Progress in Cardiology Antiplatelet drug nonresponsiveness Patrick Gladding, FCSANZ, Mark Webster, FCSANZ, John Ormiston, FCSANZ, Sarah Olsen, MBCHB, and Harvey White, DSc, FCSANZ Auckland, New Zealand The response to most medication, including antiplatelet drugs, is highly variable between individuals. Observational studies have shown that nonresponders to antiplatelet agents appear to have an increased incidence of vascular events. This review article reviews the background, mechanisms, and evidence in support of the clinial significance of this phenomenon. (Am Heart J 2008;155:591-9.) The concept of antiplatelet drug “resistance” or nonresponsiveness has received increasing attention over recent years. “Aspirin resistance” has received the most attention,1 but a variable response to other antiplatelet drugs, including clopidogrel and glycoprotein IIb/IIIa (GpIIb-IIIa) inhibitors,2,3 may also be clinically important. The evidence for the use of antiplatelet drugs in the treatment and prevention of acute coronary syndromes is extensive. A meta-analysis found that aspirin reduced the incidence of nonfatal myocardial infarction (MI), nonfatal stroke, and vascular death by 22% in those with vascular disease.4 As an alternative single agent, clopidogrel has a similar benefit,5 and the combination is about 20% more effective than aspirin alone. Clopidogrel is an established adjuvant to aspirin in patients undergoing percutaneous coronary intervention (PCI) with stent deployment6 and in those presenting with acute non–STsegment elevation and ST-elevation acute coronary syndromes.7,8 Individuals not responsive to antiplatelet drugs may be more likely to have recurrent vascular events, including an increased risk of stent thrombosis.9,10 The new point-of-care platelet function technology has made platelet drug response evaluation easier to perform,11 but the field remains constrained by considerable differences between testing methods. There is also the limitation of extrapolating from an ex vivo test result to real-world platelet thrombosis, including arbitrary thresholds for nonresponsiveness12 and limited data correlating nonresponsiveness with clinical outcomes. From the Green Lane Cardiovascular Service, Auckland City Hospital, Auckland, New Zealand. Submitted October 14, 2007; accepted December 31, 2007. Reprint requests: Patrick Gladding, FCSANZ, Green Lane Cardiovascular Service, Auckland City Hospital, Private Bag 92024, Auckland Mail Centre, Auckland 1142, New Zealand. E-mail: PatrickG@adhb.gov.nz 0002-8703/$ - see front matter © 2008, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2007.12.034 Antiplatelet agents Platelet activation plays a pivotal role in the pathogenesis of at least the later stages of atherothrombotic disease. Platelets are activated by a number of physiologic agonists including thromboxane, adenosine diphosphate (ADP), thrombin, serotonin, and collagen. Shear stress, a physical property of blood flow, also plays an important role. Platelets are capable of inducing their own aggregation, predominantly via thrombin generation, leading to an amplification reaction. Despite the wide range of platelet agonists, only 4 pathways are targeted by drugs in the marketplace ( Figure 1). There are thromboxane pathway inhibitors such as aspirin and nonsteroidal anti-inflammatory drugs; P2Y12 receptor antagonists such as ticlopidine and clopidogrel; phosphodiesterase inhibitors such as dipyridamole and cilostazol; and GpIIb-IIIa inhibitors including abciximab, eptifibatide, and tirofiban.13 Thrombin is the most potent agonist of platelet activation, and despite blockade of antiplatelet pathways with aspirin and clopidogrel, persistent thrombin generation poses a considerable continued stimulus for platelets.14 Blocking the platelet protease-activated receptor 1 leaves other thrombin-mediated hemostatic functions intact, so theoretically, bleeding events may not be increased. A protease-activated receptor 1 (thrombin receptor) antagonist is entering a phase III trial and may be available in the near future.15 The GpIIb-IIIa inhibitors are considered the most potent antiplatelet agents as they inhibit the final common pathway of platelet aggregation. However, they do not mitigate the upstream effects of platelet activation that result in the release of vasoactive substances in highrisk patients with PCI.16 Clinical trials suggest that combination therapy of GpIIb-IIIa inhibitors and P2Y12 receptor antagonists may reduce periprocedural myonecrosis and improve long-term ischemic outcomes, compared with either drug alone.17,18 Definitions Clinically significant antiplatelet drug failure can be defined as a recurrent vascular atherothrombotic event American Heart Journal April 2008 592 Gladding et al Figure 1 Agonists to platelet activation and antiplatelet agents. occurring despite drug adherence. Given the multiple pathways of platelet activation, it is difficult to be certain that a drug has “failed.” For instance, reinfarction after aspirin administration for an acute coronary syndrome may be due to ADP-mediated thrombus propagation rather than ongoing thromboxane-induced platelet aggregation.19 “Resistance” might best be defined as residual post treatment activity in the target pathway of an antiplatelet agent.20 Definitions based on output values from platelet function tests are still widely debated. The recent ASPECT study had 7 different definitions for aspirin resistance, spanning 5 forms of platelet function testing. These definitions were cutoff values for the different instruments used. Three were for light transmittance