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Aspirin “resistance”
Blood Cells, Molecules, and Diseases 36 (2006) 171 – 176 www.elsevier.com/locate/ybcmd
Aspirin “resistance” Karsten Schrör ⁎, Artur-Aron Weber, Thomas Hohlfeld Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Universitätsstr. 1, Geb. 22.21, D-40225 Düsseldorf, Germany Submitted 6 December 2005 Available online 15 February 2006 (Communicated by M. Lichtman, M.D., 19 December 2005)
Abstract A variable responsiveness to antiplatelet drugs is a clinical phenomenon that does not principally differ from other drug treatments in other therapeutic fields. The pharmacological part is to clarify whether a “true” resistance exists in pharmacological terms, i.e., a reduced potency of the compound to work as suggested and to find out the underlying cellular mechanism(s). Two principally different methods of laboratory control for platelet sensitivity to aspirin (ASA) are available: measurement of platelet function (ex vivo) or measurement of inhibition of thromboxane formation. Both methods have limitations and did not yet result in a generally accepted definition of a pharmacological ASA “resistance”. The new typological approach of Weber et al. [A.A. Weber, B. Przytulski, A. Schanz, et al., Towards a definition of aspirin resistance: a typological approach. Platelets 13 (2002) 37. [1]] helps to identify different subtypes of ASA resistance in pharmacological terms by combining in vitro aggregometry with thromboxane measurement. Using this method, a “true” pharmacological resistance, associated with a reduced antiplatelet response to ASA and reduced inhibition of thromboxane formation, was found in patients undergoing coronary artery bypass surgery. Platelets of these patients expressed a hitherto unknown isoform of COX-2-COX-2a which might generate a different gene product. In this context, it is interesting that CABG patients express transiently an immunoreactive COX-2 protein with lower molecular weight. Studies on the significance of this finding for ASA resistance are in progress. © 2006 Elsevier Inc. All rights reserved. Keywords: Aspirin resistance; Thromboxane; Platelet function; Myocardial infarction; Stroke
Introduction Aspirin (ASA) “resistance” is currently an issue of clinical concern although the problem of treatment failure is neither new [2] nor specific for ASA. A comparable number of treatment failures—though on a different background-are also seen with clopidogrel [3]. Dependent on the method of measurement and the definition used, approximately 5–45% of cardiovascular [4] and 5–65% of stroke patients [5] are considered ASA resistant. This large variation already suggests that there are probably many more treatment failures with ASA (including missing compliance) than can be explained by a reduced antiplatelet effect for pharmacological reasons [5]. With other words,
⁎ Corresponding author. Fax: +49 211 81 14781. E-mail address: [email protected] (K. Schrör). 1079-9796/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.12.017
clinical treatment failure and a failure of ASA to work pharmacologically are no synonyms [6] but, unfortunately, are frequently mixed up in an inappropriate way [7]. Table 1 is an overview on mechanisms of pharmacological ASA resistance. Antiplatelet effects of ASA are variable, especially at lower doses [8–10]. At least in some studies, preferentially stroke patients [11,12] and patients with peripheral arterial occlusive disease [13], a reduced ASA responsiveness was described which was overcome by increasing the dose. While this statement may be at variance with the overall data and recommendations of the Antithrombotic Trialists' Collaboration [14], one should also consider that the disease-related efficacy of ASA in secondary prevention varies between 0 and 50%—on average 25%. Consequently, the efficacy of atherothrombotic prophylaxis by ASA could possibly be improved by positively identifying sensitive patients and to transfer subjects who do not respond sufficiently to an alternative antiplatelet therapy. This
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K. Schrör et al. / Blood Cells, Molecules, and Diseases 36 (2006) 171–176
Table 1 Mechanisms of pharmacological ASA resistance Drug related Pharmacokinetics Insufficient bioavailability Prevention of binding to the Ser529 by other NSAID (ibuprofen, indomethacin, naproxen) Pharmacodynamics Impaired sensitivity of platelet COX-1 (CABG) Gene polymorphism(s) Disease related Platelet hyperreactivity due to ASA-insensitive mechanisms Changes in the collagen receptor Platelet “sensitizing” by isoprostanes
would also avoid unnecessary exposition of the patient to possible side-effects of ASA. The current status of ASA “resistance” A mechanistic approach for ASA resistance There is general agreement that (i) the inhibition of (plateletdependent), COX-1-mediated thromboxane A2 (TXA2) formation determines the antiplatelet and antithrombotic efficacy of ASA [15], and (ii) that this inhibition has to be more than 95% in terms of platelet thromboxane forming capacity, as assessed from determination of serum thromboxane levels. Less inhibition is clinically ineffective [16]. Thus, measurement of serum thromboxane may be useful to detect ASA resistance. Surprisingly, this test is not often used in clinical trials, attempted to determine ASA resistance. Although no definite norm values exist, any serum thromboxane formation above 25 ng/ml may be considered insufficient [17]. Another, more popular approach is to measure thromboxane metabolites, such as 11-DH-TXB2, in urine. While this assay will provide information on thromboxane metabolism, it is clearly less specific to ASA-related inhibition of platelet-dependent thromboxane formation and, therefore, highly variable: no standardized normal values exist (see below).
