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Appropriate Assessment of the Functional Consequences of Platelet Cyclooxygenase-1 Inhibition by Aspirin in vivo
Thrombosis Research 133 (2014) 697–698
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Editorial
Appropriate Assessment of the Functional Consequences of Platelet Cyclooxygenase-1 Inhibition by Aspirin in vivo
Introduction Chronic use of low dose aspirin (acetylsalicylic acid) is generally an effective and relatively low cost strategy for preventing and treating cardiovascular disease [1]. John Vane reported the inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs in 1971 by demonstrating a dose-dependent inhibition of prostaglandin synthesis by aspirin, sodium salicylate and indomethacin [2]. Thromboxane A2 (TxA2) was subsequently identified by Samuelsson’s group as the potent vasoconstrictor and platelet agonist the synthesis of which was inhibited by aspirin [3]. The ability of aspirin to prevent platelet activation initiated with arachidonate, and thus TxA2dependent activation of platelets form the basis of the antithrombotic action of aspirin [3]. Aspirin achieves this antiplatelet action by selectively acetylating the hydroxyl group of serine in platelet cyclooxygenase-1 (COX-1) or prostaglandin endoperoxidase synthase to irreversibly inactivate COX-1. This prevents platelet TxA2 synthesis from arachidonate that is released from platelet membranes by phospholipase A2 in response to platelet agonists [4]. All effective antithrombotic drugs can have major adverse effects, and clinically significant drawbacks of chronic aspirin use include gastrointestinal bleeding [5,6] and insufficient antithrombotic efficacy [7] in some patients. Both undesirable effects of aspirin may be attributable to the fact that aspirin also effectively inactivates COX-2 as reported in a recent study demonstrating effective inhibition of both thromboxane A2 and PGI2 inhibition by aspirin of recombinant human COX-1 and COX-2 his study also reported aspirin to dose-dependently inhibit TxA2 and PGI2 synthesis by guinea pig platelet-rich plasma and isolated aortic strips of guinea pigs, respectively [8]. Inactivation of human gastrointestinal COX-1 and COX-2 may partly contribute to the gastrointestinal bleeding associated with chronic use of aspirin [5,6]. The causes of the insufficient antithrombotic efficacy of aspirin found in some patients [7] are not clear. Inactivation of endothelial cell COX-2 by aspirin will prevent endothelial cell synthesis of PGI2, a prostanoid able to prevent platelets from responding to all agonists [9]. The reduced antithrombotic efficacy of chronic aspirin use in some patients has naturally led to ex vivo and in vitro studies designed to identify low responders to chronic aspirin use and establish the likely causes of reduced or non-response to aspirin [10–17]. Specifically, these studies have determined how aspirin ingestion affects the ex vivo responses of patients’ and/or volunteers’ platelets to agonists. This is accomplished by assessing how platelets recovered from the individuals under study respond to several platelet agonists ex vivo [10–17]. In some cases, how aspirin ingestion influences synthesis of thromboxane A2 and prostacyclin in vivo was also assessed by measuring the levels of stable metabolites of thromboxane A2 and PGI2 in blood 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.11.025
and urine. However, these results may not accurately reflect the specific effects of aspirin on platelets and endothelium as there are several sites for in vivo synthesis of thromboxane A2 and PGI2 The reduced clinical response of some patients’ platelets to aspirin in vivo has been called “aspirin resistance”. Several causes, including patient non-compliance, COX-1 haplotypes of patients[10], myonecrosis in patients with STsegment elevation myocardial infarction [12], myocardial infarction [13], the presence of increased numbers of immature platelets in patients with acute coronary syndromes [14],thrombus burden in patients with ST-elevation myocardial infarction [15], and low grade inflammation [17]. It is worth noting that different methods for determining platelet function and platelet agonists were used to assess platelet responses in the studies above. Therefore, an extrapolation of the information in these studies to arrive at specific conclusions establishing the common cause(s) of decreased platelet responses observed may not be justified. The direct way for demonstrating that chronic