• aspirin;
  • cerebrovascular diseases;
  • coronary heart diseases;
  • cyclooxygenase;
  • platelets


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary. Objectives: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin-treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. Results: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase-1 (COX-1) activity by measuring collagen-induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser-light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen-induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX-1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter-individual differences; variation in platelet COX-1 activity only accounted for 6–20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX-1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen-related peptide. We then prospectively enrolled 136 aspirin-treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30–11.9, P = 0.015–0.033). Conclusions: Aspirin resistance expressed as unsuppressed platelet COX-1 activity is a rare condition in an out-patient population. Other factor(s) affecting collagen-induced platelet aggregation may influence early outcomes in aspirin-treated patients.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aspirin reduces the risk of cardiovascular events by approximately 25% in a broad category of patients with arterial vascular disease [1,2]. Aspirin exerts its anti-thrombotic effects through the inhibition of platelet cyclooxygenase-1 (COX-1) by the irreversible acetylation of a specific serine moiety, thereby blocking the formation of thromboxane (Tx) A2 for the lifetime of the platelets [3–5]. The term ‘aspirin resistance’ has been used to describe the clinical inability of aspirin to protect individuals from arterial thrombotic events or when laboratory methods indicate the failure of aspirin to inhibit platelet activity [3,6]. Previous studies have estimated that between 8–45% of patients who suffered an ischemic stroke or cardiovascular disease are aspirin resistant [2,5,6]. Although the problem of aspirin resistance has been greatly emphasized in the medical literature, its precise definition and even its frequency are still unknown. Indeed, the term ‘aspirin resistance’ has been given different definitions by different researchers.

It was shown that those with a higher concentration of urinary 11-dehydro TxB2, a stable marker of TxA2 production, had a 3.5-fold higher risk of cardiovascular death [7]. Furthermore, aspirin resistance defined by aggregation tests was associated with a greater than 3-fold increase in the risk of major adverse events [8]. These data indicate that screening for aspirin resistance may optimize the anti-platelet treatment for the prevention of cardiovascular events. Accordingly, we must clarify the mechanism(s) by which some patients’ platelets are resistant and thereby establish a concise definition of aspirin resistance. In the present studies to investigate the mechanism(s) of aspirin resistance in aspirin-treated patients, we measured platelet function and COX activity by directly assaying collagen-induced TxB2 production and determined the level of urinary 11-dehydroTxB2. For platelet aggregometry, we used both the conventional light transmission (LT) method and a laser-light scattering method to quantitatively evaluate aggregate sizes and numbers. Finally, we prospectively enrolled 136 aspirin-treated patients to assess whether platelet aggregation is an attractive test for determining clinical outcome.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients and study protocol

The institutional review board of Minobusan Hospital (Yamanashi, Japan) approved the study protocols and informed consent was obtained from all participants. The first part of these series of investigations was a study called ‘Mechanisms of Aspirin Resistance: the Relationship Between TxB2 Production and Platelet Aggregation’; 51 aspirin-treated individuals (81 mg), who had all given informed consent, and 50 control individuals took part in this study. The baseline characteristics, including age; sex; additional medication use; plasma fibrinogen; the prevalence of hypertension (HT), diabetes mellitus (DM), and hyperlipidemia (HL); and additional treatment such as antihypertensive drugs were not significantly different between the two groups (data not shown). Collagen-induced TxB2 production, platelet aggregation induced by 0.3, 1, and 3 μg mL−1 collagen; 2 μmol L−1 adenosine diphosphate (ADP); and 0.03 and 0.1 μg mL−1 collagen-related peptide (CRP) were measured in all patients. Urinary 11-dehydroTxB2 was measured in urine samples from 40 aspirin-treated patients that had given informed consent. One patient who did not comply with taking aspirin was excluded from the analysis.