changes in platelet sensitivity by ASA would not capture any potential antiinflammatory effects of the compound although there are relationships to clinical outcome, for example the reduction in C-reactive protein [19]. Platelet tests have also technical limitations. For example, the comparison of two different methods (PFA-100 and optical aggregometry) yielded different results, even in the same patients [20]. In the absence of appropriate controls, i.e., platelet assays prior to ASA ingestion, results may even misqualify patients [3]. The PFA-100 preferentially measures vWFdependent platelet activation at high shear stress and, therefore, is useful for detection of hemophilics. Thus, while measurement of platelet function will help to determine platelet reactivity to standard stimuli and to detect ASA non- or low-responders in vitro [21], the transfer of data to the clinics should be done with extreme caution. Measurement of thromboxane formation as an alternative is available but poorly defined A significant elevation of plasma and urinary 11-DH-TXB2 levels in platelet activation syndromes, such as unstable angina, severe atherosclerosis, peripheral arterial disease and pulmonary embolism is well known [22–24]. This suggests that urinary 11-DH-TXB2 might be a valuable tool to determine thromboxane formation within the cardiovascular system. In 2002, Eikelboom and colleagues [25] were the first to present data from a subgroup of patients from the HOPE trial which have found considerable attention and generated concerns regarding the efficacy of ASA in cardiocoronary prevention. However, this study has several limitations. For example the differences in thromboxane metabolite excretion described by Eikelboom et al. for cases and controls were very small as opposed to the large variation of urinary excretion rates of 11DH-TXB2 including ASA pretreatment. About one third (with large variations) of 11-DH-TXB2 was not of platelet origin. It is also not known as to what extent urinary 11-DH-TXB2 excretion has to be reduced by ASA treatment in order to guarantee antiplatelet ASA efficacy.
Platelet function assays are available but have limitations
A new typological approach to identify subtypes of ASA resistance
A number of laboratory platelet assays are available and increasingly used to study ASA “resistance” [3,18]. The attraction of this approach is the fact that these tests directly study the target, i.e., alterations in platelet function after ASA treatment. However, there are several possible pitfalls that may overweight the benefits and may result in over-interpretations of the results. For example, any study of blood platelets ex vivo or in vitro is done under conditions which differ fundamentally from the in vivo situation. In all of these assays, platelet are removed from this natural environment and the numerous platelet-active factors that circulate in the blood but are labile and already inactivated (NO, prostacyclin), when the measurement of platelet function is performed. In addition, an analysis of
Weber et al. [1] have made a proposal to classify subtypes of ASA “resistance” in pharmacological terms by simultaneous measurement of thromboxane formation and thromboxanedependent platelet aggregation in one and the same sample in vitro (Fig. 1). Type I resistance, i.e., a failure of ASA to work in vivo but normal responses to added ASA in vitro in terms of both, inhibition of aggregation and thromboxane formation, suggests a pharmacokinetic type of ASA resistance. A likely explanation is missing compliance [26]. Another pharmacokinetic reason for insufficient antiplatelet effects of ASA might be an interference of certain NSAIDs, for example, indomethacin, ibuprofen, and naproxen, with the ASA binding at Ser529 in the human COX-1. Binding of one of the
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Fig. 1. Typology of ASA resistance according to Weber et al. [1].
