| | Aspirin resistance among long-term aspirin users after carotid endarterectomy and controls: Flow cytometric measurement of aspirin-induced platelet inhibitionReceived 14 December 2006; accepted 24 January 2007. BackgroundNumerous studies have indicated that some patient subpopulations do not respond to the antithrombotic effects of aspirin. The objective of this study was to evaluate aspirin-induced inhibition of platelet cyclooxygenase (COX) using a flow cytometric technique in long-term aspirin users after carotid endarterectomy (CEA) and controls with newly diagnosed carotid stenosis not taking aspirin and to compare these results with platelet function analyzer measurements. MethodsThe study included 86 patients with a history of CEA on long-term aspirin therapy (100 mg daily) and 29 age-matched patients with newly diagnosed carotid artery stenosis not taking aspirin. Platelet-rich plasma diluted with phosphate-buffered saline was incubated with arachidonic acid (ARA) at a final concentration of 80 μmol/L. After staining with phycoerythrin-labeled anti-P-selectin (CD62p) antibody, platelet CD62p-antigen expression was measured on a flow cytometer. ResultsFlow cytometric measurement of ARA-induced platelet activation showed an inhibition of ARA-induced platelet stimulation in all patients on aspirin therapy, whereas all but two controls (95%) showed expected platelet reactivity. In contrast, results of the platelet function analyzer measurements were normal in 16% of aspirin-treated patients. ConclusionsFlow cytometric measurement of CD62p expression on platelets after incubation with ARA proved to be a practicable tool to monitor aspirin-induced inhibition of platelet COX. Results in patients on long-term low-dose aspirin therapy show that the inability of aspirin to inhibit platelet COX for both symptomatic and asymptomatic patients with high-grade internal carotid artery stenosis is a very rare event. So-called aspirin resistance detected quite frequently by platelet function analyzer measurement is most likely from COX-independent mechanisms. Aspirin (acetylsalicylic acid) is the most widely used antiplatelet drug for the primary and secondary prevention of arterial thrombotic events. Aspirin reduces platelet activity by irreversibly acetylating cyclooxygenase (COX), thus inhibiting the production of platelet-derived thromboxane A2.1, 2, 3, 4 Aspirin therapy has been shown to reduce the risk of arterial thrombotic events in all vascular beds,3, 4 yet in some patients, there is a lack of expected effects. This has lead to the use of the term aspirin resistance. Unfortunately, the term aspirin resistance has been applied to describe several quite different entities. Sometimes it is used to describe the inability of aspirin to reduce platelet production of thromboxane A2,5 others use the term for a clinical failure of the drug or the inability to produce anticipated effects on in vivo (bleeding time) or in vitro tests of platelet functions.3, 6, 7 Some important questions need to be addressed, however. Is aspirin capable of blocking platelet COX and thus ultimately the production of thromboxane A2 in long-term aspirin users? And if yes, why do patients receiving sufficient aspirin therapy experience ischemic events? Can these patients be identified and treated alternatively? In platelets, arachidonic acid (ARA) is converted to thromboxane A2 by a reaction catalyzed by the enzymes COX and thromboxane synthetase.5 Therefore, incubation with ARA leads to a COX-dependent activation of platelets that is inhibited by aspirin. Measuring ARA-induced platelet aggregation has been used to evaluate the effect of aspirin on platelets,8 mostly by aggregometry. However, the method has limited reproducibility, is extremely labor intensive, and is unavailable in most laboratories.6 The objective of this study was to evaluate aspirin-induced inhibition of platelet COX using a flow cytometric technique in long-term aspirin users after carotid endarterectomy and in controls with newly diagnosed carotid stenosis not taking aspirin. Recent studies have demonstrated that patients have transient aspirin resistance during carotid endarterectomy that lasts about 24 hours.9, 10 Yet to our knowledge, no investigations so far have addressed a possible long-term effect. The present study used a simple flow cytometric method to evaluate the activity of COX in platelets by measuring the activation antigen CD62p (P-selectin) on platelet surfaces