June 2007 Clinical Laboratory News: Aspirin Resistance

 
June 2007: Volume 33, Number 6

Aspirin Resistance
Ready for Routine Testing?
By Thomas S. Kickler, MD

One of the most important and cost-effective drugs for the secondary prevention of cardiovascular disease, aspirin reduces the rate of acute arterial thrombotic events in high-risk patients by at least 25%. But even though many patients take a daily dose of aspirin, acute arterial thrombosis may recur. Normally, aspirin inhibits platelet activation and reduces thrombotic complications; however, cardiologists now recognize that some patients are resistant to the therapeutic effects of aspirin. If cardiologists could identify these patients, they could use other therapeutic options.

A variety of laboratory tests measure platelet function, and there has been much interest in applying them to assess the risk for future arterial thrombotic events. In addition, investigators are seeking laboratory measures that cardiologists could use to adjust aspirin therapy, similar to the approach used in therapeutic monitoring of oral anticoagulants. However, before cardiologists can use this information to adjust therapy, it will be important to define the role of such tests in monitoring the antiplatelet effect of aspirin. Clinically, the key issues for treatment decisions will be whether a patient’s arterial event resulted entirely from a failure of aspirin to inhibit platelet activation (pharmacologic resistance) or whether some other clinical factor contributed to the vascular event (treatment failure). Clinicians and laboratorians will first need to determine what laboratory measurements of platelet activation to use that are predictive of a vascular event. Studies will also be needed to standardize and validate these applications in diverse patient populations.

The purpose of this article is to describe platelet function, the effect of aspirin upon platelet tests, and the clinical conditions associated with arterial thrombosis that confound test result interpretation. In addition, I present a list of the laboratory tests that measure the antiplatelet effect of aspirin and give recommendations for how laboratorians should approach testing for aspirin resistance.

What is Aspirin Resistance?

Patients on aspirin therapy may experience a thrombotic event due to pharmacologic resistance or some other clinical failure. Consequently, different definitions of aspirin resistance have been proposed. Some investigators believe that resistance should be defined strictly on the basis of aspirin’s effect upon platelet activation. In other words, aspirin resistance would reflect the failure of aspirin to inhibit thromboxane A2 (TxA2) production or some other aspect of platelet function, such as thrombin generation. In fact, this definition parallels the practice of labeling patients “heparin resistant” when their activated partial thromboplastin times do not increase as expected in response to heparin therapy.

In contrast, clinical aspirin resistance represents the failure of aspirin to prevent arterial thrombotic events in patients who are compliant with therapy prescribed by their cardiologist or other healthcare professional. These clinical events have also been called aspirin failure events. Typically, cardiologists diagnose clinical resistance after a patient has experienced an arterial event while on aspirin therapy; however, this diagnosis is nonspecific. Clearly, there are multiple factors that can cause arterial thrombotic events (Table 1, below).

Possible Causes of Recurrent
Arterial Events in Patients Taking Aspirin

Pharmacologic Considerations

  • Non-compliance
  • Under dosing
  • Inadequate absorption
  • Drug interference from nonsteroidal anti-inflammatory drugs

Genetic Considerations

  • COX-1, COX-2, thromboxane synthetase polymorphisms
  • Platelet glycoprotein polymorphisms in GP1a/IIa, IB/IX, and/or IIa/IIIb receptors
  • Genetic hyper responsiveness to epinephrine
  • Factor XIII polymorphisms
  • Prothrombin gene mutation and enhanced thrombin formation

Enhanced Platelet Function Considerations

  • Greater percentage of newly formed platelets with enhanced COX- 2 levels
  • Inflammation and macrophage activation

Stress, smoking, and/or hypercholesterolemia

  • Endovascular devices, such as grafts, prosthetic valves, and stents
  • Red cell-induced platelet activation

Non-atheromatous Causes of Vascular Events

  • Embolism from the heart, such as from a prosthesis or tumor
  • Arthritis
  • Thrombus from extremities with co-existent patent foramen ovale

Platelet Biology

In order to understand the antiplatelet effect of aspirin, it helps to have a basic understanding of platelet function. Vascular injury, whether from trauma or atheromatous changes, results in endothelial disruption. This leads to exposure of collagen and the release of tissue factor. The former starts the process whereby platelets adhere to the subendothelium. von Willebrand factor also promotes platelet adhesion. The increase in tissue factor at the site of injury promotes the activation of the clotting cascade, which generates thrombin. It is the action of thrombin that converts fibrinogen to fibrin to form a clot.