reversible inhibitors to this site will prevent the (irreversible) acetylation by ASA. Because of the short half-life of ASA (15 min), the active compound will be degraded to salicylic acid by aspirinases in the blood. When the reversible inhibitor leaves the binding site and becomes degraded, no active ASA may be available anymore [27]. This explains the observation by Rao et al. [28] that ibuprofen pretreatment makes platelets insensitive to ASA when given shortly before (Fig. 2). This mechanism is not restricted to ibuprofen but has also been shown for indomethacin but not diclofenac. The possible clinical significance of this finding is underlined by the fact that three epidemiological studies provided evidence for a clinically
important pharmacological interaction between ASA and ibuprofen [29]. Type II resistance was found in platelets of CABG patients which are largely resistant to conventional oral doses of ASA, i.e., 100 mg/day shortly after the surgical procedure [21]. It was also shown that these ASA “resistant” platelets could be blocked by the combined inhibition of thromboxane synthase and thromboxane receptors (Fig. 3). This suggested that the inhibition of thromboxane formation by ASA was incomplete at the daily dose of 100 mg in vivo, and that this was an explanation for the pharmacodynamic failure. Interestingly, this was not COX-2-related; although human platelets exhibit
Fig. 2. Prevention of ASA-induced inhibition of Arachidonic acid (AA)-induced platelet aggregation and thromboxane formation by previous ibuprofen. Ibuprofen (5 mg/kg) completely prevented AA-induced platelet aggregation and thromboxane formation after 1 h (A) but failed to do so after 24 h (B). ASA (650 mg single dose) also completely prevented AA-induced platelet aggregation and thromboxane formation both after 1 and 24 h (C). This antiplatelet effect of ASA was prevented by previous ibuprofen treatment (D). The number in brackets indicates the % conversion of AA into thromboxane. The circle (●) marks addition of AA (Modified after Rao et al. [28]).
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Fig. 3. Arachidonic acid (AA)-induced platelet aggregation before (day 0) and after (day 5) coronary artery bypass surgery. Different effects of ASA and Terbogrel (combined thromboxane synthase inhibitor and receptor antagonist) on platelet aggregation (optical assay) in vitro (modified after Zimmermann et al. [21]).
immunoreactive COX-2 [30]. This largely ASA-resistant isoform of immunoreactive COX-2 was temporarily 10–15 times over-expressed in platelets of CABG patients [1,30]. The molecular mechanisms are still under investigation but possibly involve expression of a new COX-isoform (COX-2a) which becomes up-regulated in the platelets of these patients [31] (Fig. 4). These data suggest that the clinical outcome of patients, i.e., possible treatment failure of ASA, may have some relation to a true pharmacological “resistance” against ASA in terms of inhibition of platelet function or thromboxane formation. In that case, one might expect that increase in dosing may overcome a reduced responsiveness of the prothrombotic cascade. There is preliminary clinical evidence for this in bypass surgery [32] but also in patients with stable coronary heart disease [33], stroke [11,12], and
peripheral arterial disease [13]. This suggests that higher doses of ASA may be more effective than lower in certain subgroups of patients. These patients can be identified and transferred to another antiplatelet or anticoagulant therapy, respectively. Type III resistance identifies platelets where ASA does work as suggested but does not inhibit platelet function or thromboxane formation. The explanation is stimulation of platelets by factors that are ASA-independent. Formation of isoprostanes is one explanation. Isoprostanes are arachidonic acid metabolites that are formed by non-enzymatic, free radical catalyzed reactions and cause platelet activation via the platelet thromboxane (TP) receptor [34]. These compounds are increasingly generated in vivo when cyclooxygenase activation and oxidant stress coincide, for example, in stable and unstable angina [24] or type II diabetes [35].
Fig. 4. Expression of a smaller sized COX-2 mRNA (COX-2a) in patients at day 5 after coronary artery bypass grafting (A). This is paralleled by expression of an immunoreactive COX-2 protein with slightly smaller molecular weight (COX-2a) than recombinant COX-2 (rCOX-2) (B). Cloning of cDNA of COX-2 and COX-2a into pUC19 vector yields two different clones (C). Sequencing of these clones demonstrates a loss of ca. 100 bp in exon 5 resulting in a frame shift (D) (modified after Censarek et al. [31]).