aspirin ingestion has achieved the desired antiplatelet effect in vivo, namely that aspirin has acetylated all the COX-1 of circulating platelets, is to demonstrate the presence of only acetylated (inactive) COX-1 in the patients’ platelets and which, therefore, no longer respond to stimulation with arachidonate ex vivo. This would be evident from both the failure of the aspirin-treated platelets to aggregate and synthesize TxB2 (the stable metabolite of TxA2) in response to arachidonate ex vivo. Novel Approaches for Demonstrating Complete Acetylation of Platelet COX-1 by Aspirin in vivo and its Functional Consequences In this and a previous issue of this journal, Kovacs et al., report a combination of methods for demonstrating the complete acetylation, and thus complete inactivation of platelet COX-1 in vivo after the daily ingestion of 100 mg of aspirin for 7 days by a group of healthy and relatively young volunteers. A direct result of aspirin fully acetylating platelet COX-1 is the inability of the treated volunteers’ platelets to synthesize TxA2 or to aggregate ex vivo in response to arachidonate [18,19]. A monoclonal anti-(human COX-1) antibody that reacts with native (thus non-acetylated) human COX-1 but not with aspirin-inactivated (acetylated COX-1) was used to demonstrate (by Western blotting) that only native COX-1 was found in the platelets of all the healthy volunteers prior to any aspirin ingestion. All the pre-aspirin platelets also aggregated and synthesized TxA2 normally in response to arachidonate. In contrast, only acetylated COX-1 was found in all the healthy volunteers’ platelets after daily ingestion of low dose aspirin for 7 days. Specifically, only the monoclonal anti-(acetylated human COX-1) antibody detected COX-1 protein in the platelets after the volunteers had ingested aspirin daily for 7 days. Consistent with this observation, the platelets obtained after 7 daily low-dose aspirin ingestion no longer
698
Editorial
aggregated or synthesized TxA2 ex vivo in response to arachidonate. An important aspect of the first study [18] was that complete acetylation (and thus inactivation) of platelet COX-1 was achieved in all the volunteers. Further, complete acetylation of platelet COX-1 prevented the synthesis of TxA2 by the platelets following their incubation with arachidonate. Combined use of the two monoclonal anti-(human COX-1) monoclonal antibody-based assays above and the associated abrogation of platelet TxA2 synthesis and platelet aggregation in response to arachidonate allowed the authors to identify noncompliance as the basis for the one apparent “aspirin resistance” that was initially observed. This volunteer’s platelets no longer responded to arachidonate when he/she was retested after daily lowdose aspirin ingestion for 7 days was confirmed. Therefore, the authors found no evidence for aspirin resistance arising solely from an inability of aspirin to fully acetylate platelet COX-1 in this group of healthy volunteers. Following up from the above observations, the authors have compared how native or pre-aspirin COX-1and fully acetylated or post-aspirin COX-1 influence the responses of platelets to a variety of agonists. The PFA-100 closure time (using the collagen plus epinephrine cartridge) VerifyNow Aspirin methods and the following single platelet agonists (ADP, arachidonate, collagen, and epinephrine) were used to assess platelet responses. The results unambiguously show that arachidonate is the best agonist for distinguishing fully acetylated (or inactive) from active platelet COX-1. The reason for this is probably because arachidonate is the weakest of the most commonly used platelet agonists. As reported by Kovacs et al. [19], 1.53 mmol/L arachidonate was the concentration used to stimulate platelets, compared to 54.6 μmol/L epinephrine, 10 μmol/L ADP and ~ 1.0 nmol/L collagen. Therefore, nearly a million-fold, a thousand-fold, and a 200-fold molar excess of arachidonate over collagen, ADP and epinephrine, respectively, were required to aggregate platelets to similar extents. Based on their relative potencies on platelet aggregation, it should be evident that the contribution of platelet-derived TxA2 to the propagation of intraplatelet signaling reactions that cause platelet aggregation in response to collagen, ADP or epinephrine is relatively small. Therefore, it is probably reasonable not to use PFA-100, collagen, ADP or epinephrine as platelet agonists when evaluating the functional consequences of full and partial COX-1 acetylation by aspirin. As