Residual Arachidonic Acid–Induced Platelet Activation via an Adenosine Diphosphate–Dependent but Cyclooxygenase-1– and Cyclooxygenase-2–Independent Pathway
A 700-Patient Study of Aspirin Resistance
Background— Thrombotic events still occur in aspirin-treated patients with coronary artery disease.
Methods and Results— To better understand aspirin “resistance,” serum thromboxane B2 (TXB2) and flow cytometric measures of arachidonic acid–induced platelet activation (before and after the ex vivo addition of aspirin and indomethacin) were analyzed in 700 consecutive aspirin-treated patients undergoing cardiac catheterization. In 680 of 682 evaluable patients, serum TXB2 concentrations were reduced compared with nonaspirinated healthy donors. Twelve patients had serum TXB2 that was lower than nonaspirinated healthy donors but >10 ng/mL. Arachidonic acid stimulated greater platelet activation in patients with high serum TXB2 (>10 ng/mL) than in patients with low serum TXB2. Addition of ex vivo aspirin reduced arachidonic acid–induced platelet activation to similar levels regardless of serum TXB2 concentrations, which suggests that patients with high residual serum TXB2 concentrations were either noncompliant or underdosed with aspirin. Among the remaining 98% of patients, ex vivo administration of either aspirin or indomethacin failed to prevent platelet activation across all degrees of arachidonic acid–induced platelet activation and aspirin doses. Although the patients were not randomized with respect to clopidogrel treatment, multivariate analysis showed that arachidonic acid–induced platelet activation was less in patients receiving clopidogrel.
Conclusions— There is a residual arachidonic acid–induced platelet activation in aspirin-treated patients that (1) is caused by underdosing and/or noncompliance in only &2% of patients and (2) in the remaining patients, occurs via a cyclooxygenase-1 and cyclooxygenase-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate–induced platelet activation.
Received October 20, 2005; revision received April 14, 2006; accepted April 24, 2006.
Aspirin, via its antiplatelet effect, reduces the odds of an arterial thrombotic event in high-risk patients by &25%.1 However, 10% to 20% of patients with an arterial thrombotic event who are treated with aspirin have a recurrent arterial thrombotic event during long-term follow-up.1 The occurrence of arterial thrombotic events in patients receiving aspirin is often referred to as aspirin resistance or treatment failure.2 Patients demonstrate a marked variability in their laboratory responses to aspirin.3,4 True aspirin resistance has been suggested to be the insufficient inhibition of cyclooxygenase (COX)–dependent thromboxane A2 synthesis from arachidonic acid in subjects receiving aspirin.3,4 However, most clinical studies describing aspirin resistance have assessed platelet function using assays in which the COX pathway is just one of several pathways leading to platelet activation, and therefore, aspirin inhibition of COX could not be evaluated specifically.3,4 Moreover, no steps were taken to differentiate true aspirin resistance from the potentially confounding problems of patient noncompliance or underdosing. In an attempt to better understand the mechanisms of aspirin resistance, in the present study of 700 aspirin-treated patients, we therefore measured COX-dependent arachidonic acid–induced platelet function before and after the ex vivo addition of aspirin or indomethacin.
Editorial p 2865
Clinical Perspective p 2896
Patient Study Population
The study was approved by the Committee for the Protection of Human Subjects at the University of Massachusetts Medical School. All patients presenting to the Cardiac Catheterization Laboratories at UMass Memorial Medical Center for diagnostic cardiac catheterization between the hours of 7 am and 3 pm on weekdays from July 2002 through July 2004 were evaluated, and those who met the enrollment criteria of self-reported intake of either 81 or 325 mg of aspirin per day for 3 or more days were invited to participate. Patients receiving glycoprotein (GP) IIb/IIIa antagonists were excluded from the study. A total of 700 consecutive patients were enrolled. Fewer than 3% of eligible patients declined participation. After patients provided written informed consent, peripheral arterial or venous blood was drawn before angiography from the femoral artery or vein after sheath insertion. The blood was immediately placed in Becton Dickinson (BD Biosciences, San Jose, Calif) evacuated tubes containing either 3.2% sodium citrate or no anticoagulant. In anticoagulated blood drawn by standard venipuncture from patients, there is typically a low incidence of small clots observed in the samples. Consistent with this, in the present study, 17 (2.4%) of a total of 700 patients were excluded on this basis. In addition, 1 patient requested to be withdrawn from the study after originally consenting to it. Evaluable results were therefore obtained from 682 subjects.