K. Schrör et al. / Blood Cells, Molecules, and Diseases 36 (2006) 171–176
The activation of the platelet thromboxane receptor by isoprostanes is not sensitive to ASA although the circulating concentrations of these compounds are probably too low to cause platelet activation. Possibly, isoprostanes synergize with other platelet activating factors in conditions of enhanced oxidative stress. Finally, several gene polymorphisms relevant to platelet activation have been described [36] including modifications of COX-1 [37] but also the platelet GPIIIa receptor and the receptors for collagen and vWF. All of these may influence the platelet sensitivity to ASA but not necessarily antagonize the inhibitory action of ASA on thromboxane formation. No definition of aspirin resistance From a semantic point of view, the term Aspirin “resistance” is a misnomer and does not address the issue adequately. In particular, clinical treatment failure is not a sufficient surrogate parameter for the pharmacological failure of the drug to act. Furthermore, there is no mono-causal ASA “resistance”, neither in clinical nor in pharmacological terms. Thus, it seems to be premature and unwarranted to suggest that measurement of aggregation or thromboxane generation after dosing with ASA could be used to reliably predict clinical outcome [6]. Open questions There are only a few trials connecting the clinical outcome of patients at elevated vascular risk with a possible ASA “resistance” as outlined above [13,25,38]. A recent clinical meta-analysis of ASA treatment after coronary artery surgery has shown that medium dose ASA (300–325 mg) was about twice as effective as low dose ASA (75–150 mg)—45% vs. 26% risk reduction in preventing graft occlusion in bypass patients [32]. Gum and colleagues [20,39] reported ASA “resistance” as seen by platelet aggregometry in 17 out of 326 patients with stable cardiovascular disease. These patients were found to have a three-fold higher risk of a cardiovascular event than those who were not “resistant” according to the study criteria. However, this difference was only seen with optical aggregometry but not by the PFA-100 assay. Although a correlation was also found by others between reduced ASA sensitivity and coronary heart disease [40], it is questioned whether a variable degree of hyperreactivity or reduced sensitivity against weak platelet agonists, such as ADP, really indicates a possible therapeutic failure of ASA or just a different severity of the disease. Thus, it is uncertain, whether, and if so, for what extent ASA resistance, i.e., a failure of ASA to work in vivo that is to inhibit thromboxane formation and to prevent platelet-dependent thrombus formation, can be predicted from laboratory assays, measuring platelet function. Measurement of thromboxane formation is an alternative. However, the significance of these data has to be established in prospective, randomized controlled clinical trials in predefined groups of patients.
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K. Schrör et al. / Blood Cells, Molecules, and Diseases 36 (2006) 171–176 insensitive thromboxane biosynthesis in severe unstable angina, Circulation 102 (2000) 1007. J.W. Eikelboom, J. Hirsh, J.I. Weitz, et al., Aspirin-resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke or cardiovascular death in patients at high risk for cardiovascular events, Circulation 105 (2002) 1650. K.A. Schwartz, D.E. Schwartz, E. Ghosheh, et al., Compliance as a critical consideration in patients who appear to be resistant to aspirin after healing of myocardial infarction, J. Am. Cardiol. 95 (2005) 973. F. Catella-Lawson, M.P. Reilly, S.C. Kapoor, et al., Cyclooxygenase inhibitors and the antiplatelet effect of aspirin, N. Engl. J. Med. 345 (2001) 1809. G.H. Rao, G.G. Johnson, K.R. Reddy, J.G. White, Ibuprofen protects platelet cyclooxygenase from irreversible inhibition by aspirin, Arteriosclerosis 3 (1983) 383. G.A. FitzGerald, Parsing an enigma: the pharmacodynamics of aspirin resistance, Lancet 361 (2003) 542. A.A. Weber, K.C. Zimmermann, J. Meyer-Kirchrath, K. Schrör, Cyclooxygenase-2 in human platelets as a possible factor in aspirin resistance, Lancet 353 (1999) 900. P. Censarek, K. Freidel, M. Udelhoven, S.-J. Ku, et al., Cyclooxygenase COX-2a, a novel COX-2 mRNA variant, in platelets from patients after coronary artery bypass grafting, Thromb. Haemost. 92 (2004) 925. E. Lim, Z. Ali, A. Ali, et al., Indirect comparison meta-analysis of aspirin therapy after coronary surgery, Br. J. Med 327 (2003) 1309.