aggregometry (LTA), ranging from ≥20% aggregation using an agonist of arachidonic acid to ≥70% using ADP.21 Platelet function analyzers Traditional methods of platelet function testing are complicated to perform, requiring skilled and experienced phlebotomy and laboratory staff working under carefully controlled conditions. Laboratory-based LTA is accepted by most as the “gold standard” but is laborintensive, operator-dependent, and expensive, which has restricted its clinical use.22 These limitations have led investigators to use surrogates for platelet function including biochemical markers such as serum or urine thromboxane B2 for aspirin activity and vasodilatorstimulated phosphoprotein (VASP) for P2Y12 receptor inhibition ( Table I).23,24 Platelet function assays can be classified according to the method of analysis. Biochemical assays use either an enzyme-linked immunosorbent assay (thromboxane) or flow cytometry (VASP). Nonbiochemical platelet function analyzers typically use light transmittance or electrical impedance to measure platelet aggregation directly, either in isolated platelets or in whole blood.25 The platelet agonist may differ between assays, making interassay comparisons difficult. Few analyzers incorporate increased shear stress as a nonbiochemical means of platelet stimulation.26 Point-of-care platelet function devices have simplified testing with a rapid result available at the bedside or in the cardiac catheterization laboratory. The 3 most widely evaluated point-of-care devices are Dade-Behring's (Deerfield, IL) platelet function analyzer, PFA–100, which measures platelet function under high shear stress by drawing blood through a small aperture and measuring the “closure time” of that aperture by a platelet plug; Accumetric's (San Diego, CA) VerifyNow assay, which uses a light-based, whole blood aggregometry system; and the Haemoscope's (Niles, IL) Thromboelastograph (TEG), which measures clot tensile strength and has been most widely evaluated and used in patients undergoing cardiac surgery. The PFA-100 has been used clinically in the diagnosis of platelet function disorders.27 Its cartridges have collagen American Heart Journal Volume 155, Number 4 Gladding et al 593 Table I. Platelet function analyzers and clinical correlates Platelet function analyzer Advantages Bleeding time ⁎ Widely available, in vivo PFA-100 ⁎ Rapid, whole blood, hypothesized to mimic small vessel, measures shear stress effect Closed system, rapid, correlates with gold standard, small footprint Small footprint, has advantages for cardiac anesthesia VerifyNow ⁎ Thromboelastogram ⁎ Light transmittance aggregometry Historic gold standard Urinary 11-dehydrothromboxane B2 VASP Specific to COX-1 activity Specific to P2Y12 activity Disadvantages Highly variable, nonspecific activation, scarring Requires pipetting of blood, interinstrument variability, noncontinuous output, dependent on vWF levels Older model influenced by ambient light Requires pipetting of blood (operator dependent results), difficult to interpret output variables, minimal clinical studies Operator dependent, requires preparation of plasma and pipetting, costly, time consuming Not specific to platelet COX-1, dependent on renal function and urinary concentration Expensive, requires flow cytometer, technical experience required Clinical outcome studies Monitors Aspirin and clopidogrel No No Yes No Yes Yes Yes Yes with platelet mapping Yes Yes Yes No Yes No Other platelet function measures include CD40L, P-selectin, and platelet-derived microparticles. WF, von Willebrand Factor; COX, cyclooxygenase. ⁎Point-of-care assay. and either arachidonic acid or ADP as the agonist. The VerifyNow device has 3 pathway-specific cartridges that test for the effects of aspirin, clopidogrel, and GpIIb-IIIa inhibitors, all validated against light transmittance aggregometry.28-30 These devices test a single pathway of platelet activation.21 A study of 700 patients found that residual arachidonic acid–induced platelet aggregation in patients on aspirin was due to ADP-dependent rather than cyclooxygenase (COX) pathways.31 The authors concluded that “aspirin resistance,” as measured by a pointof-care platelet function analyzer specific to aspirin, is either a limitation of the device due to pathway nonspecificity or simply a measure of noncompliance with aspirin.31 This hypothesis is supported by the recent ASPECT study, a comprehensive assessment of a range of platelet function analyzers, which concluded that aspirin nonresponsiveness is rare, is overcalled by some analyzers such as the PFA-100, and may be related to non COX1 pathways such as those mediated by ADP.21 PFA-100, using epinephrine as the agonist, and light transmittance aggregometry in patients taking low dose aspirin; 9.5% of individuals were considered nonresponders using the PFA-100, compared with 5.5% nonresponders and 23.8% semiresponders by light transmittance aggregometry.37 Although population response to medication often fits a normal distribution, it is uncertain whether the same is true of aspirin responsiveness. The aspirin response assessed with the VerifyNow device may follow a bimodal distribution with a value of 550 aspirin resistance units separating responders from nonresponders.41 A bimodal response has not been found with other platelet function analyzers, so this finding needs to be replicated. Clopidogrel response appears to follow a normal distribution.42 Varying definitions of nonresponsiveness have been proposed, including a change in platelet response from baseline, and an absolute threshold.42,43 As there is no consensus