Coronary artery disease (CAD) was diagnosed by the presence of any intracoronary luminal irregularities suggestive of atherosclerosis on angiography. Angiography results were interpreted by cardiologists blinded to the assay results.
Normal Subject Population
After they provided written informed consent, 30 healthy adult subjects were studied before and after 7 days of aspirin 81 mg/d. Exclusion criteria for the healthy subjects were ingestion of aspirin in the 10 days before study; ingestion of a nonsteroidal antiinflammatory drug in the 3 days before study; history of bleeding disorder, gastrointestinal bleeding, hemorrhagic stroke, or allergy to aspirin; pregnancy; thrombocytopenia; anemia; or leukopenia. This sample size provides 80% power to detect an absolute difference of 6% P-selectin–positive platelets. A separate healthy subject population (n=34, age 36.9±1.8 years [mean±SEM], 17 males) not taking aspirin or any other antiplatelet drug (including nonsteroidal antiinflammatory drugs) during the previous 10 days was used for the serum thromboxane measurements shown in Figure 1.
Serum Thromboxane B2
Serum was separated from the nonanticoagulated blood and stored at −80°C until analysis. Serum TXB2 (the stable metabolite of thromboxane A2) was measured by an ELISA kit from R&D Systems (Minneapolis, Minn) according to the manufacturer’s recommendations.
Flow Cytometric Markers of Platelet Activation
Platelet fibrinogen receptor (GP IIb/IIIa, integrin αIIbβ3) activation and platelet surface P-selectin were measured by flow cytometry as described previously,5 with the following modifications. Fluorescein isothiocyanate–conjugated PAC1, phycoerythrin (PE)-conjugated CD62P, peridinin chlorophyll protein–conjugated CD61, and control Immunoglobulin G–PE were from Becton Dickinson. Aspirin (Sigma; St. Louis, Mo) and indomethacin (Sigma) were dissolved in ethanol at 56 and 10 mmol/L, respectively, and then diluted in 10 mmol/L HEPES, 0.15 mol/L NaCl, pH 7.4, before use. Arachidonic acid was a lyophilized preparation of sodium arachidonate (Bio/Data Corporation, Horsham, Pa) that was rehydrated with water before use, according to the manufacturer’s recommendations. Aliquots of 3.2% citrate anticoagulated blood were incubated with vehicle (0.1% ethanol vol/vol), aspirin 56 μmol/L, or indomethacin 10 μmol/L for 5 minutes at 37°C and then stimulated with arachidonic acid 125 μg/mL for 5 minutes in the presence of monoclonal antibody PAC1–fluorescein isothiocyanate (which recognizes the activated conformation of GP IIb/IIIa6). Samples were then fixed by the addition of 1% formaldehyde in 10 mmol/L HEPES, 0.15 mol/L sodium chloride, pH 7.4. After 30 minutes’ fixation, the samples were diluted with 0.5% bovine serum albumin in 10 mmol/L HEPES, 0.15 mol/L sodium chloride, pH 7.4, and stained with monoclonal antibodies CD61–peridinin chlorophyll protein (as a platelet identifier) and CD62P-PE (P-selectin specific). Sample analysis was performed in a Becton Dickinson FACSCalibur flow cytometer with CellQuest software (Becton Dickinson) as described previously.5 Positive analysis regions for P-selectin were set with appropriate nonspecific controls. PAC1 was analyzed by mean fluorescence intensity.
In preliminary experiments, aspirin 5.6 μmol/L was found to maximally inhibit arachidonic acid–stimulated platelet activation. Therefore, in the present study, a 10-fold higher concentration, ie, aspirin 56 μmol/L, was used. A higher aspirin concentration was not used because we wanted to fully inhibit COX-1 while limiting inhibition of COX-2, to enable us to dissect the mechanism of the residual arachidonic acid–induced platelet activation.