[33] P.Y. Lee, W.H. Chen, W. Ng, et al., Low-dose aspirin increases aspirin resistance in patients with coronary artery disease, Am. J. Med. 118 (2005) 723. [34] L.P. Audoly, B. Rocca, J.E. Fabre, et al., Cardiovascular responses to the isoprostanes iPF (2α)-III and iPE (2)-III are mediated via the thromboxane A (2) receptor in vivo, Circulation 101 (2000) 2833. [35] E. Vericel, C. Januel, M. Carreras, et al., Diabetic patients without vascular complications display enhanced basal platelet activation and decreased antioxidant status, Diabetes 53 (2004) 1046. [36] J.A. Cambria-Kiely, P.J. Gandhi, Aspirin resistance and genetic polymorphism, J. Thromb. Thrombolysis 14 (2002) 51. [37] M.K. Halushka, M.P. Walker, P.V. Halushka, Genetic variation in cyclooxygenase 1: effects on response to aspirin, Clin. Pharmacol. Ther. 73 (2003) 122. [38] W.H. Chen, P.Y. Lee, W. Ng, et al., Aspirin resistance is associated with a high incidence of myonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment, J. Am. Coll. Cardiol. 43 (2004) 1122. [39] P.A. Gum, K. Kottke-Marchant, P.A. Welsh, et al., A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease, J. Am. Coll. Cardiol. 41 (2003) 961. [40] J.C. Wang, D. Aucoin, D. Manuelian, et al., Incidence of aspirin nonresponsiveness using the Ultegra Rapid Platelet Function AssayASA, J. Am. Cardiol. 92 (2003) 1492.
Aspirin “resistance” Karsten Schrör ⁎, Artur-Aron Weber, Thomas Hohlfeld Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Universitätsstr. 1, Geb. 22.21, D-40225 Düsseldorf, Germany Submitted 6 December 2005 Available online 15 February 2006 (Communicated by M. Lichtman, M.D., 19 December 2005)
Abstract A variable responsiveness to antiplatelet drugs is a clinical phenomenon that does not principally differ from other drug treatments in other therapeutic fields. The pharmacological part is to clarify whether a “true” resistance exists in pharmacological terms, i.e., a reduced potency of the compound to work as suggested and to find out the underlying cellular mechanism(s). Two principally different methods of laboratory control for platelet sensitivity to aspirin (ASA) are available: measurement of platelet function (ex vivo) or measurement of inhibition of thromboxane formation. Both methods have limitations and did not yet result in a generally accepted definition of a pharmacological ASA “resistance”. The new typological approach of Weber et al. [A.A. Weber, B. Przytulski, A. Schanz, et al., Towards a definition of aspirin resistance: a typological approach. Platelets 13 (2002) 37. [1]] helps to identify different subtypes of ASA resistance in pharmacological terms by combining in vitro aggregometry with thromboxane measurement. Using this method, a “true” pharmacological resistance, associated with a reduced antiplatelet response to ASA and reduced inhibition of thromboxane formation, was found in patients undergoing coronary artery bypass surgery. Platelets of these patients expressed a hitherto unknown isoform of COX-2-COX-2a which might generate a different gene product. In this context, it is interesting that CABG patients express transiently an immunoreactive COX-2 protein with lower molecular weight. Studies on the significance of this finding for ASA resistance are in progress. © 2006 Elsevier Inc. All rights reserved. Keywords: Aspirin resistance; Thromboxane; Platelet function; Myocardial infarction; Stroke
Introduction Aspirin (ASA) “resistance” is currently an issue of clinical concern although the problem of treatment failure is neither new [2] nor specific for ASA. A comparable number of treatment failures—though on a different background-are also seen with clopidogrel [3]. Dependent on the method of measurement and the definition used, approximately 5–45% of cardiovascular [4] and 5–65% of stroke patients [5] are considered ASA resistant. This large variation already suggests that there are probably many more treatment failures with ASA (including missing compliance) than can be explained by a reduced antiplatelet effect for pharmacological reasons [5]. With other words,
⁎ Corresponding author. Fax: +49 211 81 14781. E-mail address: [email protected] (K. Schrör). 1079-9796/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.12.017