was noted previously, only platelets obtained from healthy and relatively young volunteers were evaluated in the two recent studies above [18,19]. Therefore, similar studies evaluating the responses to aspirin of platelets from healthy older volunteers and all ages of patients with confirmed arterial thrombosis will probably be required to definitively establish the absence and presence of aspirin resistance. The absence and presence of aspirin resistance is strictly defined here as complete and incomplete acetylation of platelet COX-1, respectively, after chronic aspirin ingestion. If aspirin resistance or partial acetylation of platelet COX-1 is confirmed after the anticipated studies are completed, the relative contributions of non-compliance, COX-1 haplotypes, myonecrosis, inflammation, and increased numbers of immature platelets to aspirin resistance may then be more readily and accurately quantified [10–17]. On the other hand, if the studies confirm complete acetylation (inactivation) of platelet COX-1 by aspirin in vivo, then inadequate clinical response to chronic aspirin ingestion in patients with
cardiovascular disease cannot be reasonably attributed to an inability or reduced ability of aspirin to acetylate the patients’ platelets in vivo. Conflict of Interest Statement The author declared no conflict of interest. References [1] Campbell CL, Smyth S, Montalescot G, Steinhubl SR. Aspirin dose for prevention of cardiovascular disease: a systemic review. JAMA 2007;297:2018–24. [2] Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirinlike drugs. Nat New Biol 1971;231:232–5. [3] Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci U S A 1975;72:2994–8. [4] Fuster V, Sweeny JM. Aspirin: a historical and contemporary therapeutic overview. Circulation 2011;123:768–78. [5] Garcia-Rodriguez LA, Hernandez-Dias S. Risk of complicated peptic ulcer among users of aspirin and nonaspirin nonsteroidal anti-inflammatory drugs. Am J Epidemiol 2004;159:23–31. [6] Tomisato W, Tsutsumi S, Hoshino T, Hwang HJ, Mio M, Tsuchiya T, et al. Role of direct cytotoxic effects NSAIDs in the induction of gastric lesions. Biochem Pharmacol 2004;67:575–85. [7] Patrono C, Garcia-Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med 2005;353:2373–83. [8] Sakata C, Kawasaki T, Kato Y, Abe M, Suzuki K-L, Ohmiya M, et al. ASP6537, a novel highly selective cyclooxygenase inhibitor, exerts potent antithrombotic effect without “aspirin dilemma”. Thromb Res 2013;132:56–62. [9] Gorman RR, Fitzpatrick FA, Miller OV. Reciprocal regulation of human platelets cAMP levels by thromboxane A2 and prostacyclin. Adv Cyclic Nucleotide Res 1978;9: 597–609. [10] Maree AO, Curtin RJ, Chubb A, Dolan C, Cox D, O’Brien J, et al. Cyclooxigenase-1 haplotype modulates platelet response to aspirin. J Thromb Haemost 2005;3:2340–5. [11] Valles J, Santos TM, Fuset MP, Moscardo A, Ruano M, Perez F, et al. Partial inhibition of platelet thromboxane A2 synthesis by aspirin is associated with myonecrosis in patients with ST-segment elevation myocardial infarction. Am J Cardiol 2007; 99:19–25. [12] Poulsen TS, Kristensen SR, Korsholm L, Haghfelt T, Jorgensen B, Licht PB, et al. Variation and importance of aspirin resistance in patients with known cardiovascular disease. Thromb Res 2007;120:477–84. [13] Sweeny JM, Grong DA, Fuster V. Antiplatelet drug “resistance”. Part 1. Mechanisms and clinical measurements. Nat Rev Cardiol 2009;6:273–83. [14] Grove ER, Hvas A-M, Kristensen SD. Immature platelets in patients with acute coronary syndromes. Thromb Haemost 2009;101:151–6. [15] Alexopoulos D, Xanthopoulou I, Tsigkas G, Damelou A, Theodoropoulos KC, Makris G, et al. Intrinsic platelet reactivity and thrombus burden in patients with STelevation myocardial infarction. Thromb Res 2013;131:333–7. [16] Kasmeridis C, Apostolakis S, Lip GYH. Aspirin and aspirin resistance in coronary artery disease. Curr Opin Pharmacol 2013;13:242–50. [17] Larsen SB, Grove EL, Kristensen SD, Hvas A-M. Reduced antiplatelet effect of aspirin is associated with low-grade inflammation in patients with coronary artery disease. Thromb Haemost 2013;109:920–9. [18] Kovacs EG, Katona E, Bereczky Z, Homorodi N, Balogh L, Toth E, et al. New direct and indirect methods for the detection of cyclooxygenase 1 acetylation by aspirin: the lack of aspirin resistance among healthy individuals. Thromb Res 2013;131:320–4. [19] Kovacs EG, Katona E, Bereczky Z, Homorodi N, Balogh L, Toth E, et al. Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods. Thromb Res 2014;133:811–6.
