Aspirin Resistance: Current Status

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1 REVIEW ARTICLE JIACM 2007; 8(1): 72-7 Aspirin Resistance: Current Status Aniket Puri* Abstract Aspirin resistance is considered to be an enigma, and the data available on it is scarce. Various laboratory parameters assessing the efficacy of aspirin like bleeding time, platelet reactivity, thromboxane-a2 (TX-A2) production, and measurement of platelet aggregation have confirmed the lack of its uniform effect on the platelets. Studies have estimated that 5% to 45% of patients with vascular disease are aspirin resistant. The exact mechanism of aspirin resistance also remains elusive, and there are no specific guidelines for management of aspirin resistance. We studied 50 patients and found that 41.66% showed inadequate response to aspirin. Thus, we need to formulate a policy on aspirin and ascertain whether all patients taking aspirin need to be investigated and whether all patients with so-called aspirin resistance be put on other anti-platelet drugs. Introduction Since the time of introduction of aspirin more than a hundred years ago it has become a cornerstone in the treatment of coronary artery disease (CAD) 1. Aspirin s beneficial role in the secondary prevention of vascular events is well established 2, 3. The anti-thrombotic trialist collaboration s meta-analysis with approximately 100,000 aspirin treated subjects, showed a 25% reduction in death, MI, and stroke in high risk patients 4, 5. However, it now appears that the effect of aspirin may not be uniform in all patients. Various laboratory parameters assessing its efficacy like bleeding time, platelet reactivity, thromboxane-a 2 (TX-A 2 ) production, and measurement of platelet aggregation have confirmed the lack of its uniform effect on the platelets, among patients who manifest breakthrough events with thrombotic and embolic complications despite being on therapeutic doses. It has been suggested that one out of every eight high risk individual will experience an event despite being on aspirin therapy 6. Based on this fact, the concept of aspirin resistance has emerged. Few studies have estimated that 5% to 45% of patients with vascular disease are aspirin resistant This variability in incidence is due to non-standardisation of the method and definition of aspirin resistance. Optical platelet aggregation, which, although tedious to accomplish, is considered the gold standard; it utilises a modified spectrophotometer in which platelet-rich plasma is assessed using optical density changes which detect photo electrically as platelets begin to aggregate 10. Aspirin resistance is widely defined as a mean aggregation of 70% with 10 µm adenosine diphosphate (ADP), and a mean aggregation of 20% with 0.5 mg/ml arachidonic acid (AA). Aspirin semi-responders are defined as those meeting only one of the criterias 10. This is a stringent definition, as other studies have defined aspirin resistance by merely a lack of aggregation inhibition by ADP. Mechanisms of aspirin resistance The exact mechanism of aspirin resistance remains elusive. Some factors that have been proposed are increased platelet sensitivity to collagen, increased COX-2 activity, and the platelet alloantigen 2 (PLA2) polymorphism of platelet glycoprotein IIIa. Other mechanisms that have been proposed are poor patient compliance, poor gastrointestinal aspirin absorption, increased isoprostane activity, and a COX-1 polymorphism. However, absorption and compliance present a bioavailability issue and should be distinguished from purported issues of pharmacodynamic resistance. Collagen is a potent platelet agonist and is often the first to initiate the process of platelet aggregation as platelets bind to exposed intimal collagen in an area of vascular injury. Therefore, platelets that are oversensitive to collagen supersede any inhibitory effect conferred by the aspirin. Altered expression of COX-2 has been implicated as a possible source of aspirin-insensitive TXA 2 production. COX-2 is upregulated fold by inflammatory stimuli in most tissues, including monocytes, macrophages, vascular epithelium, and platelets 11. A dose of aspirin 650 mg is required to reduce COX-2-mediated eicosanoid production. The association of aspirin resistance with COX- * Department of Cardiology, King George Medical University, Lucknow , Uttar Pradesh.