clinical treatment failure and a failure of ASA to work pharmacologically are no synonyms [6] but, unfortunately, are frequently mixed up in an inappropriate way [7]. Table 1 is an overview on mechanisms of pharmacological ASA resistance. Antiplatelet effects of ASA are variable, especially at lower doses [8–10]. At least in some studies, preferentially stroke patients [11,12] and patients with peripheral arterial occlusive disease [13], a reduced ASA responsiveness was described which was overcome by increasing the dose. While this statement may be at variance with the overall data and recommendations of the Antithrombotic Trialists' Collaboration [14], one should also consider that the disease-related efficacy of ASA in secondary prevention varies between 0 and 50%—on average 25%. Consequently, the efficacy of atherothrombotic prophylaxis by ASA could possibly be improved by positively identifying sensitive patients and to transfer subjects who do not respond sufficiently to an alternative antiplatelet therapy. This
172
K. Schrör et al. / Blood Cells, Molecules, and Diseases 36 (2006) 171–176
Table 1 Mechanisms of pharmacological ASA resistance Drug related Pharmacokinetics Insufficient bioavailability Prevention of binding to the Ser529 by other NSAID (ibuprofen, indomethacin, naproxen) Pharmacodynamics Impaired sensitivity of platelet COX-1 (CABG) Gene polymorphism(s) Disease related Platelet hyperreactivity due to ASA-insensitive mechanisms Changes in the collagen receptor Platelet “sensitizing” by isoprostanes
would also avoid unnecessary exposition of the patient to possible side-effects of ASA. The current status of ASA “resistance” A mechanistic approach for ASA resistance There is general agreement that (i) the inhibition of (plateletdependent), COX-1-mediated thromboxane A2 (TXA2) formation determines the antiplatelet and antithrombotic efficacy of ASA [15], and (ii) that this inhibition has to be more than 95% in terms of platelet thromboxane forming capacity, as assessed from determination of serum thromboxane levels. Less inhibition is clinically ineffective [16]. Thus, measurement of serum thromboxane may be useful to detect ASA resistance. Surprisingly, this test is not often used in clinical trials, attempted to determine ASA resistance. Although no definite norm values exist, any serum thromboxane formation above 25 ng/ml may be considered insufficient [17]. Another, more popular approach is to measure thromboxane metabolites, such as 11-DH-TXB2, in urine. While this assay will provide information on thromboxane metabolism, it is clearly less specific to ASA-related inhibition of platelet-dependent thromboxane formation and, therefore, highly variable: no standardized normal values exist (see below).
changes in platelet sensitivity by ASA would not capture any potential antiinflammatory effects of the compound although there are relationships to clinical outcome, for example the reduction in C-reactive protein [19]. Platelet tests have also technical limitations. For example, the comparison of two different methods (PFA-100 and optical aggregometry) yielded different results, even in the same patients [20]. In the absence of appropriate controls, i.e., platelet assays prior to ASA ingestion, results may even misqualify patients [3]. The PFA-100 preferentially measures vWFdependent platelet activation at high shear stress and, therefore, is useful for detection of hemophilics. Thus, while measurement of platelet function will help to determine platelet reactivity to standard stimuli and to detect ASA non- or low-responders in vitro [21], the transfer of data to the clinics should be done with extreme caution. Measurement of thromboxane formation as an alternative is available but poorly defined A significant elevation of plasma and urinary 11-DH-TXB2 levels in platelet activation syndromes, such as unstable angina, severe atherosclerosis, peripheral arterial disease and pulmonary embolism is well known [22–24]. This suggests that urinary 11-DH-TXB2 might be a valuable tool to determine thromboxane formation within the cardiovascular system. In 2002, Eikelboom and colleagues [25] were the first to present data from a subgroup of patients from the HOPE trial which have found considerable attention and generated concerns regarding the efficacy of ASA in cardiocoronary prevention. However, this study has several limitations. For example the differences in thromboxane metabolite excretion described by Eikelboom et al. for cases and controls were very small as opposed to the large variation of urinary excretion rates of 11DH-TXB2 including ASA pretreatment. About one third (with large variations) of 11-DH-TXB2 was not of platelet origin. It is also not known as to what extent urinary 11-DH-TXB2 excretion has to be reduced by ASA treatment in order to guarantee antiplatelet ASA efficacy.
