Laboratory Methods for Evaluating Effectiveness of Anti-platelet Therapy
By Brad S. Karon, MD, PhD and Elizabeth Jaben, MD
The number one killer in the U.S. and worldwide, cardiovascular disease is responsible for approximately 30% of all deaths. Research has now shown that atherosclerotic plaques develop over time. When a plaque ruptures, however, it exposes the arterial extracellular matrix and initiates platelet aggregation or atherothrombosis. At the same time, tissue factor produced by macrophage-derived foam cells also initiates the blood system’s coagulation cascade. These events are responsible for clot propagation and can cause clinical disease, ranging from stroke to myocardial infarction (MI).
Today, platelet inhibitors are an integral part of the prevention and treatment regimen for acute vascular events. There are three major classes of platelet inhibitors: aspirin; the theinopyridine derivatives, including clopidogrel (Plavix); and GP IIb/IIIa receptor antagonists. Because the GP IIb/IIIa antagonists are only available in intravenous forms, they are not used for long-term treatment of patients outside the hospital setting. On the other hand, aspirin and clopidogrel are commonly taken by patients to reduce the long-term risk of heart disease.
Recently, there has been a growing debate about whether clinical laboratories should measure the effects of aspirin and clopidogrel on platelet function. In this article, we discuss the pharmacology and lab tests available to measure the effects of these antiplatelet medications.
Platelets and Clot Formation
Platelets play an integral role in normal hemostasis. They lack nuclei, but contain alpha granules composed of fibrinogen, platelet-derived growth factor, von Willebrand factor (vWF), P-selectin, platelet factor-4, and dense granules containing adenosine diphosphate (ADP), serotonin, and calcium.
When blood vessel damage occurs, the exposed vWF and collagen bind to circulating platelets and activate them. During this activation, the contents of the alpha and dense granules are released (degranulation) to recruit more platelets to the site of vessel damage and to potentiate platelet activation. The platelets also synthesize and release thromboxane, another mediator of activation.
During platelet activation, the GP IIb/IIIa receptor on the cell surface undergoes a shape change that allows fibrinogen (Factor I) to bind to the platelets. At the same time, thrombin converts fibrinogen into fibrin as part of the blood coagulation cascade. Factor XIII cross-links fibrin in one of the final steps of the cascade. This complex sequence of interactions causes a stable platelet plug to form and constitutes the basis of hemostasis (Figure 1).
Pathways Leading to Clot Formation
This simplified graphic shows the fundamental steps in clot formation. In vivo the clot process occurs as a result of two distinct pathways, the blood coagulation cascade (left) and platelet activation (right).
Aspirin: Mechanism of Action
Aspirin (acetylsalicylic acid) has a long history as an analgesic to relieve minor aches and pains. This inexpensive and widely available drug also reduces the risk of a first MI in men and stroke in women. In patients at high risk for vascular disease, it has been associated with up to a 25% reduction in death related to vascular causes, MI, and stroke. Aspirin is also indicated as part of a regimen for the secondary prevention of cardiovascular events in patients with a history of coronary artery disease, cerebrovascular disease, or peripheral vascular disease.
Aspirin works by irreversibly acetylating serine 530 of cyclooxygenase 1 (COX-1). This enzyme plays a key role in the pathway that converts arachidonic acid into agents that can either increase platelet aggregation (agonist) or decrease platelet aggregation (antagonist). Research suggests that aspirin reduces the risk of cardiovascular disease by decreasing production of thromboxane, a potent platelet agonist, to a greater extent than antagonists, such as prostacyclin and other arachidonic acid metabolites. Therefore, aspirin’s net effect is a reduction in platelet aggregation.
Definitions of Aspirin Resistance
There are a number of different schemes for defining the effects of aspirin on platelet function and likewise for defining an individual’s resistance to aspirin therapy. Researchers have proposed various laboratory definitions of aspirin resistance based on platelet function tests, measures of serum or urinary thromboxane metabolites, and the pharmacological mechanism of resistance.
In individuals with Type I, or pharmacokinetic resistance, aspirin fails to inhibit COX-1, despite their having ingested adequate doses. Research indicates that the level of salicylate in these individuals’ blood may not be high enough to inhibit COX-1 due to malabsorption or genetic polymorphisms in COX-1. In Type II, or pharmacodynamic resistance, continued thromboxane production takes place despite adequate COX-1 inhibition. Other enzymes, such as COX-2, can also produce thromboxane, which may explain this type of aspirin resistance. In pseudoresistance resistance, or Type III, aspirin effectively shuts down thromboxane production. Platelet activity can still occur via thromboxane-independent pathways, such as thrombin, ADP, or epinephrine activation of platelets. While these definitions are useful for understanding the various biological mechanisms that lead to platelet activation and atherothrombosis even when patients take aspirin, it is important to realize that no standard definition of aspirin resistance exists.
Tests to Measure Aspirin Effect on Platelet Function
Laboratories have several options for measuring platelet function in patients receiving aspirin therapy.