Statistical analyses were performed with Microsoft Excel, GraphPad Prism (GraphPad, San Diego, Calif), or SAS (SAS Institute, Cary, NC) software. ANOVAs were performed for multiple comparisons, with adjustments for differences between groups. Medical records for all enrolled patients were reviewed. Variables that required adjustment included age, gender, body mass index, white cell count, hematocrit, presence of diabetes mellitus, previous percutaneous coronary intervention, previous coronary artery bypass grafting, current smoking, nonsteroidal antiinflammatory drug use, heparin use, and insulin use. Patient demographics were analyzed with unpaired t test for continuous variables and χ2 test for categorical variables. Serum TXB2 ELISA results below the limit of detection were assigned an arbitrary low value and analyzed by nonparametric Kruskal-Wallis test with Dunn multiple comparisons. Log transformation of serum TXB2 concentrations was performed before analyses because these concentrations were not normally distributed. For all statistical tests, a probability value of 0.05 or less was considered significant. Results reported are mean±SEM unless otherwise stated.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
Of the 682 evaluable patients, 593 had angiographic evidence of coronary atherosclerosis. Compared with patients without CAD, patients with CAD were, as expected, older (P<0.0001), more often male (P<0.0001), and more likely to have a history of hyperlipidemia (P<0.0001), hypertension (P<0.0001), and diabetes mellitus (P=0.0027). Patients with CAD were also more likely than patients without CAD to be taking aspirin 325 mg/d (compared with 81 mg/d; P<0.001), clopidogrel (P=0.034), β-Blockers (P=0.032), statins (P<0.0001), angiotensin-converting enzyme inhibitors (P=0.017), calcium channel blockers (P=0.006), insulin (P=0.016), and oral hypoglycemic agents (P=0.044). Patients taking 325 mg of aspirin compared with those taking 81 mg of aspirin were more often male (P=0.006), younger (P=0.04), current smokers (P=0.04), and taking a statin (P=0.03), were less likely to be taking warfarin (P<0.0001), and were more likely to have a history of acute myocardial infarction (P=0.004), percutaneous coronary intervention (P<0.0001), or coronary artery bypass grafting (P=0.009).
Pharmacological Efficacy of Aspirin: Inhibition of Serum TXB2 Generation
Aspirin inhibits COX-1, thereby preventing the generation of thromboxane A2 and its stable metabolite, TXB2.1 We therefore measured serum TXB2 to estimate residual COX-1 function in aspirin-treated patients and in healthy control subjects. Figure 1 shows the serum TXB2 concentrations for healthy control subjects, the entire study population, patients without CAD who were taking 81 or 325 mg/d of aspirin, and patients with CAD who were taking 81 or 325 mg/d of aspirin. Demographic characteristics of these groups are shown in Table 1. The mean serum TXB2 concentration for the healthy control subjects was 242±18.8 ng/mL. In patients without CAD, the mean serum TXB2 concentration was 1.7±0.4 ng/mL for those taking aspirin 81 mg/d and 0.8±0.1 ng/mL for those taking 325 mg/d (P=NS by multivariate analysis, with adjustment for demographic differences between patient groups). In patients with CAD, the mean serum TXB2 concentration was 2.6±0.5 ng/mL for those taking 81 mg of aspirin and 1.1±0.2 ng/mL for those taking 325 mg of aspirin (P<0.001). Two patients from the study population fell into the normal range of serum TXB2 in the present study (Figure 1). These patients demonstrated normal platelet function in response to various agonists (including arachidonic acid), and this platelet function was inhibited by the ex vivo addition of aspirin 56 μmol/L to the same degree as in healthy aspirin-free control subjects (data not shown). Therefore, these 2 subjects were deemed to be noncompliant with aspirin therapy and were not included in subsequent analyses.