Platelet function assays are available but have limitations
A new typological approach to identify subtypes of ASA resistance
A number of laboratory platelet assays are available and increasingly used to study ASA “resistance” [3,18]. The attraction of this approach is the fact that these tests directly study the target, i.e., alterations in platelet function after ASA treatment. However, there are several possible pitfalls that may overweight the benefits and may result in over-interpretations of the results. For example, any study of blood platelets ex vivo or in vitro is done under conditions which differ fundamentally from the in vivo situation. In all of these assays, platelet are removed from this natural environment and the numerous platelet-active factors that circulate in the blood but are labile and already inactivated (NO, prostacyclin), when the measurement of platelet function is performed. In addition, an analysis of
Weber et al. [1] have made a proposal to classify subtypes of ASA “resistance” in pharmacological terms by simultaneous measurement of thromboxane formation and thromboxanedependent platelet aggregation in one and the same sample in vitro (Fig. 1). Type I resistance, i.e., a failure of ASA to work in vivo but normal responses to added ASA in vitro in terms of both, inhibition of aggregation and thromboxane formation, suggests a pharmacokinetic type of ASA resistance. A likely explanation is missing compliance [26]. Another pharmacokinetic reason for insufficient antiplatelet effects of ASA might be an interference of certain NSAIDs, for example, indomethacin, ibuprofen, and naproxen, with the ASA binding at Ser529 in the human COX-1. Binding of one of the
K. Schrör et al. / Blood Cells, Molecules, and Diseases 36 (2006) 171–176
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Fig. 1. Typology of ASA resistance according to Weber et al. [1].
reversible inhibitors to this site will prevent the (irreversible) acetylation by ASA. Because of the short half-life of ASA (15 min), the active compound will be degraded to salicylic acid by aspirinases in the blood. When the reversible inhibitor leaves the binding site and becomes degraded, no active ASA may be available anymore [27]. This explains the observation by Rao et al. [28] that ibuprofen pretreatment makes platelets insensitive to ASA when given shortly before (Fig. 2). This mechanism is not restricted to ibuprofen but has also been shown for indomethacin but not diclofenac. The possible clinical significance of this finding is underlined by the fact that three epidemiological studies provided evidence for a clinically
important pharmacological interaction between ASA and ibuprofen [29]. Type II resistance was found in platelets of CABG patients which are largely resistant to conventional oral doses of ASA, i.e., 100 mg/day shortly after the surgical procedure [21]. It was also shown that these ASA “resistant” platelets could be blocked by the combined inhibition of thromboxane synthase and thromboxane receptors (Fig. 3). This suggested that the inhibition of thromboxane formation by ASA was incomplete at the daily dose of 100 mg in vivo, and that this was an explanation for the pharmacodynamic failure. Interestingly, this was not COX-2-related; although human platelets exhibit
Fig. 2. Prevention of ASA-induced inhibition of Arachidonic acid (AA)-induced platelet aggregation and thromboxane formation by previous ibuprofen. Ibuprofen (5 mg/kg) completely prevented AA-induced platelet aggregation and thromboxane formation after 1 h (A) but failed to do so after 24 h (B). ASA (650 mg single dose) also completely prevented AA-induced platelet aggregation and thromboxane formation both after 1 and 24 h (C). This antiplatelet effect of ASA was prevented by previous ibuprofen treatment (D). The number in brackets indicates the % conversion of AA into thromboxane. The circle (●) marks addition of AA (Modified after Rao et al. [28]).