The next study, entitled ‘The Prospective Determination of the History of Aspirin Sensitivity Measured by Aggregometry’, was performed in April, 2002 to determine whether inter-individual aggregation differences influenced the clinical outcomes. Finally, we prospectively enrolled 140 stable, aspirin-treated out-patients (81 mg) with a previous history of cerebral infarction or ischemic heart diseases in December 2003. At the time of enrollment, platelet aggregation induced by 0.3 and 1 μg mL−1 collagen and 2 μmol L−1 ADP was examined. Exclusion criteria included the following: ingestion of ticlopidine, dipyridamole, anti-inflammatory drugs, or other drugs affecting platelet function; platelet count <10 × 107 mL−1 or 40 × 107 mL−1; myeloproliferative disorders; atrial fibrillation. Compliance on aspirin was determined by patient interview both at study enrollment and follow-up. The primary endpoint was the composite of myocardial infarction (MI), cerebrovascular infarction, or death from cardiovascular events. Follow-up was performed by telephone interview and from medical records. Persons performing follow-up were unaware of the aspirin sensitivity status.

Platelet aggregation

There are many pre-analytical and analytical variables that affect the results of platelet aggregation [3]. In addition, it is difficult to compare the results obtained in one laboratory to those of another because of the lack of standardization. Therefore, we carefully standardized the conditions of blood collecting and time to measurement, and only two experienced laboratory staff members who were kept unaware of the patient information performed assays to increase the precision.

Fasting venous blood was carefully collected using a 21-guage needle into a syringe containing 1/10 sodium citrate. Blood collection was carried out at 08.30–10.00 hours to minimize the change of platelet activation with circadian variation. Platelet-rich plasma (PRP) was obtained by centrifuging the whole blood at 200 × g for 12 min. The platelet count of the PRP was measured and then adjusted to 20 × 107 mL−1 with platelet-poor plasma. Time to measurement was standardized to 1–1.5 h from blood collection. The aggregation response was measured simultaneously using two methods: the conventional method, which is based on changes in LT [9], and light scattering intensities with a PA-20 platelet aggregation analyzer (Kowa Co., Ltd., Tokyo, Japan) [10]. This device is particularly sensitive for detecting the size of small platelet aggregates and can subdivide platelet aggregates according to size into small, medium, or large aggregates [10,11]. Platelet aggregation was performed with collagen (Hormon-Chemie, Munich, Germany), ADP (MC Medical Co., Tokyo, Japan) or CRP (collagen-related peptide), a specific agonist for platelet glycoprotein (GP) VI [12], at 37°C under continuous stirring at 1000 rpm (0.82 dyn/cm2) for 5 min. CRP (GCP*[GPP]10 GCP*G, where P* represents hydroxyproline) was kindly provided by Toray Co. Ltd. (Tokyo, Japan), and was cross-linked as described previously [13].

Measurement of thromboxane metabolites

Although serum TxB2 concentration is widely used in the assessment of COX activity in aspirin-treated patients, we wanted to examine the correlation between Tx biosynthesis and platelet aggregation under the same condition, and to minimize the influence of TxB2 derived from other hematopoietic cells. Accordingly, we measured the TxB2 concentrations and the supernatant from collagen-stimulated PRP in patients. When serum TxB2 concentration and collagen-stimulated TxB2 were simultaneously measured, both measurement values correlated with each other [R = 0.97, P < 0.0001 (n = 20)].

The PRP (20 × 107 mL−1) was stimulated with 3 μg mL−1 collagen for 5 min and then centrifuged at 2000 × g for 15 min to remove the platelets. Supernatants were immediately stored at −30°C. Samples were shipped to the laboratory of BML, Inc. (Tokyo, Japan) in dry ice, and the concentration of TxB2 (stable metabolite of TxA2) was measured by radioimmunoassay [14]. Urinary 11-dehydroTxB2 was measured using enzyme-linked immunosorbent assay (ELISA) (Neogen Co., Lexington, KY, USA) according to the manufacturer's instructions. All investigators were kept unaware of the sample information. To reduce the possibility of systematic bias of the control and aspirin-treated subjects, the samples were assayed in random order.


All data analyses were performed with StatView 4.5 (SAS Institute Inc., Cary, NC, USA) for the Macintosh computer. Normally distributed variables were presented as mean ± SD and compared with Student's t-test or one-way analysis of variance (anova). Non-normally distributed variables were analyzed with the Mann–Whitney U-test. The correlation coefficient was obtained by simple regression analysis. Factor analysis consisting of (i) extraction of the initial components by use of principal-component analysis and (ii) interpretation of factors with loadings >0.1 (P < 0.05) was used to assess the relationship between several inter-correlated variables. Kaplan–Meier product limits were computed for the freedom from endpoint, and the Breslow–Gehan–Wilcoxon test was used for screening univariable group results regarding the outcomes. Multivariate Cox regression models were used to investigate the association of cardiovascular risk factors (age, HT, DM, HL, and platelet aggregation status) with the incidence of endpoints.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effects of aspirin treatment on platelet aggregation