after stimulation of platelets with ARA. Results were compared with those of a platelet function analyzer, PFA-100 (Behring, Marburg, Germany), a device frequently used to detect aspirin resistance.11 Materials and methods  Patients The study initially included 100 patients. To ensure accurate aspirin use, all patients were tested for compliance. Patients were given a 4-week supply of aspirin and asked to return within 4 weeks with the box and the remaining pills. Then, the remaining tablets were counted. Patients not having taken the amount of tablets ± three tablets for the follow-up period of 24 days (range, 21 to 27 days) were not estimated as sufficiently compliant. The study evaluated all patients who had high grade internal carotid artery (ICA) stenosis >70% according to North American Symptomatic Carotid Endarterectomy Trial12 criteria, who had undergone carotid endarterectomy at least 3 months preceding inclusion into the study, and who had no interventions during the last 3 months. All patients had been taking low-dose aspirin therapy for at least 3 months (Thromboass, Lannacher Heilmittel Ltd, Lannach, Austria) before surgery, and all patients with symptomatic carotid disease had been taking low-dose aspirin therapy at least 1 month before their ischemic neurologic event. Patients taking other antiplatelet medications or those receiving oral anticoagulation with vitamin K antagonists or heparin were excluded. An age-matched group of 29 patients with newly diagnosed carotid artery stenosis taking no antiplatelet or anticoagulatory medication served as controls. All patients had a stenosis of 70% to >90%, indicating revascularization. The study was approved by the local Ethics Committee. Blood collection and routine laboratory analyses A cubital vein was punctured after application of a light tourniquet. Immediately after venipuncture, the tourniquet was released. First, blood was collected into a 5 mL K3-ethylenediaminetetraacetic acid standard vacuum tube (Vacuette, Greiner Bio-One, Kremsmünster, Austria), which was used to determine the complete blood count. Another 5 mL of blood was drawn in a Vacuette tube containing sodium citrate (3.2% trisodium citrate; Greiner Bio-One) to be used for flow cytometric measurements. Then, 5 ml blood was drawn in a tube containing 3.8% trisodium citrate for analysis of closure-time on the PFA-100. Finally, blood for routine blood chemistry analyses was drawn in a serum tube containing standard gel. Blood samples were used for platelet analysis ≤20 minutes after venipuncture. Routine hematology and chemistry analyses were performed ≤2 hours after venipuncture. Platelet stimulation and staining of P-selectin (CD62p) First, platelet-rich plasma (PRP) was prepared by centrifuging the citrated blood samples for 15 minutes at 100g. PRP was diluted 1:10 with phosphate-buffered saline (PBS), and 20 μL of the diluted PRP was mixed with 10 μL of ARA (final ARA concentration 80 μmol/L; Diamed, Cressier sur Morat, Switzerland) and incubated for 10 minutes in a waterbath at 37°C. Then, 10 μL of phycoerythrin (PE)-labeled anti-CD62p (P-selectin) antibody (Beckmann-Coulter, Miami, Florida) or the corresponding isotype control of the same manufacturer was added and samples were incubated for another 20 minutes at room temperature in the dark. The reaction was stopped by adding 500 μL cold PBS. To minimize in vitro artifacts, fixation or washing procedures were omitted. To determine ARA-independent platelet activation, samples were also incubated with PBS instead of ARA. Results of PBS-stimulated activation were subtracted from ARA-stimulated results to yield the true ARA-induced effect. To prove platelet reactivity, samples were also incubated with adenosine diphosphate (ADP; final ADP concentration, 2.5 μmol/L) instead of ARA. In analogy to establishing a reference range, a range was defined encompassing 95% of a group of subjects without aspirin therapy (29 control patients and 80 healthy volunteers). To categorize platelet reactivity to ARA, a subject was considered reactive if ARA stimulation induced expression of CD62p within this range; that is, exceeded the 0.05 percentile of subjects without aspirin therapy (ie, >15% CD62p-positive platelets). Flow cytometric analyses Prepared samples were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif). Instrument stability was monitored by daily measurement of calibrations beads (Calibrite 3 Beads, Becton Dickinson). Side scatter, forward