definition, the incidence of nonresponse to clopidogrel is uncertain. Incidence Etiology The definition of antiplatelet drug response using these devices is somewhat arbitrary. There are 7 different thresholds defining an aspirin response with the PFA-100 reported in the literature.32-38 The incidence of nonresponse to aspirin varies with the platelet function test used and the threshold chosen to determine response; reported rates range between 9.5% and 33%.37,39,40 There is also a weak correlation between different testing methods.21 One study found no association between the The cause of antiplatelet drug resistance is multifactorial. An obvious but difficult to measure reason for lack of drug effect is noncompliance. Undetected noncompliance may have led to an overestimate of the rate of aspirin nonresponsiveness.44 Some possible causes for aspirin nonresponse are outlined in Table II. Risk factors include increasing age, smoking, and gender;35 many are related to drug pharmacokinetics and pharmacodynamics. Diabetes is a particular problem as it American Heart Journal April 2008 594 Gladding et al Table II. Possible causes of antiplatelet drug nonresponsiveness Origin Patient factors Drug factors Drug-drug interactions Example Behavioral • Nonadherence to treatment • Smoking Pharmacodynamic Pharmocokinetic Increasing age Altered binding site e.g. COX-1 polymorphisms Reduced absorption e.g. p-glycoprotein polymorphisms Reduced CYP3A4 biotransformation e.g. CYP3A4 polymorphisms • Increased volume of distribution • Reduced absorption e.g. enteric coating • Reduced biotransformation of clopidogrel • CYP3A4 substrates and clopidogrel • NSAIDs and aspirin • Overactive alternative pathways • Rapid platelet turnover • • • • Delivery vehicle Decreased metabolism Competitive substrate Steric hindrance Indirect mechanisms NSAIDs, Nonsteroidal anti-inflammatory drugs; COX, cyclooxygenase; CYP, cytochrome P450. is associated with generalized heightened platelet reactivity. The cause is multifactorial but includes increased platelet receptor expression, reduced platelet-derived formation of nitrous oxide, and increased sensitivity to ADP.45 Despite treatment of diabetic patients with dual antiplatelet therapy, there is still an increased rate of cardiovascular events, particularly in those that have measurably higher platelet reactivity.46 Pharmacokinetics Pharmacokinetics includes the bioavailability, that is, absorption and the first-pass effect, volume of distribution, and clearance of a drug. The first-pass effect on aspirin is considerable and the drug is rapidly cleared by carboxylesterases 1 and 2 in the liver.47 The antiplatelet effect of aspirin occurs predominantly in the portal circulation.48 Although aspirin inhibits both COX-I and COX-II, the systemic COX-II effect is minimal in doses b1200 mg.49,50 Platelets are anucleate and have no ability to regenerate COX-I; therefore, aspirin-mediated platelet inhibition is permanent for the life of the platelet. However, platelets are continually formed, and COX-I sources such as nucleated cells can contribute prostaglandin precursors leading to a recovery of platelet activity within 24 hours.51 Clopidogrel is a prodrug that requires hepatic biotransformation by cytochrome P450 3A4 and 2C19 to an active metabolite with a short half-life, which irreversibly antagonizes the ADP receptor.52-54 Drugs coadministered with clopidogrel that inhibit the metabolism of CYP3A4 will, at least in theory, diminish the antiplatelet effect of clopidogrel. Although both atorvastatin and erythromycin inhibit CYP3A4 and reduce the ex vivo antiplatelet response to clopidogrel,55 this interaction may not be clinically significant as post hoc analysis of the CREDO study found no increase in vascular events in those on atorvastatin and clopidogrel.56 The ISAR-CHOICE study identified intestinal absorption as another factor limiting clopidogrel efficacy.57 The intestinal P-glycoprotein efflux transporter, which has recently been implicated as an important pathway in clopidogrel absorption, is involved in a number of wellrecognized drug interactions.58 Pharmacodynamics and pharmacogenomics Coadministered medications can alter the pharmacodynamics of antiplatelet agents. Concurrent use of some nonsteroidal anti-inflammatory drugs including ibuprofen, naproxen, tiaprofenic acid, and indomethacin may block the antiplatelet effect of aspirin by steric hindrance at the COX-1 receptor site.59-62 The effect of the drug on its target site is also a point at which “resistance” may occur, an effect influenced more by an individual's response rather than by the properties of the drug. For this reason, pharmacogenetics has promise in identifying resistant individuals. Using a candidate gene approach, several studies have implicated a number of polymorphisms associated with aspirin resistance including a haplotype within the COX-I enzyme63 and within the ADP receptor P2Y1.64 As with most genetic analysis to date, these results need validating in other cohorts.65 Whole genome wide scanning and linkage analysis in larger cohorts may reveal polymorphisms in other genes of interest. The site of action of clopidogrel, the P2Y12 receptor, has only recently been defined and the gene mapped.66 A polymorphism of the P2Y12 gene, denoted haplotype H2, is associated with increased platelet responsiveness to ADP but does not appear to influence the response to clopidogrel.67 Further attention has focused on the metabolic activation of clopidogrel and possible genetic influences. Sequence variations within the hepatic CYP3A4 enzyme gene and the related