2 2 expression was investigated in 93 patients who underwent coronary artery bypass graft (CABG) surgery 12. Compared with baseline, a significant increase in platelet aggregation despite the presence of aspirin was noted. This increase coincided with a 15-fold increase in COX-2 protein expression. By day 10, platelet aggregation and COX-2 expression returned to baseline levels. These results suggest a relationship between upregulation of COX-2 expression and transient aspirin resistance after CABG. However, similar studies involving long-term aspirin therapy have not been conducted, and the role of COX-2 in the development of aspirin resistance remains unclear. PLA2 polymorphism has been identified in glycoprotein IIIa, which constitutes half the fibrinogen receptor glycoprotein IIb-IIIa complex. The hyperaggregability conferred by the PLA2 allele has been attributed to increased surface expression of glycoprotein IIb-IIIa receptors and an increased affinity for fibrinogen 13,14. In addition, the presence of the PLA2 allele has also been associated with a greater risk of coronary events 15, 16, although this is not universally accepted 17. These studies indicate a possible role of the PLA2 allele in aspirin resistance based on measures of platelet function, particularly in patients homozygous for PLA2. The proposed mechanisms for the aspirin resistance can also be broadly classified into extrinsic and intrinsic factors 18 (Table I). Table I: Proposed mechanisms of aspirin resistance. I. Extrinsic Mechanisms A. Accentuation of platelet thrombosis by exogenous substance (e.g., smoking). B. Drugs, e.g., NSAIDs may interact with aspirin s acetylation of COX-I. C. Increased platelet turnover. D. Inadequate aspirin dosing. I. Intrinsic Mechanisms A. Inducible COX-2, not adequately inhibited by low dose aspirin, thereby allowing for platelet thromboxane A 2 production despite inhibition of COX-1. B. Polymorphisms in the COX-1 gene. C. Uninhibited COX-1 in nucleated cells, e.g., macrophage and vascular endothelium producing PGH 2 is shunted into platelet and bypasses platelet COX-1. D. Polymorphisms in the glycoprotein IIb/IIIa receptor complex. Classification of aspirin resistance Weber et al 19 classified aspirin resistance into 3 types based on biochemical and functional in vitro studies. In aspirin responders, an oral intake of 100 mg/day of aspirin for 5 days resulted in more than 95% inhibition of thromboxane synthesis and also in the inhibition of collagen induced platelet aggregation measured in vitro. In type I resistance, there was no inhibition of either thromboxane synthesis or collagen induced platelet aggregation with oral aspirin intake for 5 days. However, the in vitro addition of aspirin into the platelet rich plasma showed remarkable change in parameters. This suggests considerable variation in pharmacokinetics with low dose aspirin, and hence type I resistance is called as pharmacokinetic type resistance. In type II resistance, both the platelet functions were altered neither by the oral intake of aspirin nor by the in vitro addition of aspirin. The mechanism of this type of resistance is unclear, but may relate to genetic polymorphism of the enzymatic pathway and its sensitivity to aspirin hence called as pharmacodynamic type resistance. In type III resistance (pseudoresistance), oral treatment resulted in the inhibition of thromboxane synthesis. However, this was not accompanied by the expected inhibition of platelet aggregation in response to collagen. The lack of effect of platelet aggregation in response to collagen despite thromboxane synthesis was not corrected by the addition of aspirin in vitro. This is labelled as pseudoresistance, since aspirin did exert its putative effect of inhibition of platelet thromboxane synthesis, but failed to alter other important in vitro antiplatelet effects. This may be due to increased sensitivity of platelets to collagen. It is proposed that patients with type I resistance may benefit from increasing the dose of aspirin, while type II and III resistance need other antiplatelet drugs like clopidogrel. However, clinical significance of this classification has not been prospectively tested (Table II). Measurement of aspirin resistance No clinically validated, uniformly accepted method exists to assess the effect of aspirin on platelet aggregation at present. In vitro platelet aggregation studies using optical platelet aggregometer have traditionally been used. Another method that has been used extensively is measurement of the urinary thromboxane A2 (TXA2) Journal, Indian Academy of Clinical Medicine Vol. 8, No. 1 January-March,