scatter, and fluorescence signals were measured using the instrument’s logarithmic amplification mode. Data were acquired applying a forward scatter threshold excluding all events smaller than platelets. A minimum of 10,000 platelets was collected per sample. Platelets were defined based on their light scatter properties in a forward-scatter/side-scatter dot-plot and gated into a FL-2 (PE-channel) histogram, where P-selectin (CD62p) expression on the platelet surface was measured. The percentage of CD62p PE-positive events was determined by setting a threshold according to the isotype control. PFA-100 The PFA-100 is a platelet function analyzer that has been designed as an in vitro measure of primary hemostasis at high shear rate, mimicking flow conditions in the arteries. Shear stress–induced capillary closure time of whole blood was measured using a collagen/epinephrin-coated membrane (EPI cartridge) and a collagen/ADP-coated membrane (ADP cartridge). The reference range was defined according to the recommendations of the manufacturer, which was validated before the method was adopted into laboratory routine. Thus, an aspirin response was defined using the normal range cutoff of 165 seconds on the EPI cartridge. Patients with a collagen-epinephrine closure time (CEPI-CT) of >165 seconds were considered aspirin responders. Statistical analysis Statistical calculations were performed with Statistica 5.1 (Statsoft, Tulsa, Okla) for Windows (Microsoft, Redmond, Wash). Data are reported as medians (lower quartile, upper quartile). For results not following a normal distribution, nonparametric methods were used to statistically describe and compare groups. The nonparametric Mann-Whitney U test was used to evaluate the statistical significance of differences between the study groups. Results Of the 100 patients initially included and tested for compliance, 14 patients were excluded from the further study. Of the remaining 86 patients, 47 were men and 39 women, and their median age was 73 years (range, 67 to 80). In the 86 patients who underwent operations for carotid stenosis, 47 were symptomatic and 39 were asymptomatic. Patient demographics, risk factors, and medications are listed in Table I, Table II. Comparisons of PFA 100 results and CD62 expression according to clinical stage are listed in Table III. | | |  | Risk factor | Internal carotid stenosis, n (%) | |
|---|
| Asymptomatic (n = 39) | Symptomatic (n = 47) | |
|---|
| Hypertension | 32 (82) | 36(76) | | | Diabetes mellitus | 13(33) | 17(36) | | | Smoking | 12(31) | 17(36) | | | Hypercholesterolemia | 22(56) | 30(64) | | | Renal insufficiency | 3(7) | 3(6) | | | COPD | 5(13) | 7(15) | | | Angina | 15(38) | 19(40) | | | PAOD | 11(28) | 13(28) | | | | |
| | | | Medication | Patients, n (%) n = 86 | Controls, n (%) n = 29 | |
|---|
| Antihypertensive therapy | 67(78) | 22(76) | | | Oral antidiabetic therapy | 29(34) | 9(31) | | | Insulin | 1(1) | 1(3) | | | Statins | 58(67) | 18(62) | | | Aspirin | 86(100) | 0(0) | | | | |
The age-matched group of 29 patients with newly diagnosed carotid artery stenosis taking no antiplatelet medication consisted of 20 men and 9 women, with a median age of 72 years (range, 64 to 76 years). Of these, 13 were symptomatic, and 16 presented with asymptomatic high-grade carotid stenosis. Reactivity to arachidonic acid stimulation Patients and their respective platelets were considered reactive (and thus not demonstrating the anticipated aspirin effect) if the ARA-induced expression of CD62p exceeded the 0.05 percentile of subjects without aspirin therapy (15%; 29 matched controls and 80 tested healthy volunteers without aspirin therapy; data not shown). Applying this threshold, none of the platelets of the 86 patients receiving aspirin therapy were reactive to stimulation by ARA (Fig 1). Conversely, the platelets of 27 (93%) of the 29 patient controls were reactive to ARA, and two (7%) showed no reactivity. ADP incubation significantly increased the proportion of CD62p-positive platelets in all patients. There was no difference between aspirin-treated patients and patients without aspirin therapy (79% [72% to 87%] vs 81% [76% to 85%]; P = .252); platelets of all patients were reactive to ADP. Patients receiving aspirin therapy showed a slightly lower number of CD62-positive platelets after PBS incubation than control patients [8% (5% to 13%) vs 11% (9% to 15%); P = .017]. The two patients in the control group with a lack of reactivity to ARA both