CYP2C19 and 2C9 may identify those who do not American Heart Journal Volume 155, Number 4 respond to the drug.54,68,69 A common polymorphism of the P-glycoprotein gene (MDR1 C3435T genotype) has also recently been shown to influence the intestinal absorption of clopidogrel.70 It is unlikely that a single gene will be found that accounts for all aspirin or clopidogrel nonresponsiveness. The cause of antiplatelet drug resistance is multifactorial and influenced by both environmental and genetic factors. Translation of pharmacogenomics into clinical practice will take an increased availability of genotyping and larger studies with clinical outcome data. Clinical importance Aspirin nonresponsiveness Stable coronary artery disease. Antiplatelet drug nonresponsiveness appears to be clinically relevant. An early study of aspirin nonresponsiveness in 326 patients with stable cardiovascular disease taking aspirin found that 5.2% were resistant to aspirin, as measured by the PFA-100. At 2 years of follow-up, those individuals showing nonresponsiveness had a 4-fold increase in the incidence of death, MI, or stroke.10 The recent PROSPECTAR study, also using the PFA-100, reported a higher prevalence of aspirin nonresponsiveness (22%). The nonresponsive group had a higher rate of major adverse cardiac events over 21 months, although the difference between groups was not statistically significant.71 The HOPE trial used a high level of urinary 11dehydrothromboxane B2 as a marker of aspirin nonresponsiveness. After adjustment for baseline differences, the odds for major adverse cardiovascular events were increased with each increasing quartile of 11dehydrothromboxane B2. Those in the highest quartile had a 2-fold higher risk of MI and a 3.5-fold higher risk of cardiovascular death than those in the lowest quartile.23 A limitation of this study was that it was a post hoc analysis, and there was no rigorous assessment of drug noncompliance. A recent study using the VerifyNow device found that aspirin nonresponsiveness was present in 27% of the study population and associated with a 3-fold increase in the risk of a composite cardiovascular end point at 1 year.72 Percutaneous coronary intervention. Aspirin nonresponsiveness also appears important in patients undergoing PCI.73 In a study of 151 patients, all of whom received a 300-mg clopidogrel loading dose, 19% were nonresponsive to aspirin as assessed using the VerifyNow aspirin assay. The incidence of creatine kinase (CK)–MB or troponin I elevation post PCI was significantly higher in aspirin-resistant than aspirin-sensitive patients.73 Another study using the PFA-100 in 146 patients undergoing primary PCI for ST-elevation MI found a similar Gladding et al 595 result.74 More patients with major adverse cardiovascular events had aspirin nonresponsiveness (39% vs 23% with a normal response, P b .05). Acute coronary syndromes. Several studies have evaluated platelet function in patients presenting with an acute coronary syndrome. In 216 patients with STEMI, enhanced platelet function under high shear stress, assessed with the PFA-100, was an independent predictor of markers of cardiac necrosis.75 The STRATEGY study investigated this further in 70 STEMI patients undergoing PCI.76 Assessment with the PFA100 assessed before intervention, using an ADP agonist, predicted the response to GpIIb-IIIa inhibition and long-term outcome. At 1 year, patients with an abnormal PFA-100 ADP time showed an adjusted 5- to 11-fold increase in the risk of death, reinfarction, and target vessel revascularization.76 Further support comes from another trial in 153 patients using the PFA-100, finding that the closure time, measured after PCI, was independently associated with a higher rate of death or MI.77 In contrast, another study investigated aspirin nonresponsiveness and long-term outcome in 187 individuals with suspected acute MI.78 Although no association was seen between aspirin nonresponsiveness and adverse outcomes, only 26% of patients actually had an acute coronary syndrome. Of interest, the platelet response in those with MI differed dramatically over time; platelets displayed hyperaggregation at the time of presentation compared with later time points. Hence, aspirin nonresponsiveness at the time of MI may reflect the acute clinical state rather than an individual difference in response.78 Indirect methods of measuring platelet activity also predict outcomes in patients with acute coronary syndromes. In one study, partial inhibition of thromboxane A2 by aspirin was found in 34% of patients and was associated with significant increases in serum troponin T, CK, and CK-MB, compared with patients in whom thromboxane A2 production was blocked.79 Clopidogrel nonresponsiveness Several studies have investigated the importance of platelet reactivity after clopidogrel dosing. These studies are mostly in the context of coronary intervention with platelet function measured either before or after PCI ( Table III). A key issue in clopidogrel nonresponse is the timing and dosing of the drug, particulary in those undergoing PCI and not pretreated with clopidogrel. Two trials have shown that a 600-mg loading dose, compared with a standard 300-mg dose, is associated with more rapid and complete platelet inhibition and with reduced post-PCI myonecrosis.83,87 The ongoing CURRENT/OASIS 7 will address this definitively in a 14 000- American Heart Journal April 2008 596 Gladding et al Table III. Clopidogrel response studies in PCI Study design Definition of clopidogrel nonresponse Prevalence of clopidogrel nonresponse Gurbel et al80 192 Case control – – Gurbel et al81 120 Case control – – Geisler et al82 379 Observational Lev et al9 Observational Reference No. Hochholzer et al84 Baseline minus posttreatment aggregation ≤10% in response to 5- and 20-μmol/L ADP (LTA) 292 RCT (300 vs 600 mg 10 μmol/L ADP (LTA) induced clopidogrel) platelet aggregation N70% 802 Observational Quartiles of LTA platelet response to 5umol/L ADP Bliden et al85 100 Observational Buonamici et al86 804 Observational Cuisset et al83 150 Platelet inhibition b30% (20 μmol/L ADP LTA) – 10 μmol/L ADP ≥70% (LTA) 5.8% 24% 15% in 600-mg group, 25% in 300-mg group – – 13% Clinical outcome Higher posttreatment ADP induced LTA and clot strength by TEG in patients with CV events Patients with SAT had higher mean platelet reactivity than those without SAT Low response to clopidogrel significantly enhanced the occurrence of CV events and death Higher CK levels after coronary stenting in nonresponsive patients Lower CV event rate in 600-mg group, at 30 d Platelet aggregation before elective stenting in patients pretreated with clopidogrel correlates with early CV outcomes High platelet reactivity measured by LTA and TEG is associated with increased CV events HR 3.08 for stent thrombosis (acute, subacute, or late) in nonresponders ADP was the platelet agonist used at 5-, 10-, or 20-μmol/L concentrations). CV, Cardiovascular; RCT, randomized clinical trial; SAT, subacute stent thrombosis; TEG, thromoboelastography; LTA, light transmittance aggregometry. patient study of subjects with non–ST-elevation MI undergoing PCI. Management of antiplatelet drug nonresponsiveness There is, at present, little evidence to guide treatment of the patient with laboratory evidence of a reduced response to antiplatelet drugs or thrombosis occurring during antiplatelet therapy. Empirical strategies include increasing the dose of the antiplatelet agent or adding a second antiplatelet drug. Using laboratory assays of platelet function, there is some evidence that aspirin response may be dosedependent.36 On the other hand, meta-analysis of the randomized clinical trials indicates that, across the study populations, the most effective aspirin dose with the fewest adverse consequences is 75 to 150 mg once daily.4,88 It is possible that these large trials might include a small cohort of patients who would have benefited from a higher aspirin dosage. The response to aspirin may decline over time due to tachyphylaxis.89 Clopidogrel maintenance dosages N75 mg daily also appear to offer a greater antiplatelet effect.90 Once the problems related to measurement and definitions are overcome, clinical trials will be needed to develop and validate algorithms guiding optimal antiplatelet treatment. The ongoing RESISTOR trial will address this by randomizing patients presenting with acute coronary syndromes to GpIIb-IIIa inhibitor treatment, depending on the response to aspirin or clopidogrel as measured by the VerifyNow device.91 The GRAVITAS study, due to begin shortly, will address the issue of whether an increase in the clopidogrel maintenance dose is necessary in clopidogrel nonresponders. Newer drugs may overcome the limitations of current antiplatelet drugs. Prasugrel is a third-generation thienopyridine that is not as dependent as clopidogrel on biotransformation to an active metabolite. In preclinical studies, it was shown to have greater potency and achieve more rapid platelet inhibition than clopidogrel when given orally.92 The JUMBO-TIMI trial found prasugrel to have a comparable safety profile to clopidogrel.93 However, the recent TRITON TIMI-38 trial found that prasugrel reduced ischemic events in an ACS population undergoing PCI, at the cost of increased major bleeding.15 Those assigned to clopidogrel received a 300-mg loading dose immediately before or during PCI, whereas 600 mg is now more commonly used clinically as it may be more effective.87 Although this raised the question of dose equivalence, platelet function analysis in PRINCIPLE-TIMI 44 has shown that the dose of prasugrel used in TRITON leads to greater platelet inhibition than clopidogrel at the higher loading and maintenance doses.94 Subgroup analysis of TRITON suggested prasugrel may have the greatest benefit over clopidogrel in the highest-risk patients, such as those with diabetes. Alternatively, a possible future approach may be American Heart Journal Volume 155, Number 4 individualized antiplatelet therapy based on platelet function testing or pharmacogenomic profiling. Conclusion There is surprisingly limited published information on optimal dosages and combinations of antiplatelet agents, what constitutes a suboptimal response to those agents, whether clinical outcomes can be improved by platelet function testing on antiplatelet treatment, and whether treatment outcomes can be improved by titrating or changing the antiplatelet therapy of individuals. With some antiplatelet drugs, such as clopidogrel and GpIIbIIIa inhibitors, higher dosages can achieve greater platelet inhibition, but it remains unclear whether higher dosage regimens will further reduce thrombotic events without increasing the risk of bleeding. There remains a need for antiplatelet drugs with a more rapid onset of effect and with a more predictable degree of platelet inhibition in the populations treated. Adjusting antiplatelet drug therapy on the basis of individual response is an appealing proposition but is presently not evidence-based. Identifying those with a suboptimal response to antiplatelet drugs may also influence other aspects of their management such as the choice of a drug-eluting or bare metal stent for percutaneous revascularization. An individualized approach may reduce the adverse consequences of antiplatelet therapy, allow more cost-effective use of expensive medication, and improve patient outcomes. References 1. Helgason CM, Tortorice KL, Winkler SR, et al. Aspirin response and failure in cerebral infarction. Stroke 1993;24:345-50. 