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European Heart Journal Advance Access originally published online on April 3, 2009
European Heart Journal 2009 30(10):1279-1286; doi:10.1093/eurheartj/ehp097
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org

COX-1 sensitivity and thromboxane A2 production in type 1 and type 2 diabetic patients under chronic aspirin treatment

Fabio M. Pulcinelli1,*,{dagger}, Luigi M. Biasucci2,{dagger}, Silvia Riondino1, Simona Giubilato2, Andrea Leo2, Livia Di Renzo1, Elisabetta Trifirò1, Teresa Mattiello1, Dario Pitocco3, Giovanna Liuzzo2, Giovanni Ghirlanda3 and Filippo Crea2

1 Department of Experimental Medicine, ‘Sapienza’ University of Rome, Viale Regina Elena 324, 00161 Rome, Italy
2 Institute of Cardiology, Catholic University, Rome, Italy
3 Department of Internal Medicine, Catholic University, Rome, Italy

Received 11 September 2008; revised 18 February 2009; accepted 26 February 2009; online publish-ahead-of-print 3 April 2009.

* Corresponding author. Tel: +39 (0) 6 49973002, Fax: +39 (0) 6 4454820, Email: fabio.pulcinelli{at}uniroma1.it


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Limitations of the study
 Conclusion
 Funding
 References
 
Aims: Although aspirin treatment is useful in reducing ischaemic events in diabetic patients, recent studies suggest that it is less effective when compared with non-diabetics (ND). We sought to evaluate COX-1 sensitivity and thromboxane A2 (TxA2) production in type 1 (T1DM) and type 2 diabetic (T2DM) patients under chronic aspirin treatment, and also evaluate the association between thromboxane A2 (TxA2) production and markers of inflammation and metabolic control, such as high-sensitivity C-reactive protein, fasting blood glucose, and haemoglobin A1c (HbA1c).

Methods and results: Agonist-induced platelet aggregation (PA) and TxB2, a stable metabolite of TxA2, production, serum TxB2, and platelet COX-1 and COX-2 expression were studied in T2DM patients, T1DM patients, and high-risk ND subjects, all receiving a low dose of aspirin. TxB2 formation was studied in platelets treated in vitro with aspirin alone or with a COX-2 inhibitor (NS-398). PA, collagen-induced TxB2 production, and serum TxB2 were higher in T1DM and T2DM patients than in ND subjects. TxB2 production was reduced in diabetic patients by in vitro treatment with aspirin. COX-2 was expressed in all diabetic patients but only in 46% of ND patients. In diabetic patients significant correlations were observed between TxB2 production and both fasting plasma glucose and HbA1c.

Conclusion: COX-1 sensitivity and TxB2 production is similarly reduced in both T1DM and T2DM patients under chronic aspirin treatment. The association between TxB2 production and either fasting plasma glucose and HbA1c levels suggests that in diabetic patients hyperglycaemia is a determinant of the reduced platelet sensitivity to aspirin.

Key Words: Diabetes • Aspirin • Cyclooxygenase • Platelets • Thromboxane


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Limitations of the study
 Conclusion
 Funding
 References
 
The benefit of aspirin in diabetic patients has been consistently documented in several trials.1,2 Yet, in the meta-analysis of the Antithrombotic Trialists' Collaboration, the event rate of diabetic patients on treatment was similar to that of non-diabetic (ND) patients off treatment.3 In the Primary Prevention Project Trial4 aspirin treatment reduced cardiovascular events and deaths in high-risk ND patients, but not in patients with type 2 diabetes mellitus (T2DM). Furthermore, in the recent Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes Study,5 a low dose of aspirin in primary prevention did not reduce the risk of cardiovascular events at 4 years in diabetic patients. Subset analyses of other studies in secondary prevention similarly suggested that aspirin might be less effective in T2DM,6,7 especially in patients with poor metabolic control, than in ND patients, the underlying mechanism being still largely debated.8 It has been proposed that reduced sensitivity to aspirin in diabetic patients might be owing to accelerated thrombopoiesis9 or to reduced platelet permeability to aspirin caused by membrane glycosylation.10 Because of the different pathogenetic mechanisms, patients with type 1 diabetes (T1DM) were included to test the effects of hyperglycaemia in the presence of different degrees of activation of inflammatory cells, as shown in previous studies.11,12

In order to study the platelet sensitivity to aspirin in diabetic patients, we designed a prospective case–control study to compare platelet aggregation (PA) and thromboxane B2 (TxB2) production, a stable metabolite of thromboxane A2 (TxA2), in patients with T2DM, T1DM, and in high-risk ND subjects under chronic aspirin treatment. Correlations between fasting blood glucose, haemoglobin A1c (HbA1c), high-sensitivity C-reactive protein, and TxB2 production were performed in order to assess the relative role of hyperglycaemia and inflammation with COX1 sensitivity and TxB2 production. We also performed, in a subgroup of our population, further in vitro analyses to assess the molecular mechanisms responsible for the reduced sensitivity of diabetic patients to aspirin.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Limitations of the study
 Conclusion
 Funding
 References
 