Thrombin is also one of the most important platelet agonists. Following adhesion of the platelet to the injury site, platelet activation begins. The clotting cascade is promoted by the release of ADP and epinephrine, as well as generation of thrombin. These platelet agonists activate receptors on the platelet membrane that lead to mobilization of intracellular platelet calcium, which promotes platelet degranulation or secretion and activates phospholipase A. The phospholipase A2 enzyme cleaves arachidonic acid from membrane phospholipids and provides the substrate for the generation of prostaglandin H2, which is catalyzed by cyclooxygenase. Modification of prostaglandin H2 produces a variety of prostaglandins and TxA2. TxA2 promotes further platelet activation, as well as platelet aggregation at the vascular injury site. Nearby endothelial cells generate prostacyclin, nitric oxide, and carbon dioxide that act to down regulate platelet reactivity in the vicinity of the clot.

Possible Causes of Pharmacologic Aspirin Resistance

Aspirin irreversibly inactivates the cyclooxygenase (COX) activity of the platelet enzyme prostaglandin H-synthase 1, also referred to as COX-1 (Figure 1, below). This enzyme catalyzes the conversion of arachidonic acid to prostaglandin H2, which is the first committed step in prostanoid biosynthesis. Prostaglandin H2 is an intermediate compound and a substrate for several downstream isomerases that generate different bioactive prostanoids, including TxA2, the main product of arachidonic acid metabolism in human platelets. Aspirin first binds to an Arg120 residue and acetylates a Ser529 residue located in the narrowest section of the channel, just below the catalytic pocket. Acetylation of Ser529 prevents arachidonic acid from gaining contact with Tyr385, the first step in its cyclooxygenation.


Platelet Activation by a Variety of Activators
and Effect on Thromboxane Generation

In low doses, aspirin is considered a relatively weak antiplatelet agent since it inhibits only thromboxane-dependent activation and aggregation. TxA2 increases expression of fibrinogen receptors on the platelet membrane and acts in an autocrine fashion to trigger the activation of other platelets by activating the thromboxane receptor on the platelet membrane.

TxA2 is also a potent vasoconstrictor, and a once-a-day, 30-mg dose of aspirin suppresses TxA2 by 95% after 5 days of treatment. Aspirin-treated platelets still respond to collagen, epinephrine, and thrombin, all of which may play a role in activation of platelets in acute coronary syndromes and stroke.

Although newly formed platelets express both COX-1 and COX-2, mature platelets express only COX-1. While COX-1 is highly sensitive to low doses of aspirin, COX-2 is inhibited only by doses high enough to have analgesic or anti-inflammatory effects.

Therefore, a patient who is aspirin resistant could have COX-1 polymorphisms affecting Arg120, Ser529, or both. Moreover, in any given population, these polymorphisms also could explain why a fixed percentage of drug resistance is observed and does not change over time as a function of drug exposure. Although genetic variability in COX-1 has been described, both increased and diminished responses to aspirin have been associated with the same COX-1 haplotype. This haplotype, which is carried by 12% of the population, contains the minor allele of the promoter variant A842G and is in complete linkage disequilibrium with the C50T variant in the signal peptide.

Alternatively, enhanced platelet regeneration and an increased proportion of newly formed platelets expressing COX-2 may lead to an increase in the fraction of platelets that are able to form TxA2 in both a COX-1 and COX-2 dependent manner. In this situation, the antiplatelet effect may be decreased with standard daily aspirin dosing. It is noteworthy that increased platelet turnover, which is observed in patients during coronary artery bypass graft surgery, as well as in patients with an infection or inflammation, can result in an increase in the proportion of younger platelets that elevates COX-2.

Assessment of Platelet Function

The initial definition of aspirin’s antiplatelet effect was based upon prolongation of the bleeding time and optical platelet aggregometry. Since then, diagnostic manufacturers have developed a variety of devices to measure platelet function.