2. Muller I, Besta F, Schulz C, et al. Prevalence of clopidogrel nonresponders among patients with stable angina pectoris scheduled for elective coronary stent placement. Thromb Haemost 2003;89: 783-7. 3. Madan M, Berkowitz SD, Christie DJ, et al. Rapid assessment of glycoprotein IIb/IIIa blockade with the platelet function analyzer (PFA-100) during percutaneous coronary intervention. Am Heart J 2001;141:226-33. 4. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Bmj 2002;324:71-86. 5. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee. Lancet 1996;348:1329-39. 6. Mehta SR, Yusuf S, Peters RJ, et al. Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet 2001;358:527-33. 7. Yusuf S, Zhao F, Mehta SR, et al. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001;345:494-502. 8. Sabatine MS, Cannon CP, Gibson CM, et al. Addition of clopidogrel to aspirin and fibrinolytic therapy for myocardial infarction with STsegment elevation. N Engl J Med 2005;352:1179-89. Gladding et al 597 9. Lev EI, Patel RT, Maresh KJ, et al. Aspirin and clopidogrel drug response in patients undergoing percutaneous coronary intervention: the role of dual drug resistance. J Am Coll Cardiol 2006;47: 27-33. 10. 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: 961-5. 11. Michelson AD. Platelet function testing in cardiovascular diseases. Circulation 2004;110:e489-e493. 12. Martin CP, Talbert RL. Aspirin resistance: an evaluation of current evidence and measurement methods. Pharmacotherapy 2005;25: 942-53. 13. Goodall A. The role of platelets and antiplatelet therapy in atherothrombotic disease. Br J Cardiol 2002;9(Suppl 8):S2-S7. 14. Wiviott SD, Braunwald E, McCabe CH, et al. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2007;357:2001-15. 15. Chackalamannil S, Ahn HS, Xia Y, et al. Potent non-peptide thrombin receptor antagonists. Curr Med Chem Cardiovasc Hematol Agents 2003;1:37-45. 16. Gurbel PA. The relationship of platelet reactivity to the occurrence of post-stenting ischemic events: emergence of a new cardiovascular risk factor. Rev Cardiovasc Med 2006;7(Suppl 4):S20-S28. 17. Chan AW, Moliterno DJ, Berger PB, et al. Triple antiplatelet therapy during percutaneous coronary intervention is associated with improved outcomes including one-year survival: results from the Do Tirofiban and ReoProGive Similar Efficacy Outcome Trial (TARGET). J Am Coll Cardiol 2003;42:1188-95. 18. Gurbel PA, Bliden KP, Zaman KA, et al. Clopidogrel loading with eptifibatide to arrest the reactivity of platelets: results of the Clopidogrel Loading With Eptifibatide to Arrest the Reactivity of Platelets (CLEAR PLATELETS) study. Circulation 2005;111:1153-9. 19. Andre P, Delaney SM, LaRocca T, et al. P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries. J Clin Invest 2003;112:398-406. 20. Gurbel PA, Tantry US. Clopidogrel resistance? Thromb Res 2006;120:311-21. 21. Gurbel PA, Bliden KP, DiChiara J, et al. Evaluation of dose-related effects of aspirin on platelet function: results from the Aspirin-Induced Platelet Effect (ASPECT) study. Circulation 2007;115:3156-64. 22. Harrison P. Platelet function analysis. Blood Rev 2005;19: 111-23. 23. Eikelboom JW, Hirsh J, Weitz JI, 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 2002;105:1650-5. 24. Geiger J, Brich J, Honig-Liedl P, et al. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel. Arterioscler Thromb Vasc Biol 1999;19:2007-11. 25. Dyszkiewicz-Korpanty AM, Frenkel EP, Sarode R. Approach to the assessment of platelet function: comparison between opticalbased platelet-rich plasma and impedance-based whole blood platelet aggregation methods. Clin Appl Thromb Hemost 2005;11:25-35. 26. Schlammadinger A, Kerenyi A, Muszbek L, et al. Comparison of the O'Brien filter test and the PFA-100 platelet analyzer in the laboratory diagnosis of von Willebrand's disease. Thromb Haemost 2000;84: 88-92. 27. Hayward CP, Harrison P, Cattaneo M, et al. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function: reply to a rebuttal. J Thromb Haemost 2006;4:1432. 598 Gladding et al 28. van Werkum JW, van der Stelt CA, Seesing TH, et al. A head-to-head comparison between the VerifyNow P2Y12 assay and light transmittance aggregometry for monitoring the individual platelet response to clopidogrel in patients undergoing elective percutaneous coronary intervention. J Thromb Haemost 2006;4: 2516-8. 29. Coleman JL, Wang JC, Simon DI. Determination of individual response to aspirin therapy using the Accumetrics Ultegra RFPA-ASA system. Point of Care 2004;3:77-82. 30. Wheeler GL, Braden GA, Steinhubl SR, et al. The Ultegra rapid platelet-function assay: comparison to standard platelet function assays in patients undergoing percutaneous coronary intervention with abciximab therapy. Am Heart J 2002;143:602-11. 31. Frelinger III AL, Furman MI, Linden MD, et al. Residual arachidonic acid-induced platelet activation via an adenosine diphosphatedependent but cyclooxygenase-1– and cyclooxygenase-2–independent pathway: a 700-patient study of aspirin resistance. Circulation 2006;113:2888-96. 32. Grau AJ, Reiners S, Lichy C, et al. Platelet function under aspirin, clopidogrel, and both after ischemic stroke: a case-crossover study. Stroke 2003;34:849-54. 33. Ziegler S, Maca T, Alt E, et al. Monitoring of antiplatelet therapy with the PFA-100 in peripheral angioplasty patients. Platelets 2002;13: 493-7. 