An additional 12 patients had serum TXB2 concentrations that were lower than nonaspirinated normal donors but greater than 10 ng/mL (2 standard deviations above the mean of all aspirinated patients; Figure 1). Of these 12 patients, 3 were taking 325 mg of aspirin, and 2 of these 3 were also taking 75 mg of clopidogrel. The remaining 9 subjects were taking 81 mg of aspirin, and 2 of these 9 subjects were also taking 75 mg of clopidogrel. Although the overall incidence of underdosing (serum TXB2 >10 ng/mL) was low (12 of 682, or 1.8%), underdosed patients were more likely to be taking 81 mg of aspirin (9 of 206, or 4.4% of all patients taking 81 mg aspirin) than 325 mg of aspirin (3 of 476, or 0.6% of patients taking 325 mg aspirin; Fisher exact test P<0.002).
Despite the fact that the patients were aspirinated (81 or 325 mg/d for ≥3 days), there was a significant residual arachidonic acid–induced increase in platelet surface P-selectin in patients with a low (<10 ng/mL) TXB2 (Figure 2A, left; P<0.01) and a high (>10 ng/mL) TXB2 (Figure 2A, right; P<0.01). However, arachidonic acid elicited greater platelet activation in blood from patients with high TXB2 concentrations (>10 ng/mL) than in patients with low serum TXB2 (<10 ng/mL; Figure 2A; P<0.001). The addition of ex vivo aspirin 56 μmol/L (a concentration in excess of predicted concentration after ingestion of 81 mg of aspirin7) significantly reduced arachidonic acid–induced platelet activation of the high serum TXB2 group (Figure 2A, right; P=0.0486) to a level comparable to that of the low serum TXB2 group (Figure 2A, left), which suggests that the 14 patients (of a total of 678, or 2% of the total) with high residual serum TXB2 concentrations were either underdosed or noncompliant with aspirin.
Mechanism of Residual Arachidonic Acid–Induced Platelet Activation in Aspirin-Treated Patients and Healthy Subjects
As expected, aspirin-free healthy subjects (n=30, age 38.6±2.1 years [mean±SEM], 13 males) demonstrated marked platelet surface expression of P-selectin in response to the COX-1 pathway–specific platelet agonist arachidonic acid 125 μg/mL (Figure 2B, left; P<0.001). Also as expected, this platelet activation was inhibited by the ex vivo addition of aspirin 56 μmol/L (Figure 2B, left; P<0.001), but there was a residual arachidonic acid–induced platelet surface expression of P-selectin (P<0.001) similar to that observed in the patient group (Figure 2A), despite the use of a concentration of aspirin that was 10 times higher than the amount necessary for maximal inhibition, as determined in preliminary experiments (see Methods).
We also examined the response of healthy volunteers to oral aspirin (Figure 2B, right). As expected, oral aspirin treatment (81 mg/d for 7 days) of the normal donor group led to a reduced arachidonic acid–stimulated response compared with before aspirin (P<0.001). However, the arachidonic acid–stimulated response in the presence of oral aspirin was still elevated compared with the unstimulated samples (P<0.001; Figure 2B, right) and was not further reduced by the ex vivo addition of aspirin 56 μmol/L (P>0.05; Figure 2B, right). These results with healthy subjects (Figure 2B, right, in which each healthy subject acted as their own control) were similar to the results we observed in patients treated with aspirin (Figure 2A, in which each patient acted as their own control), with and without the ex vivo addition of aspirin, which suggests that in both healthy subjects and patients, there is an arachidonic acid–stimulated platelet response that is independent of COX-1.
The addition of a nonselective COX-1 and COX-2 antagonist, indomethacin 10 μmol/L, significantly reduced arachidonic acid–induced platelet activation in the high serum TXB2 patient group (Figure 2A, right; P=0.0171) to a level comparable to that of the low serum TXB2 group (Figure 2A, left), and to a level comparable to that after the addition of aspirin 56 μmol/L. As with the ex vivo addition of aspirin, residual arachidonic acid–induced platelet activation after the ex vivo addition of indomethacin in aspirin-treated patients was greater than levels in samples not incubated with arachidonic acid (Figure 2A; P<0.001 for patients with TXB2 <10 ng/mL and P<0.01 for patients with TXB2 >10 ng/mL). Comparable results were obtained with binding of monoclonal antibody PAC1 as the marker of platelet activation (data not shown).