Frederick A. Ofosu Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street West, HSC-3N26, Hamilton ON L8S 4K1, Canada E-mail address: [email protected]. 8 November 2013
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Editorial
Appropriate Assessment of the Functional Consequences of Platelet Cyclooxygenase-1 Inhibition by Aspirin in vivo
Introduction Chronic use of low dose aspirin (acetylsalicylic acid) is generally an effective and relatively low cost strategy for preventing and treating cardiovascular disease [1]. John Vane reported the inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs in 1971 by demonstrating a dose-dependent inhibition of prostaglandin synthesis by aspirin, sodium salicylate and indomethacin [2]. Thromboxane A2 (TxA2) was subsequently identified by Samuelsson’s group as the potent vasoconstrictor and platelet agonist the synthesis of which was inhibited by aspirin [3]. The ability of aspirin to prevent platelet activation initiated with arachidonate, and thus TxA2dependent activation of platelets form the basis of the antithrombotic action of aspirin [3]. Aspirin achieves this antiplatelet action by selectively acetylating the hydroxyl group of serine in platelet cyclooxygenase-1 (COX-1) or prostaglandin endoperoxidase synthase to irreversibly inactivate COX-1. This prevents platelet TxA2 synthesis from arachidonate that is released from platelet membranes by phospholipase A2 in response to platelet agonists [4]. All effective antithrombotic drugs can have major adverse effects, and clinically significant drawbacks of chronic aspirin use include gastrointestinal bleeding [5,6] and insufficient antithrombotic efficacy [7] in some patients. Both undesirable effects of aspirin may be attributable to the fact that aspirin also effectively inactivates COX-2 as reported in a recent study demonstrating effective inhibition of both thromboxane A2 and PGI2 inhibition by aspirin of recombinant human COX-1 and COX-2 his study also reported aspirin to dose-dependently inhibit TxA2 and PGI2 synthesis by guinea pig platelet-rich plasma and isolated aortic strips of guinea pigs, respectively [8]. Inactivation of human gastrointestinal COX-1 and COX-2 may partly contribute to the gastrointestinal bleeding associated with chronic use of aspirin [5,6]. The causes of the insufficient antithrombotic efficacy of aspirin found in some patients [7] are not clear. Inactivation of endothelial cell COX-2 by aspirin will prevent endothelial cell synthesis of PGI2, a prostanoid able to prevent platelets from responding to all agonists [9]. The reduced antithrombotic efficacy of chronic aspirin use in some patients has naturally led to ex vivo and in vitro studies designed to identify low responders to chronic aspirin use and establish the likely causes of reduced or non-response to aspirin [10–17]. Specifically, these studies have determined how aspirin ingestion affects the ex vivo responses of patients’ and/or volunteers’ platelets to agonists. This is accomplished by assessing how platelets recovered from the individuals under study respond to several platelet agonists ex vivo [10–17]. In some cases, how aspirin ingestion influences synthesis of thromboxane A2 and prostacyclin in vivo was also assessed by measuring the levels of stable metabolites of thromboxane A2 and PGI2 in blood 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.11.025
and urine. However, these results may not accurately reflect the specific effects of aspirin on platelets and endothelium as there are several sites for in vivo synthesis of thromboxane A2 and PGI2 The reduced clinical response of some patients’ platelets to aspirin in vivo has been called “aspirin resistance”. Several causes, including patient non-compliance, COX-1 haplotypes of patients[10], myonecrosis in patients with STsegment elevation myocardial infarction [12], myocardial infarction [13], the presence of increased numbers of immature platelets in patients with acute coronary syndromes [14],thrombus burden in patients with ST-elevation myocardial infarction [15], and low grade inflammation [17]. It is worth noting that different methods for determining platelet function and platelet agonists were used to assess platelet responses in the studies above. Therefore, an extrapolation of the information in these studies to arrive at specific conclusions establishing the common cause(s) of decreased platelet responses observed may not be justified. The direct way for demonstrating that chronic aspirin ingestion has achieved the desired antiplatelet effect in vivo, namely that