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Clopidogrel Platelet Function Tests

Caveats and Controversies

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Published: December 2011

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Clopidogrel, also known as Plavix, is an antiplatelet prodrug used to block platelet activation and aggregation and inhibit thrombosis. The drug is metabolized in the liver by the enzyme CYP2C19 and polymorphisms of the CYP2C19 gene influence enzyme function and drug metabolism. Platelet functions studies can determine the drug’s effect and genetic testing is available to determine which polymorphisms are present in the patient’s genes. Both may be important in management of patients treated with clopidogrel.

Presenter: Gretchen Johns, MD

  • Laboratory Section Director for Coagulation
  • Medical Director for the Department of Laboratory Medicine and Pathology at Mayo Clinic in Jacksonville, Florida.

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Gretchen Johns, M.D., Laboratory Section Director for Coagulation and Medical Director for the Department of Laboratory Medicine and Pathology at Mayo Clinic in Jacksonville, Florida. Dr. Johns will describe the various testing options for patients on the commonly prescribed medication clopidogrel, including testing for genetic polymorphisms. Thank you, Dr. Johns.

Thank you, Sharon, for that introduction.

Learning Objectives

The objectives of this presentation include an overview of the currently available testing for platelet function in patients on clopidogrel, which has the trade name Plavix in the United States. I will discuss briefly some of the characteristics of each test and how to select testing appropriate for your laboratory. I will review 2 years of data concerning clopidogrel resistance testing at our institution in Jacksonville, Florida. In conclusion I will describe the controversy concerning genetic testing for polymorphisms in the P450 gene thought to be primarily responsible for clopidogrel metabolism.

Platelet Function Tests for the Detection of Clopidogrel Resistance

This slide and the next slide contain the names of platelet function tests which can be used to test for clopidogrel resistance. Clopidogrel acts by irreversibly blocking the ADP receptor P2Y12 on platelets. The first group of tests measures change in direct response to ADP stimulus to determine the effect or lack of effect of clopidogrel on the platelets. The gold standard platelet function test currently is platelet aggregometry, also known as light transmission aggregometry or LTA. Multiplate whole blood aggregometry is a newer variation that can be done without centrifuging the blood and manipulating it to achieve platelet rich plasma. Flow cytometry can test for markers of activation on the platelet surfaces following exposure to ADP. The VerifyNow assay from Accumetrics also detects whole blood platelet aggregation but uses optical detection rather than impedance.

The Platelet Function Analyzer or PFA-100 measures the blockage of a membrane coated with collagen and ADP by platelets, mimicking damaged endothelium. Plateletworks is a unique method that utilizes a special ADP tube which is run through the laboratory’s regular CBC impedance analyzer; the platelet count result from this tube is then compared to the patient’s platelet count from a routine EDTA tube which is run immediately prior to the special ADP tube. The Impact cone and plate(let) analyzer has only recently become available for commercial use and measures the amount of stained surface area and average size of platelet aggregates coating a special thrombogenic plate. Thromboelastography has been available for years, but its use for platelet mapping is much more recent. The RoTEM is a newer rotational form of thromboelastography which has been recently introduced to the U.S. The last test uses detection of the downstream effect of ADP receptor stimulation by measuring the amount of phosphorylated vasodilator-stimulated phosphoprotein using flow cytometry.

Caveat: There Is No Perfect Platelet Function Test...

There is no widely accepted test for platelet function (for patients on or off of clopidogrel) except for light transmission aggregometry (LTA), which has limited availability, is time-consuming, requires skilled technologists and is fairly expensive.

Problems With Testing For Clopidogrel

Other problems associated with clopidogrel testing include the lack of universally accepted cutoff values for resistance, lack of standards available from national or international organizations, interference with platelet function testing from many medications and the fact that the available testing predicts thrombotic events more accurately than the risk of bleeding.

How We Choose a Testing Method

With a wide variety of tests available for platelet function, a good place to start selection of a test methodology is to find out what test(s) are used most frequently in studies performed by the clinical service likely to utilize the testing at your facility. In our case this was the neurosurgery and neurology departments and the VerifyNow test was the most frequently quoted in their literature. Next you would like to see results of the test you are considering compared with the gold standard for testing, ideally with a favorable result. If such a study is not available, you might get the instrument on loan to do the study in-house if you have access to the gold standard test method. Finally, there are multiple practical issues that will influence you selection. FDA approval makes validation and billing easier. The turn around time, need for 24 hours testing and the need for point of care availability may be discussed with the departments utilizing the testing. The cost of reagents or cartridges as well as the original purchase price of the instrument may affect your selection. Contacting laboratories which are already utilizing this method can provide insight into problems or advantages to this method of testing.

