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Annals of Clinical & Laboratory Science 38:126-131 (2008)
© 2008 Association of Clinical Scientists

Aspirin Non-Responsiveness in Korean Subjects on Dual Anti-Platelet Treatment Determined by Two Different Platelet Function Assays

Yong-Wha Lee1, Yoon-Haeng Cho2, Yong-Hyun Kim1, Jong-Sung Na1, Hee Bong Shin1, Chang-Seok Ki3, Tae-Youn Choi1 and You Kyoung Lee1
1 Departments of Laboratory Medicine and 2 Cardiology, Soonchunhyang University College of Medicine, Bucheon; 3 Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Address correspondence to You Kyoung Lee, M.D., Ph.D., Department of Laboratory Medicine, Soonchunhyang University Bucheon Hospital, 1174 Jung-dong, Wonmi-gu, Bucheon 420-767, Korea; tel 82 32 621 5941; fax 82 32 621 5944; e-mail: cecilia{at}schbc.ac.kr or lywmd{at}schbc.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several techniques are available for measuring platelet function during aspirin therapy, but none is well standardized, and the reported incidence of aspirin non-responders varies widely, from 5 to 50%. We evaluated the optical platelet aggregation test and the Platelet Function Analyzer-100 test (PFA-100) for assessing aspirin responsiveness in patients receiving dual anti-platelet therapy, and we measured the incidence of non-responders to aspirin among Koreans. The study enrolled 88 participants including 51 patients on dual anti-platelet therapy, 31 controls, and 6 other volunteers. Optical platelet aggregation in response to 4 agonists and aggregation using the PFA-100 test were determined. In addition, medical records, including the results of platelet aggregation tests, were reviewed for 351 patients receiving aspirin therapy. The results showed good correlation between the PFA-100 test using a collagen/epinephrine cartridge (CEPI) and the optical platelet aggregation test using each agonist. The platelet aggregation test using arachidonic acid revealed marked suppression of aggregation (>98% inhibition) in patients taking aspirin; this value was highly correlated with the PFA-100 results using the CEPI cartridge. Seven of 351 Korean subjects (2.0%) receiving aspirin treatment were non-responsive to aspirin. This study shows that the optical platelet aggregation test using arachidonic acid gave an accurate assessment of the response to aspirin, and that results of the PFA-100 test using the CEPI cartridge correlated well with results of the optical platelet aggregation test.

Keywords: Aspirin, clopidogrel, platelet aggregation, platelet function tests


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aspirin is the most widely prescribed anti-platelet drug and is effective in preventing myocardial infarction (MI), stroke, and death in patients at high risk for occlusive vascular events [1]. The beneficial effects of aspirin are attributable to its irreversible inhibition of cycloxygenase-1, which prevents the conversion of arachidonic acid to the unstable prostaglandin intermediate PGH2 and consequently prevents the conversion of PGH2 to thromboxane TxA2, a vasoconstrictor and proaggregant present in platelets. It has been hypothesized that patients with absent or diminished anti-platelet response to aspirin, so-called clinical aspirin non-responsiveness, have increased risk of arterial atherothrombotic events [2]. However, a cause-effect relationship between platelet insensitivity to aspirin and cardiovascular recurrence has not been well defined overall because aspirin compliance has not been adequately evaluated [3].

Several techniques are available for measuring platelet function during aspirin therapy. None of these tests has been well standardized, and the wide variety of methods makes the standardization of platelet function tests difficult [4]. The Platelet Function Analyzer-100 (PFA-100, Dade Behring, Marburg, Germany) can be used to assess platelet-related hemostasis in citrated whole blood and has been proposed as a screening test for identifying aspirin-resistant patients [5].

The reported incidence of aspirin non-responders varies from 5 to 50%, with remarkably different values resulting from different platelet function tests [6–8]. The incidence of aspirin non-responders among Koreans has not been investigated using different platelet function assays.