aspirin has acetylated all the COX-1 of circulating platelets, is to demonstrate the presence of only acetylated (inactive) COX-1 in the patients’ platelets and which, therefore, no longer respond to stimulation with arachidonate ex vivo. This would be evident from both the failure of the aspirin-treated platelets to aggregate and synthesize TxB2 (the stable metabolite of TxA2) in response to arachidonate ex vivo. Novel Approaches for Demonstrating Complete Acetylation of Platelet COX-1 by Aspirin in vivo and its Functional Consequences In this and a previous issue of this journal, Kovacs et al., report a combination of methods for demonstrating the complete acetylation, and thus complete inactivation of platelet COX-1 in vivo after the daily ingestion of 100 mg of aspirin for 7 days by a group of healthy and relatively young volunteers. A direct result of aspirin fully acetylating platelet COX-1 is the inability of the treated volunteers’ platelets to synthesize TxA2 or to aggregate ex vivo in response to arachidonate [18,19]. A monoclonal anti-(human COX-1) antibody that reacts with native (thus non-acetylated) human COX-1 but not with aspirin-inactivated (acetylated COX-1) was used to demonstrate (by Western blotting) that only native COX-1 was found in the platelets of all the healthy volunteers prior to any aspirin ingestion. All the pre-aspirin platelets also aggregated and synthesized TxA2 normally in response to arachidonate. In contrast, only acetylated COX-1 was found in all the healthy volunteers’ platelets after daily ingestion of low dose aspirin for 7 days. Specifically, only the monoclonal anti-(acetylated human COX-1) antibody detected COX-1 protein in the platelets after the volunteers had ingested aspirin daily for 7 days. Consistent with this observation, the platelets obtained after 7 daily low-dose aspirin ingestion no longer
698
Editorial
aggregated or synthesized TxA2 ex vivo in response to arachidonate. An important aspect of the first study [18] was that complete acetylation (and thus inactivation) of platelet COX-1 was achieved in all the volunteers. Further, complete acetylation of platelet COX-1 prevented the synthesis of TxA2 by the platelets following their incubation with arachidonate. Combined use of the two monoclonal anti-(human COX-1) monoclonal antibody-based assays above and the associated abrogation of platelet TxA2 synthesis and platelet aggregation in response to arachidonate allowed the authors to identify noncompliance as the basis for the one apparent “aspirin resistance” that was initially observed. This volunteer’s platelets no longer responded to arachidonate when he/she was retested after daily lowdose aspirin ingestion for 7 days was confirmed. Therefore, the authors found no evidence for aspirin resistance arising solely from an inability of aspirin to fully acetylate platelet COX-1 in this group of healthy volunteers. Following up from the above observations, the authors have compared how native or pre-aspirin COX-1and fully acetylated or post-aspirin COX-1 influence the responses of platelets to a variety of agonists. The PFA-100 closure time (using the collagen plus epinephrine cartridge) VerifyNow Aspirin methods and the following single platelet agonists (ADP, arachidonate, collagen, and epinephrine) were used to assess platelet responses. The results unambiguously show that arachidonate is the best agonist for distinguishing fully acetylated (or inactive) from active platelet COX-1. The reason for this is probably because arachidonate is the weakest of the most commonly used platelet agonists. As reported by Kovacs et al. [19], 1.53 mmol/L arachidonate was the concentration used to stimulate platelets, compared to 54.6 μmol/L epinephrine, 10 μmol/L ADP and ~ 1.0 nmol/L collagen. Therefore, nearly a million-fold, a thousand-fold, and a 200-fold molar excess of arachidonate over collagen, ADP and epinephrine, respectively, were required to aggregate platelets to similar extents. Based on their relative potencies on platelet aggregation, it should be evident that the contribution of platelet-derived TxA2 to the propagation of intraplatelet signaling reactions that cause platelet aggregation in response to collagen, ADP or epinephrine is relatively small. Therefore, it is probably reasonable not to use PFA-100, collagen, ADP or epinephrine as platelet agonists when evaluating the functional consequences of full and partial COX-1 acetylation by aspirin. As was noted previously, only platelets obtained from healthy and relatively young volunteers were evaluated in the two recent