Methodology Table

This table summarizes the platelet function tests by FDA approval, ability to test whole blood without manipulation and difficulty of testing (both of which could limit time of availability) and the turn around times. If FDA approval is important for your institution, you may wish to wait until approval is granted before purchasing an instrument with FDA approval pending.

Platelet Function Testing Available at Mayo Clinic Florida

In Mayo Clinic Florida, we have several tests available for platelet function testing. All evaluations of platelets should start with a CBC and peripheral blood smear to evaluate platelet number, morphology and granulation, as well as the morphology of the WBCs and RBCs. LTA is the gold standard and is frequently used to evaluate patients with congenital or acquired platelet abnormalities. The VerifyNow test is frequently utilized to detect resistance in patients who receive a loading dose of clopidogrel in preparation for neurosurgery such as a stent placement or for patients who have a thrombotic event while taking clopidogrel. PFA-100 is used in patients who are undergoing surgery and have a history of bleeding. Thromboelastography is used in liver transplant and other critical patients, but our institution does not currently do platelet mapping.

Light Transmission Platelet Aggregometry Studies (LTA)

Light transmission platelet aggregometry (LTA) is the gold standard of platelet function testing. It has been used for several decades for detecting hereditary platelet abnormalities such as Bernard Soulier syndrome, Glanzmann’s thrombasthenia and von Willebrand disease. Because it is time and labor intensive, it is seldom used for monitoring resistance to clopidogrel. Testing is most accurate when the patient stops using all medications prior to collection, particularly any anticoagulants. This is true of all platelet function testing (except the specific drug when testing for resistance) and is not always possible due to the patient’s co-morbidities.

Platelet Aggregation (Cont'd)

LTA uses multiple agonists to stimulate platelet aggregation and the timing, slope and percent of maximum aggregation are reviewed by a pathologist in interpreting the results.

The blood specimen cannot be collected at an offsite facility and shipped to the testing site, due to nonspecific activation of platelets. The testing is usually only performed during limited hours on dayshift in a special coagulation laboratory using highly skilled personnel.

Normal Patient

Patients with normal platelet function should reach at least 70% light transmission at the point of maximum aggregation, with no delay or reversal in platelet aggregation. Different concentrations of agonists may be used.

Accumetrics VerifyNow Test

The Accumetrics VerifyNow test uses whole blood to detect platelet aggregation optically. It is easy and quick to perform, available 24 hours a day and is moderate in cost. It is used for monitoring the effect of clopidogrel on platelets, either to find resistance in patients given a loading dose in preparation for surgery or in patients having a thrombotic event while using clopidogrel.

VerifyNow P2Y12 Test

The results are given as three numbers. The first number is a measure of the patient’s platelet function in the presence of clopidogrel. The second number measures activation of the patient’s platelets independently of the P2Y12 receptor using 2 reagents within a second channel. The last number calculates the percent inhibition of the patient’s platelets by clopidogrel using the first two numbers. Less than twenty to thirty percent inhibition of platelet activation is considered to be resistance to clopidogrel. Some studies and institutions use values over 235 P2Y12 Reaction Units (PRU) as indicative of clopidogrel resistance.

P2Y12 Test Interferences

Multiple factors can interfere with the P2Y12 testing including inherited platelet disorders and the presence of glycoprotein IIb/IIIa inhibitors or cilostazol in the bloodstream. Cartridges are also available for aspirin and glycoprotein IIb/IIIa inhibitor testing.

P2Y12 Antagonists

New P2Y12 antagonists have recently been introduced. Two years ago prasugrel was FDA approved and in July 2011, ticagrelor was FDA approved as well. Ticagrelor is the first direct antagonist, meaning that it does not require metabolism by the liver to become active as the prodrugs ticlopidine, clopidogrel and prasugrel do to form an active metabolite. Ticagrelor is also the first reversible P2Y12 antagonist. Other antagonists which can be given intravenously are now under investigation.

Can These Platelet Function Tests Be Used for Ticagrelor (Brilinta/Brilique)?

Two years ago we were concerned if prasugrel could be followed using our platelet function testing; now several studies have confirmed this. Today we have the same concerns about ticagrelor which is the latest P2Y12 antagonist available. Light transmission aggregometry, VerifyNow and VASP tests were used in three studies of ticagrelor, which look promising but data is currently limited.