To determine whether the statistically significant residual response to arachidonic acid in patients (n=668) with low serum TXB2 concentrations (<10 ng/mL) was uniform across all patients or was driven by a subset of patients with a high response to arachidonic acid, patients were separated into quartiles based on 125 μg/mL arachidonic acid–stimulated GP IIb/IIIa activation (Figure 3). There was no difference between unstimulated and arachidonic acid–stimulated platelet activation as judged by PAC1 binding in the first quartile of the patients studied (Q1 in Figure 3; P>0.05, paired t test). However, in the remaining 3 quartiles (Q2 through Q4 of Figure 3), arachidonic acid produced a significant increase in GP IIb/IIIa activation (P<0.0001, paired t test), and this activation was not prevented by the addition of ex vivo aspirin 56 μmol/L or indomethacin 10 μmol/L. Moreover, the magnitude of the arachidonic acid–induced platelet activation occurred in direct proportion to the degree of activation in circulating (non–ex vivo–stimulated) platelets (Pearson correlation coefficient, r=0.7204, P<0.0001; Figure 3). Comparable results were obtained with surface expression of P-selectin as the marker of platelet activation (data not shown). As demonstrated in Figure 4, regardless of either the dose of aspirin taken by the patient or the presence or absence of angiographically defined CAD, the ex vivo addition of aspirin did not inhibit arachidonic acid–stimulated platelet activation.
To assess whether clopidogrel (an adenosine diphosphate [ADP] P2Y12 receptor antagonist1) affected arachidonic acid–induced platelet activation in aspirin-treated patients with low residual TXB2 concentrations (<10 ng/mL), patients receiving clopidogrel were compared with those not receiving clopidogrel. Demographic differences between these patient groups (Table 2) were adjusted for in multivariate analyses. As demonstrated in Figure 5, baseline levels of platelet activation (as determined by platelet surface P-selectin) were similar regardless of clopidogrel use; however, patients receiving clopidogrel had significantly less arachidonic acid–induced platelet activation than those not receiving clopidogrel (Figure 5). Nevertheless, even in the clopidogrel-treated patients with low residual serum TXB2 concentrations, residual arachidonic acid–induced platelet surface P-selectin is present and is not inhibited by ex vivo aspirin 56 μmol/L or indomethacin 10 μmol/L (Figure 5). Therefore, residual arachidonic acid–induced platelet activation is mediated, but only in part, by ADP-induced platelet activation.
The present large (700 patients) study provides novel information that addresses 2 of the main current hypotheses in the literature about the mechanism of aspirin resistance.2 Thus, in contrast to previous clinical studies of aspirin resistance, the present study examines the effects of ex vivo addition of aspirin and indomethacin to patient samples, as a means to address (1) underdosing of aspirin (addressed by the ex vivo addition of aspirin) and (2) the contribution to thromboxane precursors by COX-2 (addressed by the ex vivo addition of indomethacin). The major conclusion of the present study is that there is a residual arachidonic acid–induced platelet activation in aspirin-treated patients that (1) is caused by underdosing or noncompliance in only a minority of patients (&2%) and (2) in the remaining patients, occurs via a COX-1– and COX-2–independent pathway, because ex vivo treatment of patient platelets with additional high concentrations of aspirin or indomethacin failed to block arachidonic acid–induced platelet activation, occurred in direct proportion to the degree of activation in circulating (non–ex vivo–stimulated) platelets, and was mediated in part by ADP-induced platelet activation, as evidenced by the results with clopidogrel.