studies above [18,19]. Therefore, similar studies evaluating the responses to aspirin of platelets from healthy older volunteers and all ages of patients with confirmed arterial thrombosis will probably be required to definitively establish the absence and presence of aspirin resistance. The absence and presence of aspirin resistance is strictly defined here as complete and incomplete acetylation of platelet COX-1, respectively, after chronic aspirin ingestion. If aspirin resistance or partial acetylation of platelet COX-1 is confirmed after the anticipated studies are completed, the relative contributions of non-compliance, COX-1 haplotypes, myonecrosis, inflammation, and increased numbers of immature platelets to aspirin resistance may then be more readily and accurately quantified [10–17]. On the other hand, if the studies confirm complete acetylation (inactivation) of platelet COX-1 by aspirin in vivo, then inadequate clinical response to chronic aspirin ingestion in patients with
cardiovascular disease cannot be reasonably attributed to an inability or reduced ability of aspirin to acetylate the patients’ platelets in vivo. Conflict of Interest Statement The author declared no conflict of interest. References [1] Campbell CL, Smyth S, Montalescot G, Steinhubl SR. Aspirin dose for prevention of cardiovascular disease: a systemic review. JAMA 2007;297:2018–24. [2] Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirinlike drugs. Nat New Biol 1971;231:232–5. [3] Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci U S A 1975;72:2994–8. [4] Fuster V, Sweeny JM. Aspirin: a historical and contemporary therapeutic overview. Circulation 2011;123:768–78. [5] Garcia-Rodriguez LA, Hernandez-Dias S. Risk of complicated peptic ulcer among users of aspirin and nonaspirin nonsteroidal anti-inflammatory drugs. Am J Epidemiol 2004;159:23–31. [6] Tomisato W, Tsutsumi S, Hoshino T, Hwang HJ, Mio M, Tsuchiya T, et al. Role of direct cytotoxic effects NSAIDs in the induction of gastric lesions. Biochem Pharmacol 2004;67:575–85. [7] Patrono C, Garcia-Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med 2005;353:2373–83. [8] Sakata C, Kawasaki T, Kato Y, Abe M, Suzuki K-L, Ohmiya M, et al. ASP6537, a novel highly selective cyclooxygenase inhibitor, exerts potent antithrombotic effect without “aspirin dilemma”. Thromb Res 2013;132:56–62. [9] Gorman RR, Fitzpatrick FA, Miller OV. Reciprocal regulation of human platelets cAMP levels by thromboxane A2 and prostacyclin. Adv Cyclic Nucleotide Res 1978;9: 597–609. [10] Maree AO, Curtin RJ, Chubb A, Dolan C, Cox D, O’Brien J, et al. Cyclooxigenase-1 haplotype modulates platelet response to aspirin. J Thromb Haemost 2005;3:2340–5. [11] Valles J, Santos TM, Fuset MP, Moscardo A, Ruano M, Perez F, et al. Partial inhibition of platelet thromboxane A2 synthesis by aspirin is associated with myonecrosis in patients with ST-segment elevation myocardial infarction. Am J Cardiol 2007; 99:19–25. [12] Poulsen TS, Kristensen SR, Korsholm L, Haghfelt T, Jorgensen B, Licht PB, et al. Variation and importance of aspirin resistance in patients with known cardiovascular disease. Thromb Res 2007;120:477–84. [13] Sweeny JM, Grong DA, Fuster V. Antiplatelet drug “resistance”. Part 1. Mechanisms and clinical measurements. Nat Rev Cardiol 2009;6:273–83. [14] Grove ER, Hvas A-M, Kristensen SD. Immature platelets in patients with acute coronary syndromes. Thromb Haemost 2009;101:151–6. [15] Alexopoulos D, Xanthopoulou I, Tsigkas G, Damelou A, Theodoropoulos KC, Makris G, et al. Intrinsic platelet reactivity and thrombus burden in patients with STelevation myocardial infarction. Thromb Res 2013;131:333–7. [16] Kasmeridis C, Apostolakis S, Lip GYH. Aspirin and aspirin resistance in coronary artery disease. Curr Opin Pharmacol 2013;13:242–50. [17] Larsen SB, Grove EL, Kristensen SD, Hvas A-M. Reduced antiplatelet effect of aspirin is associated with low-grade inflammation in patients with coronary artery disease. Thromb Haemost 2013;109:920–9. [18] Kovacs EG, Katona E, Bereczky Z, Homorodi N, Balogh L, Toth E, et al. New direct and indirect methods for the detection of cyclooxygenase 1 acetylation by aspirin: the lack of aspirin resistance among healthy individuals. Thromb Res 2013;131:320–4. [19] Kovacs EG, Katona E, Bereczky Z, Homorodi N, Balogh L, Toth E, et al. Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods. Thromb Res 2014;133:811–6.
Frederick A. Ofosu Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street West, HSC-3N26, Hamilton ON L8S 4K1, Canada E-mail address: [email protected]. 8 November 2013