We first examined the effect of aspirin treatment on platelet aggregation patterns with a newly-developed aggregometry that simultaneously measures both LT and light scattering. In patients treated with aspirin, the platelet aggregation assessed by LT was significantly decreased (Fig. 1). The aspirin treatment was more effective against collagen-induced aggregation than ADP-induced aggregation. Aspirin efficiently inhibited the formation of large aggregates; however, the number of small aggregates increased, suggesting that aspirin treatment prevented small aggregates from developing into large aggregates (Fig. 1). These data indicate that the screening of aspirin sensitivity using this type of aggregometry should be defined by the variation of the LT or the large aggregate formation induced by collagen.


Figure 1.  The effects of aspirin intake on platelet aggregation assessed by light transmission and light scattering methods. Platelets in platelet-rich plasma (PRP) obtained from the control (Control) or aspirin-treated patients (ASA) were stimulated with 2 μmol L−1 adenosine diphosphate (ADP) (A), 0.3 μg mL−1 (B), 1 μg mL−1 (C), or 3 μg mL−1 collagen (D) for 5 min. Changes in the maximum light transmission (LT) were monitored using conventional methods. Light scattering intensities were measured simultaneously to detect small, medium, and large aggregates. Data represent the mean ± SD (n = 38 for control and n = 50 for aspirin-treated patients).

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Involvement of platelet COX-1 activity in platelet aggregation and urinary 11-dehydroTxB2

In a preliminary study, we attempted to define ‘aspirin resistance’ as the failure of aspirin to inhibit platelet COX-1 activity by measuring arachidonic acid-induced platelet aggregation. However, none of the aspirin-treated patients (n = 20) responded to 10 μmol L−1 arachidonic acid (data not shown), suggesting that platelet COX-1 activity is sufficiently suppressed in all patients. Accordingly, we directly measured the production of TxB2 in collagen-activated platelets in this study to prove this hypothesis. As shown in Fig. 2A, collagen-stimulated TxB2 formation was almost completely inhibited in all aspirin-treated patients, compared with the control patients. These data suggest that oral low-dose aspirin therapy is sufficient to inhibit platelet COX-1 activity in the Japanese population. On the other hand, the results of platelet aggregation tests induced by collagen had great inter-individual differences (Fig. 2B).


Figure 2.  Platelet TxB2 production and aggregation induced by collagen. In (A), platelets in PRP obtained from control (Control) or aspirin-treated patients (Aspirin) were stimulated with 3 μg mL−1 of collagen for 5 min. The TxB2 concentration of the supernatant after stimulation was measured by radioimmunoassay. In (B), individual platelet aggregations induced by collagen in aspirin-treated patient are shown (n = 50).

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We next performed regression analysis to evaluate whether TxB2 production was associated with the differences of collagen-stimulated platelet aggregation (Table 1). Variables that were found to be associated with platelet TxB2 production were (i) small and medium aggregates (0.3 μg mL−1 collagen), (ii) medium and large aggregates (1 μg mL−1 collagen), and (iii) large aggregates and LT (3 μg mL−1 collagen) (Table 1). However, the TxB2 concentration was able to predict only 6–20% of the variation in collagen-stimulated platelet aggregations (Table 1). These data indicate the existence of other variable(s) that account for the inter-individual variation of collagen-induced platelet activation.

Table 1.   Correlation coefficients between platelet TxB2 production (independent variable) and different measurements of platelet aggregation among 50 aspirin-treated patients
Dependent variableRR2P
Collagen (0.3 μg mL−1)
 Small aggregates0.290.0840.041
 Medium aggregates0.320.100.023
 Large aggregates0.0310.000980.82
 Light transmission0.150.0230.29
Collagen (1 μg mL−1)
 Small aggregates0.180.0350.19
 Medium aggregates0.370.130.0080
 Large aggregates0.440.200.0011
 Light transmission0.220.0500.11
Collagen (3 μg mL−1)
 Small aggregates0.020.000440.88
 Medium aggregates0.210.0450.14
 Large aggregates0.320.100.025
 Light transmission0.250.060.076
Urinary 11-dehydro TxB2/creatinine0.100.010.57