showed an elevated CEPI-CT (166 seconds and 228 seconds, respectively). Thus, intake of a COX-inhibiting drug could not be excluded. Shear stress–induced capillary closure time (PFA-100) Contrary to flow cytometric results, 14 (16%) of the 86 patients receiving aspirin showed no effect of aspirin and had a normal CEPI-CT (Fig 2). There was no difference in sex, age, or other risk factors or distribution of normal PFA-100 readings between symptomatic and asymptomatic patients. Four (14%) of the 29 patient controls showed an increased CEPI-CT. Discussion Aspirin resistance has been defined in many different ways, possibly reflecting the lack of consideration of the underlying pathophysiologic mechanisms.5, 8 Accordingly, the reported prevalence of aspirin resistance ranges from <1% to >40%.13, 14 High prevalence is generally reported when clinical definitions of aspirin resistance are applied or the PFA-100 platelet function analyzer is used.14, 15, 16 Thus, a clinical outcome or a surrogate of over all platelet function is depicted without addressing the underlying biochemical mechanisms of thromboxane A2–induced, COX-dependent platelet aggregation. One of the possible mechanisms of aspirin resistance is the inability of aspirin to inhibit platelet COX. As several isoforms of COX exist, and aspirin in its low-dose formulation is mostly COX-1 selective, expression of other isoforms by megacaryocytes have been linked to treatment failure.17 This effect takes place in the presence of systemic inflammation that occurs in embolic events causing tissue damage—as for instance in brain or myocardial infarction. In the present study, we used a flow cytometric method to evaluate the activity of platelet COX by measuring platelet activation after incubation with ARA. COX catalyzes a major step in the pathway from ARA to thromboxane A2, a potent stimulator of platelet aggregation. Expression of surface CD62p was measured as a sign of ARA-induced platelet activation. Although sample manipulations should be kept to a minimum for platelet analysis18 and whole blood methods have been used successfully,19 we decided to use PRP because we detected in vitro lysis of red blood cells when PBS-diluted citrated whole blood was incubated with ARA (already at a final concentration of 125 μmol/L, data not shown). ADP derived from the hemolysate might stimulate platelets in an aspirin-independent way; therefore, the use of PRP was preferred. The use of the present method to detect aspirin resistance in patients after carotid endarterectomy under long-term low-dose aspirin therapy resulted in markedly decreased reactivity to ARA in all 86 patients, indicating suppressed COX activity. The strength of this current study is that the measured aspirin effects were underlined by controls with the same disease not using aspirin, and the expected reactivity to ARA was demonstrated in these patients. This finding is of interest because monotherapy with aspirin remains a cornerstone in stroke prevention, especially in light of recent studies.20, 21 Also, it has been suggested that point-of-care tests such as PFA-100 and the Ultegra-RPFA system (Accumetrics, San Diego, Calif) may be used for detecting aspirin resistance in patients after stroke.11 Yet, discrepant results of these tests and light transmission aggregometry were seen. In this study, neither asymptomatic nor symptomatic patients with high grade ICA stenosis, whose symptoms were under aspirin therapy, were aspirin resistant as measured by flow cytometry. This finding could indicate that stroke in these patients may not have been caused by aspirin failure. Although this contrasts with results obtained by other methods for detecting aspirin resistance (or so-called aspirin resistance) or definitions, it is in very good agreement with recent studies in which only 0.8% of patients had aspirin-resistance when ARA-induced platelet aggregation was measured.13 Also, a recent study with 700 patients undergoing coronary catheterization revealed ARA-induced platelet activation in <2% of patients.22 These data suggest that aspirin resistance defined as unsuppressed platelet COX activity is a very rare phenomenon. When analyzing the same group of aspirin-treated patients with the PFA-100, we observed a normal CEPI-CT in 14 (16%) of 86 of patients, which is in agreement with results of other studies.12, 13, 14 These patients may be regarded as “aspirin resistant” as frequently defined in studies applying the