From May 2005 to October 2007 we enrolled in our hospital 150 diabetic patients, 120 with T2DM and 30 with T1DM, under chronic treatment with low dose of aspirin (100–160 mg) for primary or secondary prevention. Overall, 20 T2DM patients and 4 T1DM patients were excluded from the study, owing to use of other drugs, apart from aspirin, known to interfere with platelet function (non-steroidal anti-inflammatory drugs, ticlopidine, clopidogrel, cilostazol) in the previous 10 days (T2DM, n = 10; T1DM, n = 3), use of oral anticoagulant or heparin (previous two days; T2DM, n = 3), chronic or acute inflammatory diseases (T2DM, n = 4), or plasma salicylate concentrations <0.02 µg/mL, as a marker of lack of compliance to aspirin treatment (T2DM, n = 3; T1DM, n = 1). Thus, a total of 100 consecutive T2DM patients and 26 T1DM patients were included in the study. They were compared with a CHARISMA-like population of 100 high-risk ND subjects without history of diabetes13 on aspirin for primary or secondary prevention, followed-up in our outpatient clinic in order to monitor antiplatelet treatment, as recommended by their family doctor. In all of the control population, a screening for diabetes was performed measuring fasting blood glucose. All controls had a fasting blood glucose <100 mg/dL. All subjects in the study were recommended to take the aspirin after lunch, between 12.00 and 02.00 p.m., i.e. 18–20 h before blood sampling, a time interval that cannot make any difference in terms of platelet response to aspirin, and they were on aspirin treatment for at least 1 month.

Baseline patient characteristics are reported in Table 1.


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  		Table 1 Baseline characteristics of patients

 
T1DM and T2DM were defined according to the criteria of American Diabetes Association (ADA), and diabetic patients without a history of cardiovascular disease were on aspirin in agreement with the position statement of the ADA.14

Patients were considered: hypertensive if blood pressure was >140/90 in three different measurements when patients were in a supine position for at least 10 min, or if they were being treated with antihypertensive drugs; hypercholesterolaemic if serum cholesterol was >220 mg/dL or if they were being treated with a lipid-lowering agent. Patient compliance to antiplatelet treatment was assessed by plasma salicylate level, evaluated by high-performance liquid chromatography (HPLC).15 As little data are available on this topic, we have based the sample size estimation on the results of a previous study by Watala that has shown a difference of about 30% in the maximal inhibition of PA between patients with and without diabetes on aspirin treatment.8 No data were found on TxB2. Therefore, we used the data by Watala and with this assumption we have calculated a population of at least 25 patients to be sufficient for our aim. The study was approved by the Ethics Committees of the Catholic and of ‘La Sapienza’ University, and all patients gave their consent to use part of their blood sample for scientific purpose. All patients underwent the laboratory assays planned for the study.

Blood sampling
Three blood samples were obtained by clean venipuncture after overnight fasting. The first sample was drawn in test tubes containing one-tenth volume of Na-citrate 3.8% and used to prepare platelet-rich plasma (PRP); the second and the third were drawn in test tubes without anticoagulant and used for serum measurements (see below).

Sample preparation
PRP was prepared by centrifugation at 200 g for 15 min at room temperature. In order to minimize the presence of white blood cells (WBC), PRP was further centrifuged at 180 g for 5 min, and the platelet count was adjusted to 2.5 x 108/mL with Tyrode's buffer at pH 7.35. Patients with WBC count above 0.1 x 103 cells/µL were excluded from the study. Cell count was analysed on a Hemalaser II SEBIA routine haematology blood counter (Sebia, Italy).

Platelet-poor plasma was obtained after further centrifugation of PRP at 2000 g for 10 min and used for the measurement of plasma salicylate level.

Peripheral blood mononuclear cells (PBMC) were prepared on a Ficoll hypaque density gradient.16

Serum was prepared by centrifugation of whole blood samples at 2000 g for 15 min; for evaluating the production of TxB2 it was kept for 1 h at 37°C prior to centrifugation.

Glycaemic control
Both glycated haemoglobin (HbA1c), as a long-term monitor of average glycaemia, and daily control of fasting plasma glucose levels were evaluated. For each patient, HbA1c mean levels were obtained using HPLC and analysis performed using Diamat BioRad (BioRad, Milan, Italy). The HbA1c reference range for healthy subjects was 4.3–5.9%. Fasting plasma glucose levels were analysed by the glucose oxidase method.