Urinary 11-dehydroTxB2 reflects in vivo platelet activation and may be useful for monitoring platelet activity when testing for aspirin resistance [7,15]. Although urinary 11-dehydroTxB2 excretion in aspirin-treated patients was significantly lower than that in the control patients (12.0 ± 10.9 vs. 20.6 ± 14.8 ng mg−1 creatinine, P = 0.03), urinary 11-dehydroTxB2 excretion was not associated with platelet COX-1 activity (Table 1). As platelet COX-1 activity was inhibited in all patients enrolled, it is possible that the measurement of urinary 11-dehydroTxB2 instead reflected the Tx biosynthesis of other cells such as endothelial cells and macrophages in aspirin-treated patients.

Determination of a factor that influences collagen-induced platelet aggregation

Aspirin resistance was reportedly caused by an increased sensitivity of platelets to collagen [16]. However, not only the platelet aggregation induced by CRP, but also that induced by ADP, was significantly higher among the patients who had increased responses to collagen (data not shown), suggesting that differences in collagen-induced aggregation in aspirin-treated patients could not be solely explained by alterations in collagen-specific signaling pathways. To explore the confounding factor(s) in platelet aggregation, we next performed factor analysis, including the platelet aggregation induced by collagen, CRP, and ADP, in addition to TxB2. This model resulted in three separate factors: factor 1, aggregation of CRP, ADP, and collagen-induced platelet aggregation (other than factor 2); factor 2, small and medium aggregates induced by 3 μg mL−1 collagen; and factor 3, TxB2 (Table 2). Factors 1, 2, and 3 explained 48.4%, 17.7%, and 9.9% of the total variance, respectively. These data suggest that platelet aggregations elicited not only by collagen but also those by CRP and ADP were greatly influenced by a factor other than TxB2 in aspirin-treated patients.

Table 2.   Factor analysis using different measurements of platelet aggregation and TxB2 in 50 aspirin-treated patients
 Factor 1Factor 2Factor 3
  1. CRP, collagen-related peptide; ADP, adenosine diphosphate.

Collagen (1 μg mL−1)
 Small aggregates0.7840.2510.229
 Medium aggregates0.826−0.0930.352
 Large aggregates0.769−0.4120.035
 Light transmission0.776−0.0590.011
Collagen (3 μg mL−1)
 Small aggregates0.0760.861−0.090
 Medium aggregates0.5740.7580.027
 Large aggregates0.8220.2770.034
 Light transmission0.8560.190−0.149
CRP (0.03 μg mL−1)
 Light transmission0.711−0.463−0.196
CRP (0.1 μg mL−1)
 Light transmission0.764−0.169−0.215
ADP (2 μmol L−1)
 Light transmission0.6170.109−0.579
 Variance expected (%)48.417.79.9

Involvement of collagen-induced platelet aggregation in cardiovascular events

Finally, we prospectively enrolled 140 patients who took 81 mg of aspirin for the secondary prevention of cardiovascular or cerebrovascular disease to determine whether the variation of platelet aggregation during aspirin treatment influences clinical outcomes. Of the 140 patients enrolled, three patients that developed atrial fibrillation and one patient who did not take aspirin were excluded from the analysis. The mean follow-up duration was 721 days. The baseline characteristics of the patients were as follows: age, 75.4 ± 9.4 years; HT, 105 (77.2%); DM, 15 (11.0%); HL, 22 (16.2%); current cigarette smoking, seven (5.1%). Major events occurred in 21 (15.4%) of the 136 patients (non-fatal MI in two, stroke in 15, and cardiovascular death in four). The patient characteristics of the study according to platelet aggregation status [1 μg mL−1 collagen (LT)] are described in Table 3. The previous history of stroke and cardiovascular diseases is 63.2% and 36.8%, respectively. The use of statins and anti-hypertensive drugs was not different among the groups (Table 3).

Table 3.   Patients’ characteristics according to quintiles of platelet aggregation induced by 1 μg mL−1 collagen (light transmission) in a prospective study
 Total (n = 136)Q1 (n = 34)Q2 (n = 34)Q3 (n = 34)Q4 (n = 34)
  1. ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor II blocker; HT, hypertension.

