Aspirin Resistance

Cardiovascular disease is a major cause of morbidity and mortality in the United States and many other countries. Aspirin, or acetylsalicylic acid (ASA), is a potent and inexpensive drug prescribed to prevent first and recurrent myocardial infarcts and thrombotic cerebrovascular events including transient ischemic attacks. It is also prescribed after thrombolytic therapy and in acute myocardial infarction (MI). In the 1990s, several large-scale clinical trials were initiated to ascertain the effectiveness of different anti-thrombotic agents including ASA.  A major finding of the Antithrombotic Trialists' Collaboration was that ASA reduced the risk of serious vascular events by an average of 25% in patients with acute or previous myocardial infarct, ischemic stroke, angina, or atrial fibrillation. 

Because some patients taking ASA suffered untoward clinical events such as a second MI, the concept of ASA resistance was proposed in the 1990s. To date there has been no agreement on a clinical or pharmacologic definition. One possible definition is the occurrence of serious vascular events despite the use of recommended doses of ASA. Another is the inability of ASA to inhibit platelet thromboxane formation. Further complicating the issue of defining the phenomenon is the fact that patients differ in their clinical responses to ASA, just as would be expected with any other drug.

Whatever the definition, there is little consensus on the actual incidence of ASA resistance. Estimates vary from 5% to 60%. ASA resistance has clearly been demonstrated in populations with platelet A2 (Pl^A2) polymorphism and those heterozygous for the A-842G/C50T haplotype. ASA resistance has also been postulated to occur in other settings. Ibuprofen and some COX-2 inhibitors such as celecoxib and rofecoxib show evidence of ASA resistance in vitro. COX-1 and COX-2 are approximately 90% homologous. The 10% structural difference renders COX-2 relatively insensitive to ASA. COX-1 expression is not inducible and COX-2 expression is. Upregulation of COX-2 occurs in platelets in response to inflammation. ASA resistance has also been observed in patients with serum cholesterol levels > 220 mg/dL.  Patients who smoke or ingest the herbal medicines ginkgo biloba and ginseng may also show ASA resistance. The previous observations suggest 3 mechanisms for ASA resistance: (1) increased COX-2 activity; (2) polymorphism of Pl^A2 platelet glycoprotein IIIa; and (3) polymorphism of the COX-1 gene.

There is no gold-standard laboratory test for assessing platelet function, although standard platelet aggregometry is the assay against which all others are compared. The newest assays for determining platelet aggregation are of 2 types. The first are surrogate aggregometry assays that use whole blood; these are screening tests. The others are biochemical assays that measure thromboxane (TX) metabolites; these tests confirm activation of the COX-1 enzyme.

There are 2 analyzers and 1 assay/procedure currently available in the United States to screen for the presence of ASA. The 2 analyzers are the PFA-100 Platelet Function Analyzer (Dade Behring; Deerfield, Illinois) and VerifyNow Aspirin Assay (Accumetrics; San Diego, California). The procedure/assay is PlateletWorks (Helena Labs; Beaumont, Texas). The biochemical assay for TX metabolites is AspirinWorks (Creative Clinical Concepts; Denver, Colorado).

The PFA-100 is US Food and Drug Administration (FDA)-approved to detect platelet dysfunction, von Willebrand disease, and aspirin-induced platelet inhibition. It is an in vitro quantitative measurement of platelet adhesion and aggregation that requires whole blood collected in 3.8% sodium citrate. The cartridges used are coated with collagen/epinephrine and collagen/adenosine diphosphate (ADP). The instrument measures collagen-induced platelet plug formation as time in seconds to occlude an aperture. The numerical result is inversely related to platelet activity, with normal defined as < 193 seconds and abnormal as > 300 seconds. Its sensitivity as a screen for platelet dysfunction is approximately 95%.

 As with any laboratory test, there are limitations to the PFA-100. Among them are that an incorrect citrate concentration, ie, 3.2%, may shorten closure time. Also, the cut-off value to determine ASA sensitivity is poorly defined. There have been very few correlation studies with clinical events.  The manufacturer's stated sample stability time of 4 hours is too long; samples can show deterioration after only 2 hours.

The VerifyNow Aspirin Assay was previously marketed as the Ultegra Rapid Function Platelet Assay -- ASA. It is FDA-approved to aid in the detection of platelet dysfunction due to aspirin ingestion. It is an in vitro semiquantitative measurement of ASA-dependent aggregation that requires whole blood collected in 3.2% sodium citrate. The cartridge contains arachidonic acid, which is thought to be more specific for ASA. The instrument tests aggregation of activated platelets through binding to human fibrinogen-coated beads. The numerical result is reported in aspirin-response units (ARU) with the ASA resistance cut-off defined as ≥ 550 ARU (aspirin-related platelet dysfunction not detected). Its sensitivity as a screen for ASA-induced platelet dysfunction is approximately 91%, according to the manufacturer's product information.