Today, the majority of studies reporting the occurrence of aspirin resistance in different clinical settings have relied on ex vivo measurement of platelet function using one or more of the following: light transmittance or impedance-lumi aggregometry in platelet-rich plasma or whole blood; Dade Behring’s (Deerfield, Ill.) PFA-100 platelet function analyzer, a bedside, rapid, whole-blood assay; the VerifyNow Rapid Platelet Function Assay (Accumetrics, Inc., San Diego, Calif.), a bedside, whole-blood assay; the Thromboelastograph Hemostasis Analyzer (Haemoscope, Niles Ill.), a device that tests platelets’ response to an aggregating stimulus, such as arachidonic acid, added at variable concentrations.

Although a wide range of methods is available, many investigators and clinicians regard optical aggregometry in citrated, platelet-rich plasma as the gold standard for assessing platelet function. Optical aggregometry measures the increase in light transmission through a platelet-rich plasma preparation when platelets are aggregated by a platelet agonist. For determining the effect of aspirin, arachidonic acid is the best platelet agonist for detecting inhibition of thromboxane formation since it is the precursor of thromboxane. Other platelet agonists, such as collagen and ADP, induce platelet aggregation through mechanisms that are less dependent upon thromboxane formation.

But there is little standardization of the technique between laboratories, despite the fact that optical aggregometry was developed 50 years ago. Several preanalytical and analytic factors are important to obtaining reproducible results, including preparation of the platelet-rich plasma, the final concentration of platelet count, and the concentration of the agonist used to stimulate the platelets. For example, 0.50 mM arachidonic acid inhibits > 90% of platelets, and 1 µg/mL collagen or 5 µM epinephrine produces >70% inhibition. It is important to note that platelet aggregation results are also affected by the patient’s race, sex, diet, as well as the sample collection technique.

An alternative to optical platelet function is whole-blood aggregometry, a method that measures the change in impedance when platelets adhere to a platinum electrode probe. This technique circumvents the need for centrifugation and permits whole-blood testing, which may reflect in vivo platelet function more closely as interactions of all cell types are involved.

Both optical and whole-blood impedance aggregometry are precise, but the methods require technical skill and interpretation. Because of this, there is much interest in dedicated instruments, such as the PFA-100 and the VerifyNow.

The PFA-100 analyzer has proven useful for monitoring the pharmacologic effect of aspirin upon platelets and for identifying aspirin-resistant patients in several clinical trails. The instrument uses small membranes coated with either collagen and epinephrine or collagen and ADP. Anticoagulated whole blood is passed through the membrane at a high shear rate to simulate the in vivo hemodynamics in the small capillaries. Platelets adhere to the membranes and gradually occlude a small aperture in the center of each membrane. The time that it takes for this to occur is called the closure time and is the measure of platelet activity. With this method, epinephrine-induced closure time is prolonged when the patient is taking aspirin. An abnormal ADP closure time reflects other causes of platelet dysfunction. In addition, the PFA-100 analyzer may be influenced by the hematocrit value and the platelet count, as well as the von Willebrand factor concentration. These considerations may account for the poor agreement between results from the PFA-100 and platelet aggregation.

In contrast, the VerifyNow platelet function analyzer is a turbidimetric-based optical system that measures platelet-induced aggregation as an increase in light transmission. Fibrinogen-coated microparticles are used to measure platelet aggregation in response to a novel platelet agonist, propyl gallate or arachidonic acid. The fibrinogen-coated microparticles agglutinate in whole blood in proportion to the number of unblocked platelet glycoprotein IIB-IIIa receptors. The easy-to-use instrument automatically calculates the results and expresses them as aspirin resistance units. It is important to note, however, that some investigators have questioned the aspirin resistance cutpoint determined by the manufacturer. The value was determined by comparison to platelet aggregation in response to administration of 325-mg dose of aspirin and epinephrine-induced platelet aggregation. But epinephrine is not a specific measure of aspirin effect, and many normal patients have a variable response to this agonist. As with the PFA-100 analyzer, the VerifyNow analyzer has only been used in studies of relatively small populations; therefore, further validation in larger populations is needed.