Age75.4 ± 9.474.2 ± 11.274.4 ± 8.876.7 ± 8.076.3 ± 9.4
Gender (female) (%)73 (53.7)12 (35.2)18 (52.9)22 (64.7)21 (61.7)
Hypertension (%)105 (77.2)24 (70.6)27 (79.4)27 (79.4)27 (79.4)
Diabetes mellitus (%)15 (11.0)3 (8.82)4 (11.8)7 (20.6)1 (2.9)
Hyperlipidemia (%)22 (16.2)4 (11.8)6 (17.6)6 (17.6)6 (17.6)
Previous history (%)
 Cardiovascular disease50 (36.8)14 (41.2)10 (29.4)14 (41.2)12 (35.3)
 Stroke86 (63.2)20 (58.8)24 (70.6)20 (58.8)22 (64.7)
Drug use (%)
 Ca2+ channel blocker62 (45.6)10 (29.4)15 (44.1)18 (52.9)19 (55.9)
 ACEI/ARB47 (34.6)10 (29.4)11 (32.4)14 (41.2)12 (35.3)
 Π21 (15.4)4 (11.8)3 (8.8)8 (23.5)6 (17.6)
 Other anti-HT30 (22.1)13 (38.2)6 (17.6)3 (8.8)8 (23.5)
 Statin25 (18.4)5 (14.7)5 (14.7)6 (17.6)9 (26.4)

Figure 3 depicts the Kaplan–Meier time-to-event curves for event-free survival based on platelet aggregation induced by 1 μg mL−1 collagen. The composite outcome increased in the upper quartile of large platelet aggregation and light transmission, but not in small or medium aggregates (Fig. 3). ADP sensitivity did not appear to predict the outcomes (data not shown). Total events of the upper quintile of light transmission, but not other indicators, were significantly higher than the lowest quintile (P = 0.029) or other groups (P = 0.045). In addition, the upper quartile of the large aggregates and light transmission were more likely to experience major clinical events within 12 months (Fig. 3). To evaluate whether the increased baseline collagen-induced platelet aggregations were associated with early rather than late cardiovascular events, we performed separate analyses in patients who had an event within 12 months of study enrollment and those whose events occurred at >12 months after study entry. As expected with Kaplan–Meier analysis, the adjusted odds for the primary endpoint within 12 months were significantly higher in the highest quartile of the large aggregates and light transmission in multivariable analysis (Table 4). On the other hand, the existence of DM seemed to be an independent risk factor for overall outcomes (HR 1.30–11.9, P = 0.015–0.033).


Figure 3.  Kaplan–Meier plot relating quartiles of small (A), medium (B), large aggregates (C), and light transmission (D) (1 μg mL−1 of collagen) to the composite risk of myocardial infarction, stroke, and cardiovascular death (Q1 = lowest quartile < Q2 < Q3 < Q4 = highest quartile).

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Table 4.   Multivariate analyses of time-to-event among patients according to aspirin sensitivity assessed by 1 μg mL−1 collagen (n = 136)
 Early events (<12 months)Total events
HR (95% CI)PHR (95% CI)P
  1. CI, confidence internal; HR, hazard ratio.

Small aggregates
 Upper quartile0.27 (0.032–2.21)0.220.34 (0.10–1.21)0.10
Medium aggregates
 Upper quartile1.98 (0.51–7.62)0.320.73 (0.26–2.01)0.54
Large aggregates
 Upper quartile7.98 (1.78–35.7)0.00661.93 (0.72–5.01)0.18
Light transmission
 Upper quartile7.76 (1.72–35.3)0.00772.32 (0.89–6.01)0.084


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aspirin is the most prescribed antiplatelet drug and its effectiveness in preventing cardiovascular events is well established [1–3]. As low-dose aspirin has been believed to be potent enough to inhibit platelet COX-1 activity, the HOPE study, showing that the inability of aspirin to inhibit Tx biosynthesis resulted in poor clinical outcomes [7], makes us re-consider whether aspirin efficiently inhibits platelet COX activity in all patients. Several mechanisms can be proposed to account for the incomplete suppression of platelet COX-1 by aspirin [2,3]: TxA2 production by the induction of aspirin-insensitive COX in platelets [17,18] and COX-1 polymorphism [19]. Aspirin is approximately 50- to 100-fold more potent in inhibiting COX-1 than COX-2 [2], it is ideally suited to act on a nucleate platelet, inducing a permanent defect in Tx-dependent platelet function. Thus the transient expression of COX-2 in newly formed platelets in a clinical setting of enhanced platelet turn-over [17,20], is a potentially important mechanism that deserves further investigation. Indeed, the incomplete inhibition of COX has been reported in specific clinical settings, such as severe unstable angina [21], coronary artery bypass surgery [18], and endarteriectomy [22]. On the other hand, low-dose aspirin treatment invariably inhibited platelet COX-1 activity in our study, suggesting that ‘aspirin resistance’, which is unsuppressed platelet COX-1 activity, is likely to be rare, at least in an out-patient population which has normal platelet turn-over.