Optical Light Transmission Aggregometry. The current gold standard method is optical light transmission aggregometry (LTA). This assay requires a citrated blood sample that has been centrifuged to create platelet-rich plasma, with a platelet count of 200–300 x 109/L). To perform the assay, the technologist places a sample of the platelet-rich plasma in an aggregometer cuvette along with a platelet agonist, such as ADP, arachidonic acid, or epinephrine, to induce platelet aggregation. As the platelets aggregate, the turbidity of the platelet-rich plasma in the cuvette decreases, which is measured as increased light transmittance over time. Aggregometers that perform LTA are available from several manufacturers.
Research has shown that age, gender, race, and hematocrit all affect platelet aggregation measured by LTA. In addition, pre-analytical variables related to platelet activation during sample collection and processing create variability in the results. While there is no universally accepted definition of aspirin resistance as measured by LTA, many studies have used ≥20% aggregation in response to arachidonic acid and/or ≥70% aggregation in response to ADP as metrics.
Many laboratories prefer LTA because it is one of the few tests that has predicted outcomes in patients on aspirin therapy. For example, individuals with more LTA-defined platelet activity had more cardiovascular events in studies. However, the test has several disadvantages, including being time- and labor-intensive, difficult to standardize, and subject to operator-induced variability.
in vitro Bleeding Time. Another technology to assess platelet function is available on a specialized instrument. The Platelet Function Analyzer-100 (PFA-100, Siemens Healthcare Diagnostics, Figure 2) uses a proprietary technology that works by passing blood through a capillary tube with a very small aperture coated with an agonist that induces platelet aggregation. As platelets activate, they stick to the aperture and stop blood from flowing through the capillary tube. The instrument measures the time it takes the aperture to clog and reports it as an in vitro clotting time. Because of its similarity to the conventional bleeding time test, the platelet function test results are sometimes referred to as an “in vitro bleeding time.”
Studies using the PFA-100 indicate that most patients on aspirin therapy have prolonged in vitro clotting times. However, the percentage of patients who have relatively normal clotting times, despite taking aspirin, has also varied widely, which is one potential disadvantage of this technology.
The PFA-100 test for aspirin resistance does have some distinct advantages, as well. Most notably, it is rapid and uses whole blood, which means patients can be tested in a variety of care settings. Furthermore, laboratories may be able to standardize PFA-100 results better than those obtained by LTA, as well as use the assay to assess inherited platelet disorders. On the negative side, studies have used various cut-offs for defining aspirin resistance, and the technology is not specific for aspirin resistance. For example, patients with von Willebrand’s disease and other platelet abnormalities will have prolonged in vitro clotting times even though they haven’t taken aspirin. Hematocrit and platelet count also affect the test’s results.
The Platelet Function Analyzer-100 tests for platelet function and uses a high shear-flow environment to mimic in vivo hemostasis. The instrument uses whole blood and produces results in 4–8 minutes.
Used with permission of Siemens Healthcare Diagnostics.
Cartridge-based Platelet Aggregation. The VerifyNow Aspirin Assay (Accumetrics, Figure 3) is another proprietary technology for assessing the effects of aspirin on platelet function. Assay cartridges contain an activator specific for thromboxane-induced platelet activation, making the test more specific for aspirin-induced platelet inhibition than the in vitro bleeding time test. Inside the assay cartridge, fibrinogen-coated microparticles aggregate in response to platelet activation, resulting in increased light transmittance similar to LTA. Accumetrics recommends a cut-off value above which patients are classified as having a sub-optimal response to aspirin therapy. This value is reported in arbitrary aggregation units defined by the company.
One of the main advantages of the VerifyNow technology is its rapid turnaround time using a whole-blood sample. In general, the test also identifies a smaller population of resistant patients compared to the PFA-100. On the other hand, the results generally show poor concordance with other tests considered to be gold standards for assessing the aspirin effect, such as LTA and measurement of serum or urinary thromboxane metabolites.
The VerifyNow Instrument
The VerifyNow instrument is an automated analyzer that uses disposable cartridges to measure platelet function. The point-of-care instrument can test for both aspirin and clopidogrel response and delivers results in 5 minutes or less.
Used with permission of Accumetrics.
Clopidogrel: Mechanism of Action
Clinicians prescribe clopidogrel to reduce the risk of MI, unstable angina, stroke, and cardiovascular death in patients with cardiovascular disease. This oral, thienopyridine class, antiplatelet drug decreases the activity of platelets, making them less likely to form blood clots.
The drug works by covalently and irreversibly binding to the P2Y12 receptor, the major receptor involved in ADP-induced aggregation of platelets. Formation of the active drug metabolite occurs in the liver; therefore, genetic polymorphisms in the CYP2C19 liver enzymes responsible for metabolism of the drug appear to play an important role in individual variability to the anti-platelet effects of clopidogrel.