34. Grundmann K, Jaschonek K, Kleine B, et al. Aspirin non-responder status in patients with recurrent cerebral ischemic attacks. J Neurol 2003;250:63-6. 35. Macchi L, Christiaens L, Brabant S, et al. Resistance to aspirin in vitro is associated with increased platelet sensitivity to adenosine diphosphate. Thromb Res 2002;107:45-9. 36. ten Berg JM, Gerritsen WB, Haas FJ, et al. High-dose aspirin in addition to daily low-dose aspirin decreases platelet activation in patients before and after percutaneous coronary intervention. Thromb Res 2002;105:385-90. 37. Gum PA, Kottke-Marchant K, Poggio ED, et al. Profile and prevalence of aspirin resistance in patients with cardiovascular disease. Am J Cardiol 2001;88:230-5. 38. Andersen K, Hurlen M, Arnesen H, et al. Aspirin non-responsiveness as measured by PFA-100 in patients with coronary artery disease. Thromb Res 2002;108:37-42. 39. Gonzalez-Conejero R, Rivera J, Corral J, et al. Biological assessment of aspirin efficacy on healthy individuals: heterogeneous response or aspirin failure? Stroke 2005;36:276-80. 40. Sadiq PA, Puri A, Dixit M, et al. Profile and prevalence of aspirin resistance in Indian patients with coronary artery disease. Indian Heart J 2005;57:658-61. 41. Coleman J, Wang J, Daniel S. Determination of individual response to aspirin therapy using the Accumetrics Ultegra RPFA-ASA system. Point of Care 2004;3:77-82. 42. Serebruany VL, Steinhubl SR, Berger PB, et al. Variability in platelet responsiveness to clopidogrel among 544 individuals. J Am Coll Cardiol 2005;45:246-51. 43. Gurbel PA, Bliden KP, Hiatt BL, et al. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation 2003;107: 2908-13. 44. Cotter G, Shemesh E, Zehavi M, et al. Lack of aspirin effect: aspirin resistance or resistance to taking aspirin? Am Heart J 2004;147: 293-300. 45. Simon DI, Schmaier AH. Sweet and sticky: diabetic platelets, enhanced reactivity, and cardiovascular risk. J Am Coll Cardiol 2007;50:1548-50. American Heart Journal April 2008 46. Angiolillo DJ, Bernardo E, Sabate M, et al. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease. J Am Coll Cardiol 2007;50:1541-7. 47. Tang M, Mukundan M, Yang J, et al. Antiplatelet agents aspirin and clopidogrel are hydrolyzed by distinct carboxylesterases, and clopidogrel is transesterificated in the presence of ethyl alcohol. J Pharmacol Exp Ther 2006;319:1467-76. 48. Cerletti C, Latini R, Del Maschio A, et al. Aspirin kinetics and inhibition of platelet thromboxane generation-relevance for a solution of the “aspirin dilemma”. Thromb Haemost 1985;53: 415-8. 49. Bucchi F, Bodzenta A, de Gaetano G, et al. Effects of 1 gram oral or intravenous aspirin on urinary excretion of thromboxane B2 and 6keto-PGF1 alpha in healthy subjects. Prostaglandins 1986;32: 691-701. 50. Frolich JC. A classification of NSAIDs according to the relative inhibition of cyclooxygenase isoenzymes. Trends Pharmacol Sci 1997;18:30-4. 51. Perneby C, Wallen NH, Rooney C, et al. Dose- and timedependent antiplatelet effects of aspirin. Thromb Haemost 2006;95:652-8. 52. Savi P, Pereillo JM, Uzabiaga MF, et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2000;84:891-6. 53. Pereillo JM, Maftouh M, Andrieu A, et al. Structure and stereochemistry of the active metabolite of clopidogrel. Drug Metab Dispos 2002;30:1288-95. 54. Brandt JT, Close SL, Iturria SJ, et al. Common polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. J Thromb Haemost 2007;5:2429-36. 55. Lau WC, Waskell LA, Watkins PB, et al. Atorvastatin reduces the ability of clopidogrel to inhibit platelet aggregation: a new drug-drug interaction. Circulation 2003;107:32-7. 56. Saw J, Steinhubl SR, Berger PB, et al. Lack of adverse clopidogrelatorvastatin clinical interaction from secondary analysis of a randomized, placebo-controlled clopidogrel trial. Circulation 2003;108:921-4. 57. von Beckerath N, Taubert D, Pogatsa-Murray G, et al. Absorption, metabolization, and antiplatelet effects of 300-, 600-, and 900-mg loading doses of clopidogrel: results of the ISAR-CHOICE (Intracoronary Stenting and Antithrombotic Regimen: Choose Between 3 High Oral Doses for Immediate Clopidogrel Effect) trial. Circulation 2005;112:2946-50. 58. Fromm MF. P-glycoprotein: a defense mechanism limiting oral bioavailability and CNS accumulation of drugs. Int J Clin Pharmacol Ther 2000;38:69-74. 59. Rao GH, Johnson GG, Reddy KR, et al. Ibuprofen protects platelet cyclooxygenase from irreversible inhibition by aspirin. Arteriosclerosis 1983;3:383-8. 60. Catella-Lawson F, Reilly MP, Kapoor SC, et al. Cyclooxygenase inhibitors and the antiplatelet effects of aspirin. N Engl J Med 2001;345:1809-17. 61. Livio M, Del Maschio A, Cerletti C, et al. Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclooxygenase activity. Prostaglandins 1982;23:787-96. 62. Capone ML, Sciulli MG, Tacconelli S, et al. Pharmacodynamic interaction of naproxen with low-dose aspirin in healthy subjects. J Am Coll Cardiol 2005;45:1295-301. 63. Maree AO, Curtin RJ, Chubb A, et al. Cyclooxygenase-1 haplotype modulates platelet response to aspirin. J Thromb Haemost 2005;3: 2340-5. American Heart Journal Volume 155, Number 4 64. Jefferson BK, Foster JH, McCarthy JJ, et al. Aspirin resistance and a single gene. Am J Cardiol 2005;95:805-8. 65. Lev EI, Patel RT, Guthikonda S, et al. Genetic polymorphisms of the platelet receptors P2Y(12), P2Y(1) and GP IIIa and response to aspirin and clopidogrel. Thromb Res 2006;119:355-60. 66. Hollopeter G, Jantzen HM, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001;409:202-7. 