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Fig. 3. Arachidonic acid (AA)-induced platelet aggregation before (day 0) and after (day 5) coronary artery bypass surgery. Different effects of ASA and Terbogrel (combined thromboxane synthase inhibitor and receptor antagonist) on platelet aggregation (optical assay) in vitro (modified after Zimmermann et al. [21]).
immunoreactive COX-2 [30]. This largely ASA-resistant isoform of immunoreactive COX-2 was temporarily 10–15 times over-expressed in platelets of CABG patients [1,30]. The molecular mechanisms are still under investigation but possibly involve expression of a new COX-isoform (COX-2a) which becomes up-regulated in the platelets of these patients [31] (Fig. 4). These data suggest that the clinical outcome of patients, i.e., possible treatment failure of ASA, may have some relation to a true pharmacological “resistance” against ASA in terms of inhibition of platelet function or thromboxane formation. In that case, one might expect that increase in dosing may overcome a reduced responsiveness of the prothrombotic cascade. There is preliminary clinical evidence for this in bypass surgery [32] but also in patients with stable coronary heart disease [33], stroke [11,12], and
peripheral arterial disease [13]. This suggests that higher doses of ASA may be more effective than lower in certain subgroups of patients. These patients can be identified and transferred to another antiplatelet or anticoagulant therapy, respectively. Type III resistance identifies platelets where ASA does work as suggested but does not inhibit platelet function or thromboxane formation. The explanation is stimulation of platelets by factors that are ASA-independent. Formation of isoprostanes is one explanation. Isoprostanes are arachidonic acid metabolites that are formed by non-enzymatic, free radical catalyzed reactions and cause platelet activation via the platelet thromboxane (TP) receptor [34]. These compounds are increasingly generated in vivo when cyclooxygenase activation and oxidant stress coincide, for example, in stable and unstable angina [24] or type II diabetes [35].
Fig. 4. Expression of a smaller sized COX-2 mRNA (COX-2a) in patients at day 5 after coronary artery bypass grafting (A). This is paralleled by expression of an immunoreactive COX-2 protein with slightly smaller molecular weight (COX-2a) than recombinant COX-2 (rCOX-2) (B). Cloning of cDNA of COX-2 and COX-2a into pUC19 vector yields two different clones (C). Sequencing of these clones demonstrates a loss of ca. 100 bp in exon 5 resulting in a frame shift (D) (modified after Censarek et al. [31]).
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The activation of the platelet thromboxane receptor by isoprostanes is not sensitive to ASA although the circulating concentrations of these compounds are probably too low to cause platelet activation. Possibly, isoprostanes synergize with other platelet activating factors in conditions of enhanced oxidative stress. Finally, several gene polymorphisms relevant to platelet activation have been described [36] including modifications of COX-1 [37] but also the platelet GPIIIa receptor and the receptors for collagen and vWF. All of these may influence the platelet sensitivity to ASA but not necessarily antagonize the inhibitory action of ASA on thromboxane formation. No definition of aspirin resistance From a semantic point of view, the term Aspirin “resistance” is a misnomer and does not address the issue adequately. In particular, clinical treatment failure is not a sufficient surrogate parameter for the pharmacological failure of the drug to act. Furthermore, there is no mono-causal ASA “resistance”, neither in clinical nor in pharmacological terms. Thus, it seems to be premature and unwarranted to suggest that measurement of aggregation or thromboxane generation after dosing with ASA could be used to reliably predict clinical outcome [6]. Open questions There are only a few trials connecting the clinical outcome of patients at elevated vascular risk with a possible ASA “resistance” as outlined above [13,25,38]. A recent clinical meta-analysis of ASA treatment after coronary artery surgery has shown that medium dose ASA (300–325 mg) was about twice as effective as low dose ASA (75–150 mg)—45% vs. 26% risk reduction in preventing graft occlusion in bypass patients [32]. Gum and colleagues [20,39] reported ASA “resistance” as seen by platelet aggregometry in 17 out of 326 patients with stable cardiovascular disease. These patients were found to have a three-fold higher risk of a cardiovascular event than those who were not “resistant” according to the study criteria. However, this difference was only seen with optical aggregometry but not by the PFA-100 assay. Although a correlation was also found by others between reduced ASA sensitivity and coronary heart disease [40], it is questioned whether a variable degree of hyperreactivity or reduced sensitivity against weak platelet agonists, such as ADP, really indicates a possible therapeutic failure of ASA or just a different severity of the disease. Thus, it is uncertain, whether, and if so, for what extent ASA resistance, i.e., a failure of ASA to work in vivo that is to inhibit thromboxane formation and to prevent platelet-dependent thrombus formation, can be predicted from laboratory assays, measuring platelet function. Measurement of thromboxane formation is an alternative. However, the significance of these data has to be established in prospective, randomized controlled clinical trials in predefined groups of patients.
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