Similar to testing for aspirin resistance, researchers have used various definitions of clopidogrel resistance. The most common laboratory definition involves ADP-induced platelet aggregation by LTA, as measured before and after taking the drug. Many investigators have used a decrease in platelet aggregation of ≥30% as evidence of the drug’s effectiveness. Definitions of resistance vary from <10% decrease in ADP-induced aggregation by LTA to <20% decrease in aggregation.
Tests to Measure Clopidogrel Effect on Platelet Function
As with aspirin resistance, multiple methodologies are available to measure clopridogrel’s effect on platelet function.
Optical Light Transmission Aggregometry. LTA generally is cited as the gold standard for defining responsiveness to clopidogrel. The advantages of this technology include its acceptance as the gold standard and its specificity for the actual pharmacologic mechanism of the drug, ADP-induced platelet activation. The method’s disadvantages are largely the same as when used to monitor aspirin therapy: lack of standardization, labor requirements, and the need for platelet-rich plasma.
Cartridge-based Platelet Aggregation. The VerifyNow P2Y12 Assay (Accumetrics) also measures the effectiveness of platelet clotting by clopidogrel. The same test can be used for other P2Y12 inhibitors as well. The principle of the device is the same as the test for aspirin, but the cartridge uses ADP to induce platelet aggregation and normalizes the results by comparing them to activation in the presence of thrombin, a potent platelet agonist. The test reports the percentage of clopidogrel’s inhibition of platelet activation by comparing ADP-induced to thrombin-induced aggregation.
This technology produces rapid results from whole blood samples and is relatively specific for clopidogrel-induced platelet inhibition. Test results, however, show relatively poor concordance with LTA. Researchers also have reported wide variability in patients’ responses, with some studies finding as high as 45–50% resistance. In addition, platelet count and use of other platelet inhibitors can affect test results.
Thromboelastography. Thromboelastography (TEG) is another means of assessing clopidogrel’s effect on platelet function (Figure 4). To perform the test, the technologist adds whole blood to a plastic cup coated with an agonist that initiates clotting. The instrument has a pin attached to a torsion wire. When the pin is lowered into the cup, the cup begins to rotate, causing the blood to clot. The clot creates tension on the wire, and the instrument graphs this variable over time, producing a curve. It is the curve’s maximum amplitude (MA) that reflects platelet function. For assessing clopidogrel response, the test is run with ADP-, fibrin-, and kaolin-coated cups. Similar to the VerifyNow P2Y12 assay, MA in response to ADP is normalized to the MA values for fibrin and kaolin to assess the extent to which a patient’s platelets are affected by an ADP receptor inhibitor.
Some laboratories prefer TEG because it works with a whole-blood sample and can also be used to assess aspirin inhibition of platelet function by using arachidonic acid, fibrin, and kaolin as agonists. In addition, the technology is relatively specific for clopidogrel’s effect on platelets, and it gives information beyond platelet function with each test, such as clotting factor and fibrinogen concentrations. On the down side, TEG is labor intensive and lacks standardization. Furthermore, the test results depend on calculations involving multiple, relatively imprecise parameters.
A thromboelastography analyzer measures platelet function by creating an artificial clotting environment in a whole-blood sample. The clot forms between a torsion wire and pin suspended in the specialized sample cup, and the instrument graphs the time to clot and maximum clot strength. Both resistance to aspirin and clopidogrel can be measured with this instrument.
Used with permission of Haemonetics.
While laboratories currently have multiple methods for assessing platelet function in patients on aspirin or clopridogrel therapy, other new technologies are coming into use. These include The Platelet Works (Helena Laboratories), Cone and Plate(let) Analyzer (Matis Medical), and flow cytometric assessment of various platelet surface receptors. Among the flow cytometric techniques, phosphorylation of vasodilator-stimulated protein (VASP, Diagnostica Stago) deserves special mention. Researchers have used this technology in preliminary studies as an alternative gold standard method to assess clopidogrel’s effect on platelet function.
Aspirin and clopidogrel therapy have become standard therapies for preventing recurrent atherothrombotic events and for preventing primary events in many high-risk patients. The two drugs affect platelet function via different mechanisms. Aspirin reduces thromboxane-mediated platelet aggregation, while clopidogrel partially prevents ADP-induced platelet activation.
Because patients exhibit substantial variability in their response to these therapeutics, laboratory measurement of platelet function may some day play a role in successful patient management. However, it is not clear today which test or device offers the most valuable assessment of inhibition of platelet activation. Therefore, laboratorians will need to keep a watchful eye for new developments in this area.
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Brad S. Karon, MD, is associate professor of Laboratory Medicine and Pathology Clinic and director of Point-of-care Testing and Hospital Clinical Laboratories at Mayo Clinic, Rochester, Minn. Email: firstname.lastname@example.org.
Elizabeth Jaben, MD, is a transfusion medicine fellow at Mayo Clinic, Rochester Minn. Email: email@example.com.