Investigators have also proposed other laboratory tests of platelet function, including thromboelastographic analysis, cone and plate analysis of platelet function, and flow cytometry to measure p-selectin activation. While all of these assays may be potentially useful, there are not enough studies to make any recommendations about their utility.

Laboratory Tests to Measure
the Antiplatelet Effect of Aspirin
TEST
METHOD
COMMENTS
Platelet Function

Optical platelet aggregation

Impedance Platelet aggregation

No standard protocols developed for concentrations of agonists

Labor and time intensive

Gold standard of platelet function

 

High shear stress platelet aggregation (PFA-100)

Optical detection of platelet

Results affected by packed cell volume, von Willebrand level

Easy to perform and well standardized reagents

Excellent preliminary studies

  VerifyNow Aspirin Now

Question about how cutoff values determined

Easy to perform, well standardized

  Thromboelastographic analysis

Uncertain sensitivity and uncertain correlation with clinical events

Generally widely available in many hospitals

  Measurement of p-selectin expression byflow cytometry

Requires careful collection of blood sample and expensive equipment

Excellent marker of platelet secretion and platelet activation status

Thromboxane Generation Serum Thromboxane B2

Requires careful blood collection

Uncertain sensitivity and specificity

  Urinary 11-dehydro thromboxane B2

Clinical assay format only recently introduced

Elevated in conditions other than aspirin resistance

Standardization available through a well organized group of laboratories

Biochemical Assessment of Platelet Inhibition

Another method to measure aspirin resistance is to assess platelet inhibition via a biochemical pathway. TxA2 has a short half-life in plasma and is rapidly hydrolyzed to thromboxane B2 (TxB2), which is then metabolized to three molecules: 11-dehydro thromboxane B2; 11-dehydro 2, 3 dinor thromboxane B2; and a number of other minor thromboxane B2 metabolites that are excreted by the kidney. These metabolites can be measured in serum or plasma using a variety of commercially available ELISAs; however, such assays require a variety of preparatory steps before the metabolites can be measured. Recently, Corgenix (Broomfield, Colo.) developed a clinical assay that measures urinary 11-dehydrothromboxsane B2 without any preparatory steps.

Researchers have also described aspirin-insensitive TxA2 biosynthesis in patients with unstable angina, as well as in patients with post-stroke dementia. One research group led by Eikelboom explored the clinical relevance of this insensitivity to aspirin. They performed a nested case-controlled study of baseline urinary 11-dehydro-thromboxane B2 excretion in relation to the occurrence of major vascular events in aspirin-treated, high-risk patients enrolled in the Heart Outcomes Prevention Evaluation (HOPE) trial. After adjustment for baseline differences, the odds for the composite outcome of myocardial infarction, stroke, or cardiovascular death increased with each increasing quartile of 11-dehydro-thromboxane B2 excretion, with patients in the upper quartile having a 1.8-fold higher risk than those in the lower quartile.

This study also revealed another important point about aspirin resistance. The researchers found that increasing levels of the thromboxane metabolite and increasing risk of arterial events is a continuum, rather than a positive or negative outcome. Consequently, a patient’s risk of an adverse vascular event goes up with increasing levels of aspirin resistance, similar to other risk factors such as obesity, blood pressure, and hyperlipidemia.

However, platelets are not the only source of TxA2 biosynthesis. Urinary excretion of 11-dehydro-thromoboxane B2 is reduced by 60–80% following aspirin administration. The underlying mechanisms include COX-2 expression in inflammatory cells endowed with thromboxane synthase and a higher percentage of COX-2-expressing platelets. Moreover, research has suggested that signal-dependent de novo synthesis of COX-1 occurs in aspirin-treated platelets after persistent activation provides an additional source of aspirin-insensitive thromboxane biosynthesis.

Recommendations for Lab Testing

In the laboratory, measurement of aspirin resistance relies on assessment of platelet inhibition. Ideally, such tests should be reproducible, easy to perform, quick, and inexpensive. In addition, the validity and predictive power of such tests should be documented in properly designed studies. While several platelet function tests are commercially available, at present none of the available tests fulfill all the criteria mentioned above.