Recent studies assessed the response to aspirin by measuring the urinary level of 11-dehydroTxB2 [3,7,21] and cardiovascular death associated with aspirin resistance as documented by the urinary TxB2 concentration [7]. A major portion of this metabolite is believed to come from platelets, but there are also additional cellular sources including monocytes and endothelial cells [2,21,23]. Our results suggest that urinary 11-dehydroTxB2 variations reflect systemic COX activity other than that from platelets in aspirin-treated patients. Recently, the polymorphism of the COX-2 gene was reportedly associated with a decreased risk of a MI and stroke [24]. It is necessary to clarify whether patients with high 11-dehydroTxB2 levels, despite the inhibition of platelet COX-1, should be treated with a higher dose of aspirin, and whether the COX-2 polymorphism is involved in differences in systemic Tx production.

Although aspirin resistance was also defined by in vitro platelet function in previous studies, there appears to be wide-spread misunderstanding that in vitro platelet function directly represents the COX-1 activity with the results of platelet aggregation tests. In reality, the inter-individual differences of platelet aggregation under aspirin-treatment were caused by a factor(s) other than COX-1. There are several explanations for the factor-influenced platelet activation. First, the threshold of platelet aggregation might decrease in some patients. It is supposed that the decreased basal levels of (or decreased sensitivity to) c-AMP or c-GMP [25,26] and the increases in the level of myosin light chain phosphorylation in platelets are possible factors causing hyperaggregability of platelets in high-risk patients such as diabetics [27]. Furthermore, ADP signaling may be involved in the inter-individual differences [28]. A strategy targeting the ADP signaling pathway is attractive because recent trials have demonstrated the superior clinical benefit of the combination of clopidogrel with aspirin, compared with aspirin alone [29,30]. The elucidation of the factor(s) leading to the inter-individual differences would lead to a new therapeutic approach, and the specific antagonists of the factor may supplement the therapeutic effect of aspirin in some aspirin-treated patients.

We found that the upper quartile of LT or large aggregate formation induced by collagen was an independent risk factor for developing cardiovascular events within 12 months in multivariable analyses, suggesting that the necessity of inhibiting platelet function to an optimized level, even if aspirin effectively abolishes platelet COX-1 activity. Unfortunately, the presented data appears to show no advantage of measuring small and medium aggregates in aspirin-treated patients. Taking into consideration that aspirin inhibits the transition from small aggregates to large aggregates, it is likely that the conventional aggregometry which preferentially detects large aggregates has proved useful in monitoring the effects of aspirin on platelet aggregation. An association between suboptimal platelet function inhibition during aspirin treatment and the heightened incidence of cardiovascular events has been also described [8]. However, this study used inadequate techniques to measure the response to aspirin: ADP at 10 μm induces full platelet aggregation, and a high concentration of arachidonic acid induces platelet lysis [3] and activates platelets through unknown signaling pathways [31]. As collagen-induced platelet aggregation is more sensitive to aspirin therapy than the aggregation elicited by ADP or other soluble agonists and reflects clinical outcomes in our study, we recommend collagen as a standard platelet agonist for assessment of aspirin treatment. Considering the small number of individuals studied, our current findings should be interpreted with some caution, and larger-scale studies are awaited to draw more conclusive answers to this issue. Furthermore, the study to evaluate additional treatment and elucidate the precise biological effects of aspirin should be performed.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to thank Drs K. Sekido, A. Hagihara, S. Kamei, and A. Maruyama (Minobusan Hospital, Yamanashi, Japan) for obtaining informed consent and following patient outcomes. This study was supported in part by a Health and Labor Sciences Research Grant from the Ministry of Health, Labor and Welfare and Grants-in-aid for Scientific Research from the Ministry of Education and Science.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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