In the present study, serum TXB2 concentrations were suppressed in almost all subjects taking either 81 or 325 mg of aspirin daily, consistent with the known ability of low-dose aspirin to completely inhibit platelet COX-1.1 Indeed, dosages as low as 30 mg/d have proved effective in reducing vascular events in at-risk patients.1 In contrast to some other studies that reported a high noncompliance rate in patients’ self-reporting of aspirin usage,8 but consistent with other studies,9 the present study demonstrates a high rate of compliance with aspirin as determined by suppression of serum TXB2 concentrations (Figure 1). Furthermore, underdosing of aspirin was not a major factor in the presently reported, residual arachidonic acid–induced platelet activation. Only 12 patients (ie, &2% of the total number of study patients) had residual TXB2 concentrations 2 times above the standard deviation of the population (Figure 1). Within this small group, a greater proportion of patients were taking 81 mg rather than 325 mg of aspirin (P<0.002), which suggests a dosage effect. Platelet activation in these 12 patients was further suppressible by the ex vivo addition of either aspirin or indomethacin (Figure 2). Whether these patients demonstrated intermittent compliance with aspirin administration or a true need for a higher dose of aspirin cannot be determined by the present study. In any event, this 2% rate of aspirin noncompliance and/or aspirin underdosing in the present study provided us with the opportunity to establish in the remaining 98% of patients the presence of residual arachidonic acid–induced platelet activation. Furthermore, we were able to investigate the mechanism of the residual arachidonic acid–induced platelet activation in this large cohort of aspirin-treated patients.
The arachidonic acid–induced platelet activation that occurred despite aspirin therapy was not further inhibited by high-dose supplemental aspirin or indomethacin, which indicates that a COX-1– and COX-2–independent pathway is responsible for this phenomenon. The COX-1 and COX-2 independence of this pathway was demonstrated by 2 different pathophysiologically relevant end points: (1) platelet surface P-selectin10 (Figure 2) and (2) activated GP IIb/IIIa (integrin αIIbβ3)11 (Figures 3 and 4⇑). Furthermore, results with healthy subjects (Figures 2B, in which each healthy subject acted as their own control) were similar to the results we observed in patients treated with aspirin (Figure 2A, in which each patient acted as their own control), with and without the ex vivo addition of aspirin; this indicates that in both healthy subjects and patients, there is a heretofore undescribed arachidonic acid–stimulated platelet response that is independent of COX-1. The presence of this pathway in normal donors indicates that the aspirin-resistant, arachidonic acid–stimulated response observed in patients is not necessarily a result of their disease or treatment with aspirin.
Although the patient population was not randomized with respect to clopidogrel treatment, multivariate analysis (in which we adjusted for differences between patients not taking clopidogrel and those taking clopidogrel) suggested that those patients taking clopidogrel had significantly reduced arachidonic acid–induced platelet activation (Figure 5). This partial inhibition of the arachidonic acid response by clopidogrel suggests that ADP, and specifically its P2Y12 receptor, is involved in the response, with the implication that like COX-1–dependent activation, the presently demonstrated COX-1– and COX-2–independent platelet activation induces platelet dense granule release. Indeed, thromboxane A2 is known to cause platelet shape change and intracellular calcium mobilization, but thromboxane A2–induced platelet aggregation is dependent on intracellular granular secretion and subsequent ADP-induced platelet activation.12 Because clopidogrel is known to incompletely block ADP-induced platelet activation,1 further inhibition, and therefore perhaps additional clinical benefit, may be possible with P2Y12 antagonists currently under development that result in more complete blockade of P2Y12 receptors.13 Alternatively, or in addition, the residual arachidonic acid–induced platelet activation in clopidogrel users may derive from a non–P2Y12-dependent pathway. The partial inhibition of the arachidonic acid response by clopidogrel suggests the release of ADP from platelet dense granules. Therefore, ADP P2Y1 receptor inhibitors14 or inhibitors of other agonists released from dense granules (eg, serotonin15) might also be effective in reducing the residual platelet activation response to arachidonic acid in aspirinated patients.
It is important to note that failure of the ex vivo addition of indomethacin to inhibit responses in whole-blood assays in the present study rules out the presence of prostaglandin H2 generated by COX-2 in blood cells other than platelets (as suggested by others2–4,16) as a potential pathway. Alternative possible pathways for aspirin-resistant platelet activation in response to exogenously added or endogenously liberated arachidonic acid include enzymatic pathways (eg, 12-lipoxygenase–derived 12-hydroperoxyeicosatetraenoic acid) and nonenzymatic pathways (eg, oxidation-dependent synthesis of arachidonic acid–derived isoprostanes).17 Finally, our finding that the baseline circulating (no ex vivo stimulation) degree of platelet activation is proportional to the arachidonic acid–induced ex vivo platelet activation (Figure 3) suggests that platelets may be activated in vivo by a similar mechanism.