67. Fontana P, Dupont A, Gandrille S, et al. Adenosine diphosphateinduced platelet aggregation is associated with P2Y12 gene sequence variations in healthy subjects. Circulation 2003;108: 989-95. 68. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, et al. Contribution of gene sequence variations of the hepatic cytochrome P450 3A4 enzyme to variability in individual responsiveness to clopidogrel. Arterioscler Thromb Vasc Biol 2006;26:1895-900. 69. Hulot JS, Bura A, Villard E, et al. Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects. Blood 2006;108: 2244-7. 70. Taubert D, von Beckerath N, Grimberg G, et al. Impact of Pglycoprotein on clopidogrel absorption. Clin Pharmacol Ther 2006;80:486-501. 71. Pamukcu B, Oflaz H, Onur I, et al. Clinical relevance of aspirin resistance in patients with stable coronary artery disease: a prospective follow-up study (PROSPECTAR). Blood Coagul Fibrinolysis 2007;18:187-92. 72. Chen WH, Cheng X, Lee PY, et al. Aspirin resistance and adverse clinical events in patients with coronary artery disease. Am J Med 2007;120:631-5. 73. Chen WH, Lee PY, Ng W, 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 2004;43:1122-6. 74. Marcucci R, Paniccia R, Antonucci E, et al. Usefulness of aspirin resistance after percutaneous coronary intervention for acute myocardial infarction in predicting one-year major adverse coronary events. Am J Cardiol 2006;98:1156-9. 75. Frossard M, Fuchs I, Leitner JM, et al. Platelet function predicts myocardial damage in patients with acute myocardial infarction. Circulation 2004;110:1392-7. 76. Campo G, Valgimigli M, Gemmati D, et al. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study. J Am Coll Cardiol 2006;48: 2178-85. 77. Jacopo G, Elisabetta V, Silverio S, et al. Identification of platelet hyper-reactivity measured with a portable device immediately after percutaneous coronary intervention predicts in stent thrombosis. Thromb Res 2007;121:407-12. 78. Poulsen TS, Kristensen SR, Korsholm L, et al. Variation and importance of aspirin resistance in patients with known cardiovascular disease. Thromb Res 2006;120:477-84. 79. Valles J, Santos MT, Fuset MP, 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. Gladding et al 599 80. Gurbel PA, Bliden KP, Guyer K, et al. Platelet reactivity in patients and recurrent events post-stenting: results of the PREPARE POST-STENTING study. J Am Coll Cardiol 2005;46:1820-6. 81. Gurbel PA, Bliden KP, Samara W, et al. Clopidogrel effect on platelet reactivity in patients with stent thrombosis: results of the CREST study. J Am Coll Cardiol 2005;46:1827-32. 82. Geisler T, Langer H, Wydymus M, et al. Low response to clopidogrel is associated with cardiovascular outcome after coronary stent implantation. Eur Heart J 2006;27:2420-5. 83. Cuisset T, Frere C, Quilici J, et al. Benefit of a 600-mg loading dose of clopidogrel on platelet reactivity and clinical outcomes in patients with non–ST-segment elevation acute coronary syndrome undergoing coronary stenting. J Am Coll Cardiol 2006;48: 1339-45. 84. Hochholzer W, Trenk D, Bestehorn HP, et al. Impact of the degree of peri-interventional platelet inhibition after loading with clopidogrel on early clinical outcome of elective coronary stent placement. J Am Coll Cardiol 2006;48:1742-50. 85. Bliden KP, DiChiara J, Tantry US, et al. Increased risk in patients with high platelet aggregation receiving chronic clopidogrel therapy undergoing percutaneous coronary intervention: is the current antiplatelet therapy adequate? J Am Coll Cardiol 2007;49:657-66. 86. Buonamici P, Marcucci R, Migliorini A, et al. Impact of platelet reactivity after clopidogrel administration on drug-eluting stent thrombosis. J Am Coll Cardiol 2007;49:2312-7. 87. Patti G, Colonna G, Pasceri V, et al. Randomized trial of high loading dose of clopidogrel for reduction of periprocedural myocardial infarction in patients undergoing coronary intervention: results from the ARMYDA-2 (Antiplatelet therapy for Reduction of MYocardial Damage during Angioplasty) study. Circulation 2005;111: 2099-106. 88. Derry S, Loke YK. Risk of gastrointestinal haemorrhage with long term use of aspirin: meta-analysis. Bmj 2000;321:1183-7. 89. Pulcinelli FM, Pignatelli P, Celestini A, et al. Inhibition of platelet aggregation by aspirin progressively decreases in long-term treated patients. J Am Coll Cardiol 2004;43:979-84. 90. Von Beckerath N, Kastrati A, Wieczorek A, et al. A double-blind randomized comparison between two different clopidogrel maintenance doses after percutaneous coronary intervention (ISARCHOICE 2 trial). (abstr) Eur Heart J 2006;27. 91. Cheng X, Chen WH, Simon DI. Aspirin resistance or variable response or both? Am J Cardiol 2006;98:11N-7N. 92. Weerakkody GJ, Jakubowski JA, Brandt JT, et al. Comparison of speed of onset of platelet inhibition after loading doses of clopidogrel versus prasugrel in healthy volunteers and correlation with responder status. Am J Cardiol 2007;100:331-6. 93. Wiviott SD, Antman EM, Winters KJ, et al. Randomized comparison of prasugrel (CS-747, LY640315), a novel thienopyridine P2Y12 antagonist, with clopidogrel in percutaneous coronary intervention: results of the Joint Utilization of Medications to Block Platelets Optimally (JUMBO)-TIMI 26 trial. Circulation 2005;111:3366-73. 94. Wiviott SD, Trenk D, Frelinger AL, et al. Prasugrel compared with high loading- and maintenance-dose clopidogrel in patients with planned percutaneous coronary intervention; the Prasugrel in Comparison to Clopidogrel for Inhibition of Platelet Activation and AggregationThrombolysis in Myocardial Infarction 44 trial. Circulation 2007;116:2923-32.