNow result. In contrast, the patient in the lower tracing had a VerifyNow P2Y12 result of 241 PRUs, indicating possible clopidogrel resistance. For this patient, it is possible that P2Y12 receptor signaling was incompletely blocked by clopidogrel.

Figure 1.

Whole-blood impedance aggregometry (WBA) tracings for two patients on dual antiplatelet therapy with aspirin and clopidogrel, showing apparent clopidogrel resistance. Aggregation to arachidonic acid is completely suppressed (trace 5), suggesting full aspirin effect, but aggregation to adenosine diphosphate greatly exceeds a cutoff of 5 ohms (traces 1 and 3), suggesting clopidogrel resistance. A, The patient's VerifyNow P2Y12 result was 119 P2Y12 reaction units (PRUs), indicating full clopidogrel effect; see discussion in text regarding this discordance. B, In contrast, the patient's VerifyNow P2Y12 result was 241 PRUs, indicating clopidogrel resistance, concordantly with the WBA result.

Large randomized trials that failed to demonstrate superiority of tailored antiplatelet regimens (ARCTIC [assessment with a double randomization of (1) a fixed dose vs a monitoring-guided dose of aspirin and clopidogrel after drug-eluting stent implantation and (2) treatment interruption vs continuation, 1 year after stenting], TRIGGER-PCI [testing platelet reactivity in patients undergoing elective stent placement on clopidogrel to guide alternative therapy with prasugrel],[15] and GRAVITAS [gauging responsiveness with a VerifyNow P2Y12 assay: impact on thrombosis and safety]) used the VerifyNow P2Y12 assay. More recently, TROPICAL-ACS [testing responsiveness to platelet inhibition on chronic antiplatelet treatment for acute coronary syndromes][16] investigated the safety and efficacy of early de-escalation of antiplatelet treatment from prasugrel to clopidogrel guided by PFT using MP impedance aggregometry. TROPICAL-ACS met its noninferiority goals and thus demonstrated that, in patients with acute coronary syndrome undergoing percutaneous coronary intervention, PFT-guided selection of P2Y12-inhibiting therapy could represent an alternative approach to a standard strategy of prasugrel use. Thus, TROPICAL-ACS suggested a "potential resurgence of a nearly abandoned instrument"—referring to PFT in general, not specifically MP, but raising the question of whether MP might be the "right" PFT.[17] While the US Food and Drug Administration–approved version of MP is not currently available in the United States, many laboratories, including ours, perform WBA (Figure 1). Both MP and WBA measure the electrical resistance (impedance) between two sensor electrodes when platelets adhere and aggregate in response to agonists.[18] WBA reports the result in ohms for each agonist. MP reports arbitrary aggregation units (AUs), where approximately 8 AUs correspond to 1 ohm. MP also reports an area under the curve, which depends on both the total height of the aggregation curve and its time course. Because they use whole blood, MP and WBA are faster and require less preanalytical work than LTA. LTA requires preparation of platelet-rich plasma to a standardized concentration before agonists are added. LTA reports percent maximum aggregation, which is the difference in light transmittance before vs after platelets aggregate.

Helten et al[19] compared PFT results on a group of 23 patients receiving dual antiplatelet therapy with aspirin and clopidogrel using three methods: ADP-induced LTA, MP, and VASP. With LTA, HPR occurred in 57% of patients; with VASP, it occurred in 43% of patients; and with MP, it occurred in 13% of patients. Remarkably, in 17% of patients, one assay showed HPR, whereas another showed low platelet reactivity (LPR), suggesting a risk of bleeding. Aradi et al[20] conducted a collaborative analysis of 17 studies including 20,839 patients, of whom 97% were treated with clopidogrel and 3% with prasugrel. Eight studies used VerifyNow, five used VASP, and four used MP. Aradi et al[20] confirmed the large methodical heterogeneity in assessing on-treatment platelet reactivity and the lack of generally applicable cutoff values to define patients with HPR and LPR. They proposed cutoffs to define an intermediate range of platelet inhibition, called "optimal," and suggested that conclusions of several of the studies would be different using these cutoffs.

Restoration of Platelet Function With Platelet Transfusion

In the APTITUDE [antagonize P2Y12 treatment inhibitors by transfusion of platelets in an urgent or delayed timing after acute coronary syndrome][21] study, both ex vivo and in vivo platelet transfusions in patients exposed to P2Y12 receptor blockers had a significant effect on restoration of platelet function measured by VASP, LTA, or thromboelastography. Overall restoration of platelet function was dependent on the potency of P2Y12 inhibition, with the most restoration for clopidogrel and the least for ticagrelor. However, platelet transfusions had no effect on restoration of platelet function measured by VerifyNow P2Y12. The authors comment that the VerifyNow assay was not designed for this setting, and LTA was not possible in some cases because of low platelet count and hemolysis of the sample cause by cardiopulmonary bypass. Similarly, Choi et al[22] found that only 33% of patients with traumatic brain injury after taking clopidogrel had reversal of platelet dysfunction on VerifyNow P2Y12 after platelet transfusion.

Bertling et al[23] examined the effects of P2Y12 blockers on healthy donor platelets by flow cytometry assessment of fibrinogen binding and externalization of CD62P in response to ADP. They showed that platelets from clopidogrel- or prasugrel-treated patients did not suppress donor platelet function upon mixing. In contrast, platelets from ticagrelor-treated patients did suppress donor platelet function, suggesting the transfer of ticagrelor from patient platelets to donor platelets. Thus, while persistent effects from ticagrelor might compromise the effectiveness of platelet transfusion therapy, their results did not support a similar concern for clopidogrel or prasugrel.