PFA-100. Interestingly, aspirin-treated patients with a normal CEPI-CT did not show a higher number of ARA-induced activated platelets than those with a prolonged CEPI-CT (3.6% [2.8% to 4.2%] vs 4.5% [2.8% to 6.3%]; P = .137). This suggests that the high number of patients with normal CEPI-CT found with the PFA-100 is not due to a more sensitive detection of residual COX activity but that the PFA-100 detects other effects of platelet activation that are based on factors other than residual platelet COX activity. And indeed, thromboxane A2–induced COX-dependent platelet aggregation is only one of many catalytic pathways of platelet aggregation. The present study has several limitations. First, the clinical relevance of elevated PFA-100 CEPI-CT in patients regarding increased risk for ischemic events could not be addressed owing to the cross-sectional design. Second, the sample size was too small to demonstrate a possible difference in platelet reactivity between symptomatic and asymptomatic patients. Third, we could demonstrate that ARA-induced expression of CD62p was suppressed in all patients; however, this effect describes only the aspirin effect on one platelet receptor. Overall assessments of platelet activity and reactivity, such as platelet-leukocyte or platelet-monocyte aggregates, may be better comparable with PFA-100 CEPI-CT and clinically more relevant. Conclusion Flow cytometric measurement of CD62p expression on platelets after incubation with ARA proved to be a practical tool to monitor aspirin-induced inhibition of platelet COX. The results of this study and previous studies13, 22 do suggest that aspirin does exert its desired effects on the target enzyme in platelets in almost all compliant patients. However, COX is only one of many, albeit important, catalytic enzymes in the process of platelet aggregation. It seems that what is described as aspirin resistance today—ischemic events under aspirin therapy—may have other underlying mechanisms than the inability of aspirin to block platelet COX. In light of these findings, it appears that PFA-100 could be more appropriate in detecting patients prone to form thrombus under adequate aspirin therapy. However, the underlying mechanism and its clinical relevance need to be investigated further in longitudinal studies. Author contributions Conception and design: AA, WH Analysis and interpretation: AA, UML, GWH, PMB, WL Data collection: JL, UML, WH Writing the article: AA, WH Critical revision of the article: AA, JL, UML, GWH, PMB, WH Final approval of the article: AA, JL, UML, GWH, PMB, WH Statistical analysis: WH Obtained funding: Not applicable Overall responsibility: AA References 1. 1Roth GJ, Majerus PW. The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein. J Clin Invest. 1975;56:624–632. MEDLINE |
CrossRef
2. 2Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci U S A. 1975;72:3073–3076. MEDLINE |
CrossRef
3. 3Patrono C, Coller B, FitzGerald GA, Hirsh J, Roth G. Platelet-active drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest. 2004;126:234S–264S. MEDLINE |
CrossRef
4. 4Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353:2373–2383.
CrossRef
5. 5Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet. 2006;367:606–617. Abstract | Full Text |
Full-Text PDF (162 KB)
|
CrossRef
6. 6Patrono C. Aspirin resistance: definition, mechanisms and clinical read-outs. J Thromb Haemost. 2003;1:1710–1713. MEDLINE |
CrossRef
7. 7Michelson AD, Cattaneo M, Eikelboom JW, Gurbel P, Kottke-Marchant K, Kunicki TJ, et al. Aspirin resistance: position paper of the Working Group on Aspirin Resistance. J Thromb Haemost. 2005;3:1309–1311. MEDLINE |
CrossRef
8. 8Szczeklik A, Musial J, Undas A, Sanak M, Nizankowski R. Aspirin resistance. Pharmacol Rep. 2005;57(suppl):33–41. 9. 9Payne DA, Jones CI, Hayes PD, Webster SE, Naylor AR, Goodall AH. Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance?. Thromb Haemost. 2004;92:89–96. MEDLINE 10. 10Webster SE, Payne DA, Jones CI, Hayes PD, Bell PRF, Goodall AH, et al. Anti-platelet effect of aspirin is substantially reduced after administration of heparin during carotid endarterectomy. J Vasc Surg. 2004;40:463–468. Abstract | Full Text |
Full-Text PDF (131 KB)
|
CrossRef
11. 11Harrison P, Segal H, Blasbery K, Furtado C, Silver L, Rothwell PM. Screening for aspirin responsiveness after transient ischemic attack and stroke (Comparison of 2 point-of-care platelet function tests with optical aggregometry). Stroke. 2005;36:1001–1005.