In this study, two different platelet function tests, a conventional optical platelet aggregation test using four agonists and the PFA-100 test, were performed to evaluate aspirin responsiveness in Korean subjects with arterial occlusive disease who were receiving dual anti-platelet therapy. The results identified the definitive agonist for screening aspirin responsiveness. In addition, the incidence of aspirin non-responders in a Korean population was investigated by reviewing the medical records, including the results of platelet aggregation tests, for 351 Korean subjects taking aspirin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects.  A total of 88 participants were enrolled in this study. Over a 3-mo period, 51 consecutive patients with arterial occlusive disease who were taking daily oral doses of 100–325 mg aspirin and 75 mg clopidogrel (Plavix) were enrolled. Thirty-one subjects with no history of a bleeding tendency or anti-platelet drug therapy were enrolled as healthy controls. An additional 6 healthy volunteers underwent the optical platelet aggregation test and PFA-100 assay before and after ingesting 100 mg of aspirin daily for 3 days. Patients with a blood platelet count <100 x 109/L, hemoglobin <9 g/dl, or hematocrit <35%, all of which are known to influence platelet function, were excluded. All of the participants were free from any known interfering medication. Patient compliance and their latest dosages of anti-platelet drugs were assessed using a detailed questionnaire. To investigate the incidence of aspirin non-responders in a Korean population, the medical records, including the results of platelet aggregation tests, of 351 Korean patients receiving aspirin were reviewed intensively. This study was approved by the institutional review board and informed written consent was obtained from each subject before enrollment in the study.

Blood samples.  Before blood samples were collected from the subjects, their clinical characteristics regarding pre-existing clotting abnormalities, disease status, and the use of drugs affecting hemostasis were determined. Blood samples were obtained from all subjects by venipuncture and were used for the PFA-100 and platelet aggregation tests. For the platelet aggregation tests, 4 samples were collected in 3-ml tubes containing 3.2% sodium citrate. For the PFA-100 test, blood samples were collected in 3-ml tubes containing 3.2% sodium citrate and analyzed independently.

Platelet aggregation test.  The whole-blood samples in citrate were centrifuged at 179 x g for 10 min to obtain platelet-rich plasma (PRP), which was kept at room temperature. The remaining blood was centrifuged at 2195 x g for 10 min to obtain platelet-poor plasma. The platelet count was adjusted to 200 x 109 to 400 x 109/L with the autologous platelet-poor plasma. After a 5-min incubation at 37°C, platelet aggregation was induced by addition of arachidonic acid (0.5 mg/ml), adenosine diphosphate (ADP) (5.0 or 14.0 µmol/L), epinephrine (500 µmol/L), or collagen (4 µg/ml). The assay was performed with constant stirring at 37°C, using a Chronolog lumiaggregometer (Labordiagnostica, Endingen, Germany). The change in turbidity due to aggregation was recorded and the result was expressed as percent aggregation.

PFA-100 assay.  The PFA-100 assay was performed in duplicate with citrated whole blood according to the manufacturer’s instructions. Aggregation was induced using either a collagen/ epinephrine (CEPI) or collagen/ADP (CADP) cartridge, each of which contained a membrane coated with 2 µg of collagen and either 10 µg of epinephrine or 50 µg of ADP, respectively. A syringe was used to aspirate citrated whole blood under high shear stress through a small aperture cut in the membrane, and a platelet plug formed as a response to the high shear stress and the agonists on the membrane. This plug ultimately blocked the blood flow, and the time to occlusion of the microscopic aperture (closure time, CT) indicated platelet function. The maximum allowable CT was 300 sec, and any time longer than this was reported as non-closure. The tests were repeated in the event of an error in measurement.

Reference ranges.  For both tests, the reference ranges were determined based on samples from 37 healthy control individuals and 6 volunteers who had not taken any anti-platelet drugs during the previous 2 weeks. The reference ranges for both tests were established at the respective 5th percentiles.

Statistics.  Results obtained with the PFA-100 and those of the platelet aggregation test were compared by Pearson’s correlation analysis. To examine whether platelet aggregation differed significantly before and after aspirin ingestion in the 6 volunteers, a paired-sample t-test was performed using SPSS 11.5 software (SPSS, Chicago, IL). A p value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reference values in healthy controls.  For the optical platelet aggregation test, the reference values for agonist-induced aggregation responses at the end of aggregation, based on the 43 healthy individuals, were: ADP (5 µmol/L), >36%; ADP (14 µmol/L), >65%; collagen, >64%; epinephrine, >58%; and arachidonic acid, >50%. The closure time (CT) reference range for the PFA-100 with the CEPI cartridge (CEPI-CT) was 94-188 sec, and with the CADP cartridge (CADP-CT) it was 60–130 sec.

Response to aspirin ingestion in volunteers.  Platelet function was determined in 6 volunteers after ingestion of aspirin. The percent platelet aggregation in response to ADP, epinephrine, or arachidonic acid was significantly decreased and the CEPI-CT was significantly increased in the post-aspirin samples compared with the pre-treatment samples in all volunteers (Fig. 1Go). The mean values differed significantly between the post- and pre-treatment samples (p < 0.05; Table 1Go). Furthermore, all of the post-treatment percent aggregation values were lower than the reference values established for the platelet aggregation test, and the CEPI-CT was higher than the 188-sec cut-off value for the PFA-100 test.