Moreover, because aspirin’s antiplatelet effect is highly variable, patients’ test results will span a broad range of values. This presents a problem because labs need to establish cutoff values that clinicians can use to assess aspirin resistance. Until more study results are available, the predictive value of any test of aspirin resistance remains highly uncertain.

For this reason, laboratorians should follow the recommendations of the International Society of Hemostasis and Thrombosis, Scientific and Standardization Committee, published in 2005 in the Journal of Thrombosis and Haemostasis. (See Suggested Reading, below). These experts unanimously recommend against measuring the antiplatelet effect of aspirin in order to assess aspirin resistance in individual patients. This statement parallels the recommendations found in the seventh American Conference on Antithrombotic Therapy, published by Patrono, et al. in Chest (See Suggested Reading, below).

The Opportunity for Improved Patient Care

For many patients, aspirin is an effective antithrombotic agent; however, patients taking aspirin may demonstrate highly variable responses to in vitro tests for platelet aggregation and may experience breakthrough thromboembolic events. Although this phenomenon has been termed aspirin resistance, the lack of a uniform definition or agreement on diagnostic criteria precludes definitive recommendations at this time.

Clearly, a test that would identify patients at risk for aspirin resistance who might benefit from alternative or combined antiplatelet therapy would provide valuable treatment information to cardiologists. However, investigators and manufacturers need to conduct more studies to evaluate and validate quantitative measures of platelet inhibition that correlate with patient outcomes.

Furthermore, in order for aspirin resistance testing to become routine, the laboratory community needs standardization and proficiency testing. To address those needs, Aspirin Works, Inc. (www.aspirinworks.com), a service of Corogenix, has organized a network of laboratories aimed at providing cutoff values verified from carefully performed clinical trials for interpretation of an individual’s test result. In order to follow the new developments in this area, laboratorians should monitor the information provided by this site.

SUGGESTED READING

Bhatt BL. Aspirin resistance: More than just a laboratory curiosity. J Am Coll Cardiol 2004; 43: 1127–1129.

Cattaneo M. Aspirin and clopidogrel. Efficacy, safety and the issue of drug resistance. Arterioscler Thromb Vasc Biol 2004; 24: 1980–1987.

Cohen M, ed. Handbook of Antiplatelet Therapy. London: Taylor and Francis, 2003.

Eikelboom JW, Hirsh J, White JI, Johnston M, Yi Q, and Yusuf S. Aspirin resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke or cardiovascular death in patients at risk of high for cardiovascular event. Circulation 2002; 105: 1650–1655.

Gum PA, Kottke-Marchant K, and Poggio ED et al. Profile and prevalence of aspirin resistance in patients with cardiovascular disease. Am J Cardiol 2001; 88: 230–235.

Hankey G, Eikelboom. Aspirin resistance. Lancet 2006; 367: 606–615.

Harrison P. Progress in the assessment of platelet function. Br J Haematol 2000; 111: 733–744.

Michaelson Alan, Coleman L, Hamlington J, Kickler TS, Bray P. Differential sensitivity to agonists and antagonists of platelets displaying the PLA polymorphisms. Circulation 2000; 101: 1013-1018.

Michelson AD, Cattaneo M, Eikelboom JW, et al. Aspirin resistance position paper of the Working Group on Aspirin Resistance. J Thromb Haemost 2005; 3: 1309–1311.

Patrono C. Aspirin resistance: definition, mechanisms and clinical read-outs. J Thromb Haemost 2003; 1: 1710–1713.

Patrono C, Coller B, FitzGerald A, Hirsh J, Roth G. Platelet-active drugs: the relationships among dose, effectiveness, and side effects—the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004; 126: 234S–264S.

Williams MS, Kickler TS, Vaidya D, Ng’alla LS, Bush DE. Evaluation of platelet function in aspirin treated patients with CAD. J Thromb Thrombolysis 2006; 21(3): 241–247.

Thomas Kickler, MD, is Director of the Hematology and Coagulation Laboratories and Co-Director of the Core Laboratory, Johns Hopkins Hospital in Baltimore, Md. He is also Professor of Pathology, Medicine and Oncology at the Johns Hopkins University School of Medicine.

Dr. Kickler has received funding for a sponsored project from Dade Behring (Deerfield, Ill.)

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