3 metabolite 11-dehydro- thromboxane B2 (TXB2) using both radioimmunoassay (RIA) and enzyme immunoassay (EIA). A two automated, point-of-care platelet function assays have also been studied, the Platelet Function Analyzer 100 (PFA-100) and the VerifyNow Aspirin Assay. metabolite of TXB2 that can be measured non-invasively in the urine and can serve as an indirect measure of TXA2 activity in vivo 26. Reduced levels of these products would be expected as a result of aspirin administration. This assay is advantageous because it is noninvasive and is normalised Table II: Typological classification of aspirin resistance. Oral aspirin treatment (100 mg/day, 5 days) Additionofinvitroaspirin(100mg) Platelet thromboxane Collagen-inducedplatelet Platelet thromboxane Collagen-inducedplatelet inhibition aggregation inhibition inhibition aggregation inhibition Aspirinresponders Yes Yes TypeIresistance(pharmacokinetic) No No Yes Yes TypeIIresistance(pharmacodynamic) No No Partial Insignificant TypeIIIresistance(pseudoresistance) Yes No Noadditiveeffect No Platelet aggregation with optical aggregometer: Traditional platelet aggregation studies are performed with platelet-rich plasma prepared from a citrated whole blood sample 20, 21. Platelet aggregation is determined using an optical platelet aggregometer. This method relies on the concept that platelet aggregation in a uniform solution of platelet-rich plasma decreases the turbidity of the solution as the platelets cross-link and clump together. The amount of platelet aggregation is directly related to the amount of light that is allowed to be transmitted through the solution. Aggregation is induced by the addition of a single platelet agonist to the platelet-rich plasma at standard concentrations; typical agonists used are ADP, arachidonic acid, collagen, epinephrine, and thrombin. Aspirin almost completely inhibits platelet aggregation induced by arachidonic acid and collagen, but aggregation in response to ADP and epinephrine is only partially inhibited Platelet aggregation is sensitive to changes in temperature and hydrogen-ion concentration, and must be conducted within hours of sample collection. The technical difficulties and cumbersome nature of the assay require a specialised laboratory setting to perform optical platelet aggregation. Urinary 11-dehydro-TXB2 levels: Another approach to quantify the activity of aspirin has been to measure the levels of the products of COX-1 enzyme action. Thromboxane A2 is rapidly hydrated to form the more stable TXB2, which is subsequently converted by the liver into two major metabolites: 2, 3-dinor TXB2 and 11-dehydro- TXB2 23, 24. Both metabolites, alongwith TXB2, are excreted unchanged in the urine dehydro-TXB2 is a stable with standard controls. However, it is based on a retrospective case-control study 27 in which the frequencies of significant risk factors for cardiovascular disease were higher in the case group than in controls. Platelet function analyzer 100: The PFA-100 is a whole blood, point-of-care platelet aggregometer that has been investigated for measurement of platelet dysfunction as a result of disease and in response to anti-platelet agents 28. The system functions by aspirating a blood sample through a capillary tube at a high shear rate, and through a small slit aperture that is cut into a membrane coated with either collagen-epinephrine (CEPI) or collagen-adp (CADP) platelet agonists 28, 29. In a study of 31 patients with peripheral arterial disease who started receiving aspirin 100 mg/day, 40% of patients were non-responsive when CEPI closure time was measured with the PFA In a subset of patients enrolled in the second Warfarin-Aspirin Reinfarction Study (WARIS), 35% of patients taking aspirin were aspirin resistant as determined by PFA-100. The PFA-100 is a convenient point-of-care device for measurement of platelet aggregation; however, its role in measurement of aspirin resistance has not been satisfactorily explored. In addition, aspirin resistance measured by the PFA-100 has been only weakly correlated with an increased risk of clinical events in one observational cohort study 31. VerifyNow aspirin assay: Like the PFA-100, the VerifyNow aspirin assay is a whole blood, point-of-care device that measures platelet aggregation using different cartridges for different applications. Unlike the PFA-100, the VerifyNow is designed only to detect platelet dysfunction as the result 74 Journal, Indian Academy of Clinical Medicine Vol. 8, No. 1 January-March, 2007