Figure 1
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  		Fig. 1. Response to aspirin ingestion in volunteers. Inhibition % of platelet aggregation and CEPI-CT of post-treatment samples were significantly increased compared with those of pre-treatment samples using platelet aggregation tests with ADP, epinephrine or arachidonic acid, and PFA-100 assay in all volunteers. CEPI-CT: Closure time of collagen/epinephrine cartridge.

 

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  		Table 1. Results of the optical platelet aggregation test and the PFA-100 test of the pre- and post-treatment samples in 6 volunteers who ingested 100 mg of aspirin daily for 3 days.
 
Comparison of the platelet aggregation and PFA-100 tests.  A comparison of the PFA-100 and platelet aggregation test results demonstrated that the CEPI-CT correlated well with the percent aggregation for each agonist in patients on dual therapy and in control individuals (Fig. 2Go). The percent aggregation in response to arachidonic acid or epinephrine was significantly inhibited and the CEPI-CT was significantly increased in patients treated with aspirin compared to control individuals. Specifically, the platelet aggregation test using arachidonic acid revealed marked suppression of aggregation, >98% inhibition, in patients taking aspirin; this value was highly correlated with the CEPI-CT (Table 2Go). These results were also in accord with the results in the volunteers.


Figure 2
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  		Fig. 2. Comparison study between platelet aggregation test and PFA-100 assay. CEPI-CT correlated well with the results of platelet aggregation tests using each agonist in patients with dual therapy and control individuals. R: Correlation coefficient, CEPI-CT: Closure time of collagen/epinephrine cartridge.

 

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  		Table 2. Comparison between healthy control individuals and patients with aspirin treatment.
 
Incidence of aspirin non-responsiveness in Koreans.  Aspirin non-responsiveness was defined as a CEPI-CT below the upper limit of the reference range, ie, <188 sec, and an arachidonic acid-induced aggregation response >50%. Two of the 51 patients taking aspirin showed aspirin non-responsiveness. In these 2 patients, the percent aggregation induced by arachidonic acid (55 and 73%, respectively) and the CEPI-CT (153 and 127 sec, respectively) were within the reference range for each test. The results in these patients were also within the reference ranges for platelet aggregation using epinephrine and collagen and for the CADP-CT.

A review of medical records revealed that 7 of 351 Korean subjects (2%) treated with aspirin showed aspirin non-responsiveness on a platelet aggregation test using arachidonic acid, and 6 of these aspirin non-responders showed a normal aggregation response to epinephrine (Table 3Go).


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  		Table 3. Clinical and laboratory characteristics of aspirin non-responders in Korea.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our study, the incidence of aspirin non-responders was low in Koreans compared with other ethnic groups. Variable rates of aspirin non-responsiveness (5-51%) have been reported using optical aggregometry or the PFA-100 assay [6,7]. Coakley et al [8] reported that a large proportion (51%) of the study population demonstrated aspirin non-responsiveness according to the PFA-100 test. However, this high rate might be attributable to the contribution of high levels of von Willebrand factor (vWF) to the CEPI-CT, because high vWF levels cause persistent aggregation of platelets despite adequate inhibition of thromboxane A2 production by aspirin. To elucidate the incidence of aspirin non-responsiveness in this study, we reviewed the platelet aggregation test results and medical records of 351 Korean patients receiving aspirin and found an incidence of 2%. The higher incidence of non-responders in other reports might be attributable to imprecision of the PFA-100 assay and not because of a standardized cut-off value or patient non-compliance.

In our study, the patients showing aspirin non-responsiveness by the PFA-100 test had compatible results by the platelet aggregation test using arachidonic acid. These findings differed from the results of Chakroun et al [9], who found that the observed number of aspirin non-responders increased when the PFA-100 test was used to monitor aspirin treatment [9]. They suggested that increased levels of vWF led to the higher observed incidence of in vitro aspirin non-responsiveness by the PFA-100 test.

Our reference CEPI-CT time (188 sec) was similar to previously reported times of 165, 176, 193, and 200 sec [5,10–12]. Some disparity in the proposed cut-off values might result from different citrate concentrations used for anticoagulation or variations in the ages of the subjects.

In our study, the lack of a correlation between the results with the PFA-100 test using the CADP cartridge and those with the platelet aggregation test using ADP or collagen might be explained by the differences in agonist concentrations: the platelet aggregation test used 2 concentrations of ADP (5 µM and 14 µM), whereas the CADP cartridge used 50 µg of ADP in combination with another platelet agonist, 2 µg of equine type I collagen (data not shown).