CrossRef
12. 12North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high grade stenosis. N Engl J Med. 1991;325:444–445. 13. 13Hillarp A, Lethagen S, Mattiasson I. Aspirin resistance is not a common biochemical phenotype explained by unblocked cyclooxygenase-1 activity. J Thromb Haemost. 2003;1:196–197. MEDLINE |
CrossRef
14. 14Sambola A, Heras M, Escolar G, Lozano M, Pino M, Martorell T, et al. The PFA-100 detects sub-optimal antiplatelet responses in patients on aspirin. Platelets. 2004;15:439–446. MEDLINE |
CrossRef
15. 15Alberts MJ, Bergman DL, Molner E, Jovanovic BD, Ushiwata I, Teruya J. Antiplatelet effect of aspirin in patients with cerebrovascular disease. Stroke. 2004;35:175–178.
CrossRef
16. 16Ohmori T, Yatomi Y, Nonaka T, Kobayashi Y, Madoiwa S, Mimuro J, et al. Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity: involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients. J Thromb Haemost. 2006;4:1271–1278. MEDLINE |
CrossRef
17. 17Mitchell JA, Warmenr TD. Cox isoforms in the cardiovascular system: understanding the activities of non-steroidal anti-inflammatory drugs. Nat Rev. 2006;5:75–85. 18. 18Schmitz G, Rothe G, Ruf A, Barlage S, Tschope D, Clemetson KJ, et al. European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost. 1998;79:885–896. MEDLINE 19. 19Panzer S, Hocker L, Koren D. Agonists-induced platelet activation varies considerably in healthy male individuals: studies by flow cytometry. Ann Hematol. 2006;85:121–125. MEDLINE |
CrossRef
20. 20Diener HC, Bogousslavsky J, Brass LM, Cimminiello C, Csiba L, Kaste M, et al.MATCH investigators Aspirin and clopidogrel compared with clopidogrel alone after recent ischaemic stroke or transient ischaemic attack in high-risk patients (MATCH): randomised, double-blind, placebo-controlled trial. Lancet. 2004;364:331–337. Abstract | Full Text |
Full-Text PDF (102 KB)
|
CrossRef
21. 21Bhatt DL, Fox KAA, Hacke W, Berger PB, Black HR, Boden WE, et al.CHARISMA Investigators Clopidogrel and Aspirin versus Aspirin Alone for the Prevention of Atherothrombotic Events. N Engl J Med. 2006;354:1706–1717.
CrossRef
22. 22Frelinger AL, Furman MI, Linden MD, Li Y, Fox ML, Barnar MR, et al. Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independant pathway (A 700-patient study of aspirin resistance). Circulation. 2006;113:2888–2896.
CrossRef
a Department of General and Vascular Surgery, Wilhelminenspital Vienna, Vienna, Austria b Department of Laboratory Medicine, Wilhelminenspital Vienna, Vienna, Austria.  Reprint requests: Afshin Assadian, MD, Department of General and Vascular Surgery, Wilhelminenspital Vienna, Montleartstr 37, A-1160 Vienna, Austria.
Competition of interest: none. PII: S0741-5214(07)00229-7 doi:10.1016/j.jvs.2007.01.064 © 2007 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved. | |
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