In accord with the literature,18 the present data (Figure 2) demonstrate that the patient group as a whole had a higher baseline platelet surface P-selectin expression than healthy subjects. However, our conclusions are independent of this difference, because each patient was used as their own control (ie, aliquots of a single blood sample were used to determine both unstimulated and arachidonic acid–stimulated response with and without ex vivo addition of aspirin and indomethacin), and, separately, each healthy subject was used as their own control (ie, aliquots of a single blood sample were used to determine both unstimulated and arachidonic acid–stimulated response with and without ex vivo addition of aspirin and indomethacin).
The mechanisms of aspirin resistance have been poorly defined.2–4 In the present study, we demonstrate a novel mechanism for aspirin resistance: an ADP-dependent but COX-1– and COX-2–independent pathway of residual arachidonic acid–induced platelet activation. Although the correct treatment of aspirin resistance remains unknown,2–4 our findings with clopidogrel (Figure 5) raise the possibility that clopidogrel therapy, in addition to its direct antiplatelet effect via the P2Y12 ADP receptor,1,3,4 may decrease aspirin resistance in both the presence and absence of CAD. Clinical outcomes studies will be required to prove this hypothesis before any clinical recommendations can be made.
Patients in the present study were not randomized to the dose of aspirin or to administration of clopidogrel. In addition, patients with CAD not taking aspirin were not used as control subjects; however, this control is not practical, because there are very few CAD patients not taking aspirin.
Sources of Funding
This study was funded in part by an unrestricted grant from Dade Behring to the Center for Platelet Function Studies.
Dr Frelinger has received research grant support from BioCytex, Dade Behring, Eli Lilly, and the TIMI Study Group. Dr Furman has received research grant support from Dade Behring and Eli Lilly and honoraria for speaking for Sanofi Aventis. Dr Michelson has received research grant support from Accumetrics, BioCytex, Dade Behring, Eli Lilly, McNeil, the TIMI Study Group, and the National Institutes of Health (pending), and honoraria from Eli Lilly and Co, Sanofi Aventis/Bristol-Myers Squibb, and Dade Behring. The remaining authors report no conflicts.
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McEver RP. P-selectin/PSGL-1 and other interactions between platelets, leukocytes, and endothelium. In: Michelson AD, ed. Platelets. New York, NY: Academic Press/Elsevier Science; 2002:chap 7.
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Wiviott SD, Antman EM, Winters KJ, Weerakkody G, Murphy SA, Behounek BD, Carney RJ, Lazzam C, McKay RG, McCabe CH, Braunwald E. 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: 3366–3373.
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The occurrence of arterial thrombotic events in patients receiving aspirin is often referred to as aspirin resistance or treatment failure. Patients demonstrate a marked variability in their laboratory responses to aspirin. True aspirin resistance has been suggested to be insufficient inhibition of cyclooxygenase (COX)–dependent thromboxane A2 synthesis from arachidonic acid in subjects receiving aspirin. However, most clinical studies describing aspirin resistance have assessed platelet function using assays in which the COX pathway is just one of several pathways leading to platelet activation, and therefore, aspirin inhibition of COX could not be evaluated specifically. Moreover, no steps were taken to differentiate true aspirin resistance from the potentially confounding problems of patient noncompliance or underdosing. In the present study of 700 aspirin-treated patients, we demonstrate that there is a residual arachidonic acid–induced platelet activation that (1) is caused by underdosing and/or noncompliance in only &2% of patients, and (2) in the remaining patients, occurs via a COX-1 and COX-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate–induced platelet activation. Although the correct treatment of aspirin resistance remains unknown, our findings suggest that duel antiplatelet therapy with a thienopyridine, in addition to its direct antiplatelet effect via the P2Y12 adenosine diphosphate receptor, may decrease residual arachidonic acid–induced platelet activation in both the presence and absence of coronary artery disease. Clinical outcomes studies will be required to prove this hypothesis before any clinical recommendations can be made.