4 of exposure to anti-platelet agents, such as aspirin, clopidogrel, and glycoprotein IIb-IIIa inhibitors. When first developed, the VerifyNow used a proprietary platelet agonist consisting of metallic cations and propyl gallate, but it was recently changed to use an arachidonic acid agonist 32. Platelet aggregation detection is based on the agglutination of platelets on fibrinogen-coated beads detected by an optical turbidimetry method. Management of aspirin resistance At present, there are no specific guidelines for management of aspirin resistance. Assessing patient s compliance will be the logical first step. The optimal dose of aspirin has been controversial in the past. There is no convincing evidence showing that the anti-thrombotic effect of aspirin is dose related. The meta-analysis by Anti-thrombotic Trialist s Collaboration refuted the claim that high doses of aspirin (500-1,500 mg/day) were effective than low doses ( mg/day). Another strategy of circumventing the aspirin resistance is by addition of other newer anti-platelet drugs. In CAPRIE trial 33 they have shown superior clinical benefit of combination of aspirin and clopidogrel compared with aspirin alone. Since clopidogrel inhibits another pathway of platelet activation, combination of both drugs are logical. However, till date, it is not clear whether the superiority of a combination of clopidogrel and aspirin over aspirin is due to clopidogrel compensation for aspirin non-responders 31. Even resistance to clopidogrel is present in recent literature 34, 35. So the question, whether a combination of anti-platelet therapy be accepted in every high risk patient, is still unanswered. Aspirin dosage According to the Anti-thrombotic Trialists Collaboration, daily doses of aspirin mg are as efficacious as higher doses for prevention of thrombotic events and involve a lower risk of bleeding 6. A dose of aspirin as low as 100 mg has completely inhibited the COX-1 enzyme 36, evidence indicates that patients with resistance established during low-dose aspirin therapy may respond to higher doses. An evaluation was conducted in patients with secondary stroke whose aspirin therapy failed 37. Results revealed that aspirin 500 mg/day significantly prolonged the time between first and second stroke (p = 0.002) compared with lower doses. In a study of aspirin resistance determined by in vitro optical platelet aggregometry, 14 patients did not become sensitive despite titration to aspirin 1,300 mg/day. A similar study by the same authors showed that an increase to aspirin 650 mg/day in five patients who were aspirin resistant with 325 mg/day yielded aspirin sensitivity 38. In another study 39, three patients remained resistant with aspirin 1,300 mg, which indicates that inadequate dose cannot explain aspirin resistance in all patients. Current evidence Till now the clinical data available on aspirin resistance is scarce. Data using varying methodologies have given inconsistent results with aspirin resistance ranging from 5 to 50% depending on the type of test used In fact a study by Tantry et al showed that only one out of 143 patients studied was aspirin resistant 40. In a study by Gum et al, aspirin resistance was found in 5.5%, while 23.3% were semiresponders, thus giving an inadequate response of 28.8% 10. The patients were taking 325 mg of aspirin and patients who showed inadequate response were more likely to be females (34.4% v/s 17.3% p = 0.001) and less likely to be smokers (0% v/s 8.3% p = 0.004). There was a trend towards increased age of patients showing inadequate response (65.7 v/s 61.3 yrs, p = 0.06). Gum et al 10 also showed that among stable patients with CAD over a mean follow-up period of 679 ±185 days, aspirin resistance was associated with an increased risk of composite end-points of death, MI, or cerebrovascular accident (p = 0.03), and the multivariate analysis showed aspirin resistance to be a significant independent predictor of long-term major adverse outcome. Hung et al 41 had shown that smoking was significantly associated with aspirin resistance (p < 0.05) 41. A recent study had also shown that aspirin resistance was significantly more in men (p = 0.02) and those using tobacco (p = 0.03) 42. A few long-term studies have suggested the clinical importance of aspirin resistance. In a cohort of stroke patients, it was found that 30% of patients were aspirin non-responders, and after a follow-up of 2 years, major clinical vascular end-points were higher in this group compared to responders (p < ) 9. Another study showed aspirin non-responder status was seen in 34% of patients with recurrent cerebrovascular ischaemic events, despite regular use of aspirin for more than 5 years 31. A subgroup analysis from the HOPE trial, reported higher Journal, Indian Academy of Clinical Medicine Vol. 8, No. 1 January-March,