There are 2 major limitations to the VerifyNow Aspirin Assay. The first is that, according to the manufacturer, this test cannot be used in patients with inherited platelet defects or in patients receiving many other anti-platelet drugs. The second is that the few evaluation studies to date are flawed. In some studies, ASA resistance has been correlated with biochemical cardiovascular injury, but there has been no systematic clinical outcomes data. In those studies that did attempt to correlate test results with clinical outcomes, there were confounding variables, such as other anti-platelet drugs.

PlateletWorks is FDA-approved to detect platelet dysfunction due to inhibition secondary to diet, ASA, and/or other drugs. It is an in vitro quantitative measurement of platelet activation that requires whole blood collected in 3.2% sodium citrate. This assay uses separate tubes containing collagen and ADP. This method uses traditional cell counting principles (electronic impedance). Both pre- and postactivation platelet counts are obtained and the numerical result represents percent inhibition. The formula is:

Agonist platelet count x 100/baseline platelet count

PlateletWorks also has limitations. There is a very short time allowed -- 10 minutes -- between sample collection and assay. Some studies have indicated that the assay's precision may be a cause for concern. Some laboratories have found an unacceptably high false-positive rate because of interference by dietary substances such as chocolate and red wine. Obtaining a dietary history that includes chocolate, wine, herbal medicines, and prescribed and over-the-counter medications is prudent before performing any of these assays.

AspirinWorks is FDA-approved to detect ASA-induced inhibition of TX metabolites. It is an in vitro quantitative measurement of ASA-induced inhibition of TXA₂ generation. The assay requires 10 mL of freshly voided urine collected in a preservative. The urine sample may be frozen for assay later. The test, either a radio-immunoassay (RIA) or an enzyme immunoassay (EIA), measures levels of 11-dihydro-thromboxane-B₂, a relatively stable breakdown product of TXA₂. The assay therefore indirectly measures in vivo activity of TXA₂. Reduced levels are interpreted as due to ASA effect. The numerical results are expressed in quartiles. The first quartile is less than 132 pg/mg creatinine, the second quartile 133 to 193 pg/mg creatinine, the third quartile 194 to 296 pg/mg creatinine, and the fourth quartile greater than 296 pg/mg creatinine. Different quartiles represent differing degrees of risk for clinically significant events. A patient whose results are in the first quartile has a relative risk of an untoward cardiovascular event defined as 1. A patient whose results are in the second quartile has a 1.3 times greater risk of myocardial infarction (MI) than a patient in the first quartile. A patient whose results are in the third quartile has a 1.5 times greater risk, and a patient in the fourth quartile has twice the risk of MI.

There are 2 potential limitations with the AspirinWorks assay. The first is that reference ranges of RIA assays differ from those of EIA assays. Similar to tests for lipid levels, the method for a particular patient should be the same or the results may not be interpretable. The second major possible limitation is that the results interpretation depends on a demarcation of the quartiles. However, patients whose results comprised the second, third, and fourth quartiles appeared to be different from patients in the control population. Specifically, the cardiovascular risk factors were higher in the case group than in the control group. Despite this possible limitation, the study authors did conclude that a clear association existed between urinary 11-dihydro-thromboxane-B2 levels and cardiovascular outcomes.

In conclusion, there are no perfect assays for a clinical phenomenon for which there is no agreed-upon definition.  At the present time, there is insufficient evidence to prove that these assays can assess ASA resistance or predict clinical outcomes. 

Suggested Reading

• Cambria-Kiely JA, Gandhi PJ. Possible mechanisms of aspirin resistance. J Thromb Thrombolysis. 2002;13:49-56. 
• Catella-Lawson F, Reilly MP, Kapoor SC, et al. Cyclooxygenase inhibitors and the antiplatelet effects of aspirin. N Engl J Med. 2001;345:1809-1817. 
• de Gaetano G, Cerletti C. Aspirin resistance: a revival of platelet aggregation tests? J Thromb Haemost. 2003;1:2048-2050. 
• Gum PA, Kottke-Marchant K, Welsh PA, et al. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol. 2003;41:961-965. 
• Patrono C, Coller B, Dalen JE, et al. Platelet-active drugs: the relationships among dose, effectiveness, and side effects. Chest. 2001;119:39S-63S. 
• Serebruany VL, Malinin AI, Sane DC. Risk of bleeding complications with antiplatelet agents: a meta-analysis of 338,191 patients enrolled in 50 randomized controlled trials. Am J Hematol. 2004;75:40-47. 
• Weber AA, Przytulski B, Schanz A, et al. Towards a definition of aspirin resistance: a typological approach. Platelets. 2002;13:37-40. 
• Zimmerman N, Wenk A, Kim U, et al. Functional and biochemical evaluation of platelet aspirin resistance after coronary artery bypass surgery. Circulation. 2003;108:542-547.