Although we recruited patients receiving both aspirin and clopidogrel as anti-platelet therapy, clopidogrel non-responsiveness could not be evaluated. Clopidogrel is an effective and specific inhibitor of the P2Y12 ADP receptor, but it can also bind to a second ADP receptor, P2Y1. The extent of residual P2Y1-dependent platelet aggregation inducible by ADP in the presence of clopidogrel varies widely among individuals.

Therefore, use of ADP alone to induce platelet aggregation would not be a suitable test for evaluating clopidogrel non-responsiveness [13]. In addition, aspirin affects ADP-induced platelet aggregation to some extent, as seen in our volunteer study and other studies [14,15], and thus may mask clopidogrel non-responsiveness, making ADP-induced aggregation inappropriate for measuring clopidogrel non-responsiveness in the presence of aspirin.

As the cyclooxygenase (COX)-1 pathway is strongly inhibited by aspirin and is not inhibited by clopidogrel, we could evaluate the effect of aspirin in patients on dual therapy in our study [16]. Previous studies reported no significant effect of clopidogrel on the PFA-100 CEPI-CT and optical platelet aggregation percentage using arachidonic acid as an agonist [16,17]. There is increasing interest in finding a platelet function test that permits the evaluation of platelet responsiveness in patients on dual anti-platelet therapy (aspirin and clopidogrel).

In conclusion, the platelet aggregation test using arachidonic acid gave an accurate assessment of the response to aspirin, and the results of the PFA-100 test using the CEPI cartridge correlated well with the optical platelet aggregation results.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Anon. 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.[Abstract/Free Full Text]
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  4. Breddin HK, Harder S. The value of platelet function tests. Vasa 2003;32:123–129.[Medline]
  5. Feuring M, Schultz A, Losel R, Wehling M. Monitoring acetylsalicylic acid effects with the platelet function analyzer PFA-100. Semin Thromb Hemost 2005;31:411–415.[Medline]
  6. Gum PA, Kottke-Marchant K, Welsh PA, White J, Topol EJ. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol 2003; 41:961–965.[Abstract/Free Full Text]
  7. Mani H, Linnemann B, Luxembourg B, Kirchmayr K, Lindhoff-Last E. Response to aspirin and clopidogrel monitored with different platelet function methods. Platelets 2006;17:303–310.[Medline]
  8. Coakley M, Self R, Marchant W, Mackie I, Mallett SV, Mythen M. Use of the platelet function analyser (PFA-100) to quantify the effect of low dose aspirin in patients with ischaemic heart disease. Anaesthesia 2005;60:1173–1178.[Medline]
  9. Chakroun T, Gerotziafas G, Robert F, Lecrubier C, Samama MM, Hatmi M, Elalamy I. In vitro aspirin resistance detected by PFA-100 closure time: pivotal role of plasma von Willebrand factor. Br J Haematol 2004; 124:80–85.[Medline]
  10. Mammen EF, Alshameeri RS, Comp PC. Preliminary data from a field trial of the PFA-100 system. Semin Thromb Hemost 1995;21(Suppl 2):113–121.[Medline]
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  12. Svenstrup Poulsen T, Risom Kristensen S, Atar D, Mickley H. A critical appraisal of the phenomenon of aspirin resistance. Cardiology 2005;104:83–91.[Medline]
  13. Aleil B, Ravanat C, Cazenave JP, Rochoux G, Heitz A, Gachet C. Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases. J Thromb Haemost 2005;3:85–92.[Medline]
  14. Mueller MR, Salat A, Stangl P, Murabito M, Pulaki S, Boehm D, Koppensteiner R, Ergun E, Mittlboeck M, Schreiner W, Losert U, Wolner E. Variable platelet response to low-dose ASA and the risk of limb deterioration in patients submitted to peripheral arterial angioplasty. Thromb Haemost 1997;78:1003–1007.[Medline]
  15. Geiger J, Teichmann L, Grossmann R, Aktas B, Steigerwald U, Walter U, Schinzel R. Monitoring of clopidogrel action: comparison of methods. Clin Chem 2005;51:957–965.[Abstract/Free Full Text]
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H. Kim, H. K. Lee, K. Han, and H.-K. Jeon
Prevalence and Risk Factors for Aspirin and Clopidogrel Resistance in Patients with Coronary Artery Disease or Ischemic Cerebrovascular Disease
Ann. Clin. Lab. Sci., January 1, 2009; 39(3): 289 - 294.
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