5 adverse outcomes at a follow-up of 5 years in patients showing aspirin resistance 27. We studied patients who were on 150 mg of aspirin at least for the previous seven days. Fasting blood samples were assessed using optical platelet aggregation. The mean platelet aggregation with 10 µm of adenosine di-phosphate (ADP) in our patient group was 49.42% ± 23.29%, and with 0.5 mg/ml of arachidonic acid (AA) it was 13.58% ± Aspirin resistance was defined as a mean aggregation of 70% with 10 µm ADP and a mean aggregation of 20% with 0.5% mg/ml AA). Aspirin semi-responders were defined as those meeting only one of the criterias. Based on these criterias, 2.08% were found to be aspirin resistant, 39.58% were aspirin semi-responders. Thus in our group, 41.66% patients showed inadequate response to aspirin, i.e., aspirin resistant plus semi-responders 43. In our study, there was no difference related to age (p = 0.2). There was only a trend for females to be semiresponders (31.67% v/s 10.7%, p = 0.08). We could not find any statistically significant difference related to smoking among the groups( p = 0.2). All other parameters tested including diabetes, hypertension, obesity, lipid fractions, haemoglobin concentration, platelet count, ejection fraction and concomitant drug intake did not show any statistically significant difference among the groups 43. Thus, it was shown that inadequate response to aspirin is prevalent in Indian patients, and there are no predictors for this condition. The diagnosis is primarily laboratory based, and this incomplete therapeutic response may be of clinical importance. Although, much is currently known about aspirin s effect on platelets, the mechanism by which some platelets are resistant has not been established. The proposed mechanisms for the aspirin resistance can be broadly classified into extrinsic and intrinsic factors 18. The extrinsic factors like smoking have shown to accentuate platelet thrombosis 41, 42. However, some studies have also refuted this claim by showing that aspirin resistance was less likely among smokers 10. Other factors such as use of NSAIDs, which act through the same pathway, may compete with aspirin 44. Thus, we need to formulate a policy on aspirin and ascertain whether all patients taking aspirin need to be investigated, whether all patients with so-called aspirin resistance be put on clopidogrel, and lastly, whether there is a serious issue of clopidogrel resistance at hand as well. We also foresee further advancements in the diagnostic tests for aspirin resistance like PFA-100, and estimation of 17 hydroxy TXA 2 which are user friendly, and once these are commercially available, the true picture of aspirin resistance may come to light. References 1. Jack DE. One hundred years of aspirin. Lancet 1997; 350: Vane JR. Inhibition of prostaglandin syntheses as a mechanism of action for aspirin-like drugs. Nat New Biol 1971; 231: Second International Study of Infarct Survival Collaborative Group Randomised trial of intravenous steptokinase, oral aspirin, both or neither among 17,187 cases of suspected acute myocardial infarctions: ISIS-2. Lancet 1988; 2: Anti-platelet Trialists Collaboration. Collaborative overview of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. Br Med J 1994; 308: Anti-platelet Trialists Collaboration. Collaborative overview of randomised trials of antiplatelet therapy-iii: reduction in venous thrombosis and pulmonary embolism by antiplatelet prophylaxis among surgical and medical patients. Br Med J 1994; 308: Anti-platelet Trialists Collaboration. Meta analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Br Med J 2002; 324: Helgason CM, Bolin KM, Hoff JA et al. Development of aspirin resistance in persons with previous ischaemic stroke. Stroke 1994; 25: Pappas JM, Westengard JC, Bull BS. Population variability in the effect of aspirin on platelet function. Implications for clinical trials and therapy. Arch Pathol Lab Med 1994; 118: Grotemeyer KH, Scharafinski HW, Husstedt IW. Two-year follow-up of aspirin responders, and aspirin nonresponders. A pilot-study including 18 post-stroke patients. Thromb Res 1989; 20: Gum PA, Kottke-Marchant K, Poggio ED et al. Profile and prevalence of aspirin resistance in patients with cardiovascular disease. American Journal Cardiology 2001; 88: Weber AA, Zimmerman KC, Myerer-Kirchrath J, Schror K. Cyclooxygenase-2 in human platelets as a possible factor in aspirin resistance. Lancet 1999; 353: Zimmerman N, Wenk A, Kim U et al. Functional and biochemical evaluation of platelet-aspirin resistance 76 Journal, Indian Academy of Clinical Medicine Vol. 8, No. 1 January-March, 2007

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