PFA-100

The PFA-100 is designed to measure platelet adhesion and aggregation under high shear conditions, mimicking injured endothelium. The time necessary to block a membrane coated in either collagen and epinephrine or collagen and ADP is measured and called the closure time. The collagen/ADP cartridge would be the one used for P2Y12 antagonist testing.

The PFA-100 can be run 24 hours a day, is relatively quick and easy to operate and is moderately priced. It is fairly adept at screening patients for mild congenital bleeding disorders such as Type 1 von Willebrand disease. It is also useful for following aspirin resistance or looking for platelet dysfunction from other medications. However, the results with clopidogrel were variable and it was not recommended for this purpose.

INNOVANCE PFA P2Y

Recently a new cartridge became available in Europe for testing for the P2Y12 antagonists. This test is similar to the current PFA-100 tests in that it uses whole blood and a small sample. It does have an incubation step but the test is fairly rapid. A small correlation study has been performed with the gold standard LTA and also with VASP. It appears most useful for detection of poor responders of clopidogrel metabolism. The updated model of the instrument is called the PFA-200 and may soon be available in the U.S.

TEG — Platelet Mapping

Thromboelastography has been around for several years with some improvements over time. A new use for TEG is called platelet mapping. It requires two TEG instruments per patient test and skilled personnel to run this. It is moderate in cost and may be useful if your institution has the extra capacity to use your TEGs for this testing.

Results of VerifyNow Testing

This slide summarizes a little over 2 years of VerifyNow testing in Mayo Clinic Florida, looking at selected patients suspected of being resistant to clopidogrel or who received a loading dose of clopidogrel in preparation for surgery, often for stent placement with the goal of prevention of stent thrombosis. If 20% or less inhibition is used as the cutoff for clopidogrel resistance, 37% of the patients tested were found to be resistant out of over 750 patients tested. If however 30% was used as the cutoff for resistance, slightly over 50% of patients were resistant.

2009 – 2011 Clopidogrel Response at 30%

This is a graphic representation of the same data. The percent resistance was fairly consistent from year to year. The number of tests performed doubled from 2009-10 to 2010-11.

Genetic Testing: Clopidogrel Metabolism

Genetic testing is available for identifying the patient’s CYP2C19 genotype. As with all genetic testing, informed consent for DNA testing is required. The turnaround time is approximately one week and the testing is fairly expensive currently. Patients who have undergone liver or bone marrow transplantation or have recently received non-leukoreduced blood products require special consideration.

Clopidogrel Metabolism

Clopidogrel is a prodrug that is metabolized to the active metabolite in the liver by 2 oxidation steps mainly using the enzyme produced by the CYP2C19 gene, which is part of the cytochrome P450 system. Several polymorphisms of this gene exist which cause patients to have less function (more common) or more function of their enzyme and hence less or more amount of the active drug present within their bloodstreams. Genetic testing is available to determine which polymorphisms are present in the patient’s genes.

Number of Gene Polymorphisms

The normal genotype for the CYP2C19 gene is designated as CYP2C19*1. Patients who possess two copies of this have normal metabolism of clopidogrel, which is referred to as extensive metabolism. Most of the polymorphisms of the CYP2C19 gene result in less activity of the enzyme and patients with one copy of the normal genotype and one of the polymorphisms have decreased metabolism of clopidogrel which is called intermediate metabolism. Patients with two copies of the same polymorphism or one copy each of two different polymorphisms may have markedly decreased activity and are known as poor metabolizers. CYP2C19*2 is the most common of the polymorphisms and both CYP2C19*2 and CYP2C19*3 result in no or very little activity of the enzyme produced by that gene.

Clopidogrel Metabolism and Genetic Polymorphisms

The CYP2C19 gene polymorphisms are thought to account for about 12% of the variability in clopidogrel metabolism. Other genetic polymorphisms also affect the metabolism and these include polymorphisms in the gut, liver and platelets. The ABCB1 polymorphism decreases drug absorption in the duodenum, the CYP2C19*17 polymorphism increases metabolism of clopidogrel, the CYP3A5*3 and other CYP genes polymorphisms influence clopidogrel metabolism and the ADP receptor gene P2RY12 platelet polymorphismdecreases effectiveness of the drug.

Why Do Genetic Testing?

Patients with two loss of function alleles are at risk of experiencing thrombosis while taking clopidogrel. Increasing the dose for these patients may not be adequate and they may require a change of medication. The number of poor metabolizers is approximately 4% of European Americans and African Americans and is somewhat higher in Asian Americans. People from Polynesia or Micronesia have a much higher likelihood of being poor metabolizers.

Clopidogrel Genetics (Cont'd)

Several medications are also metabolized by the CYP2C19 enzyme and would be affected by these CYP2C19 gene polymorphisms as well. Prasugrel, like clopidogrel, is a prodrug converted in the liver to the active metabolite, but prasugrel uses multiple different CYP enzymes and thus is not affected by the polymorphisms of the CYP2C19 gene.

Caveats — Other Reasons for Poor Response to Clopidogrel

There are several other factors which are known to affect clopidogrel metabolism. Smoking, caffeine and use of the herb St. John’s wort will affect clopidogrel metabolism. Medications that are metabolized by CYP enzymes other than the CYP2C19 enzyme can cause variability in clopidogrel metabolism. Some medications are known to increase the activity of the CYP2C19 enzyme, while other will decrease the activity of the enzyme. Both diabetes and high body mass index are independent risk factors for poor metabolism of clopidogrel regardless of CYP2C19 status. There is another enzyme which may affect clopidogrel variability more than the CYP2C19 enzyme and this is the paroxonase-1 (PON1) enzyme.

CYP2C19 Polymorphism May Require Another Factor

Not all patients with CYP2C19 polymorphisms are resistant to clopidogrel. Of 6 patients with one polymorphism at our institution, only 2 were resistant by VerifyNow testing. In one study, even some patients with 2 polymorphisms were not resistant by VASP testing. Studies have shown that patients with diabetes, high BMI or age over 70 years with a CYP2C19 polymorphism have poorer metabolism of clopidogrel than expected from the CYP2C19 polymorphism alone. We have already mentioned the platelet ADP receptor polymorphism, the duodenal absorption polymorphism and the PON1 enzyme activity and these in combination with a CYP2C19 polymorphism, may decrease clopidogrel metabolism more profoundly.

Controversy for Clopidogrel

In March 2010, the FDA added a "Boxed Warning" to the drug label for clopidogrel based on a study of healthy subjects – 10 each of ultrarapid, extensive (normal), intermediate and poor metabolizers.

Warning: Diminished Effectiveness In Poor Metabolizers

The warning from the FDA stated that clopidogrel metabolism depends primarily on the enzyme CYP2C19. Patients who were poor metabolizers treated with clopidogrel at the usual dose were at higher risk for a cardiovascular event after acute coronary syndrome or percutaneous coronary intervention than patients with normal metabolism. There is genetic testing available to determine the patient's CYP2C19 genotype and aid in guiding therapy. Alternative dosing or drug treatment should be considered in patients identified as poor metabolizers.

What Does Warning Mean For Clinicians With Patients On Clopidogrel?

The FDA did not clarify what this warning meant for patients or clinicians. Should all patients on clopidogrel be tested or just new patients? Perhaps only patients with an adverse event while on clopidogrel should be tested or maybe all patients should be screened with a platelet function test first and the ones deemed resistant could go on to genetic testing. I sent this question along with several other questions to the FDA to the email address they provided in their boxed warning bulletin.

FDA Answer

The FDA replied that they were not making any recommendation concerning the use of genetic testing; they were only informing health care professionals that such genetic testing is available to identify CYP2C19 polymorphisms and were leaving genetic testing decisions up to the practitioner’s professional judgment.

More Questions

Likewise, the FDA was not making any recommendations regarding the legal or financial implications of the decision to use genetic testing. However, we as health care practitioners must consider the best strategy for our patients and for the use of genetic testing. Many times genetic testing is not covered by insurance or government agencies, leaving a large financial burden for the patient and the family.

Summary

In summary, there is no perfect test for platelet function. Light transmission aggregometry is the gold standard test but has time and availability limitations. Genetic testing can identify poor metabolizers of clopidogrel, but multiple other factors contribute to the variability in response as well. Prasugrel and ticagrelor are newer medications which are believed to be less affected by genetic polymorphisms, but are more costly. Prasugrel also has some increased risk of bleeding. Clopidogrel may become generic next spring leading to a tremendous decrease in the price of this medication.

References

The final slide shows selected references that were used in preparation of this presentation and I thank you for your attention.

Disclosure

I have no financial disclosures pertaining to the content of this presentation. I discussed several instruments that are not currently FDA-approved.

 


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