C-reactive protein measurement
Serum high-sensitivity C-reactive protein levels were measured using a high-sensitivity nephelometric system (Latex BNII, Dade-Behring, Glasgow, Delaware, NJ, USA) with a detection limit of 0.1 mg/L.

Plasma salicylate levels
In all patients quantitative analysis of plasma concentrations of salicylate was performed using HPLC according to Cerletti et al.17 The threshold salicylate level for patient enrolment was fixed at 0.02 µg/mL.

Platelet aggregation
PA (Born's Method) was evaluated on PRP in an AggRAM (Helena Biosciences, Sunderland, UK) aggregometer as previously described.18 The results are reported as the maximal percentage of aggregation (Mx%) observed after 4 min stimulation in response to collagen (4 µg/mL), arachidonic acid (AA; 1 mM), and adenosine diphosphate (ADP; 2 µM) (all from Helena Laboratories).19 Concurrent controls were performed to ensure that all agonists retained the same level of activity during the whole study.

Thromboxane B2 production
TxB2 production was evaluated by blocking collagen-induced platelet activation after 4 min with indomethacin (10 µM); samples were then spun at 8000 g for 1 min. TxB2 was measured in the sample supernatants as well as in serum samples using an EIA commercial kit (Cayman Chemical Company, Ann Arbor, MI, USA). Collagen was selected as platelet agonist as it has been used to identify residual platelet activation, both TxA2-dependent and -independent.20,21 In vitro substudies were performed in 50 T2DM and all (n = 26) T1DM patients. Platelets of these patients already on aspirin treatment were further pre-incubated in vitro (10 min at 37°C prior to agonist addition) with aspirin (Sigma Chemicals Co., St Louis, MO, USA) at a concentration (100 µM) required to completely abolish AA-induced TxB2 production22,23 with or without 10 µM of the COX-2 inhibitor NS-398 (Sigma Chemicals), as COX-2-dependent TxB2 production can be studied only in cells pre-treated with aspirin.24 Control platelets were treated with the inhibitor solvent (dimethyl sulfoxide).

COX-1 and COX-2 assessment
Cells (1.5 x 107 platelets for COX-1 studies and 7.5 x 107 platelets for COX-2 studies; 0.5–1.5 x 105 PBMC) from 50 T2DM, all (n = 26) T1DM patients, and 50 controls were lysed using RIPA buffer and resolved using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Following electrophoresis proteins were transferred to Immobilion-P (Millipore, Bedford, MA, USA) membranes and identified with anti-COX-1 or anti-COX-2 monoclonal antibodies (Cayman Chemical), followed by horseradish peroxidase-conjugated secondary antibody and detected with ECL chemiluminescence reaction reagent (both from Amersham Pharmacia, Biotech, Little Chalfont, UK) and Kodak X-ray film (X-OMAT AR). For COX-2 detection, lysates of PBMC cells (7.5 x 104) activated with lipopolysaccharide were used as positive controls.25

Statistical analysis
As the distributions of TxB2 and high-sensitivity C-reactive protein values were non-normal according to Kolmogorov–Smirnov, the differences between groups were analysed using non-parametric tests—Kruskal–Wallis or Wilcoxon signed rank—as appropriate. The remaining continuous variables were compared using analysis of variance. Corrections for multiple comparisons were performed using Dunnett's or Dunn's test as appropriate. Categorical data were compared using {chi}2 test. Non-parametric Spearman's correlation coefficient was used to assess the relationship between collagen-induced TxB2 formation and serum TxB2 levels. Tests for linear trends were computed using an ordinal variable for biomarker quartiles.

Significance was accepted at the P < 0.05 level. Data are reported as mean ± SD and, when appropriate, as medians and ranges. All tests were two-tailed and the data were analysed using Stata version 6.0 (College Station, TX, USA; Stata Corporation, 1999).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Limitations of the study
 Conclusion
 Funding
 References
 
Baseline measurements
Both T2DM and T1DM patients displayed a significantly greater Mx% PA, compared with ND subjects, following activation by ADP (P = 0.01 for both comparisons), by collagen (P = 0.02 for both comparisons), or by AA (P = 0.01 for both comparisons). Of note, all patients showed AA-induced PA <20%, except one who had an AA-induced PA of 24%. In contrast, no difference was found between T1DM and T2DM patients (Figure 1).


Figure 1
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  		Figure 1 Platelet aggregation (PA) is higher in both type 1 (T1DM) and type 2 (T2DM) patients than in non-diabetic (ND) subjects. PA in ND (closed columns), T1DM (open columns), and T2DM (dotted columns) patients under chronic treatment with aspirin. PA was analysed as maximal aggregation (Mx%) in response to adenosine diphosphate (ADP; 2 µM), collagen (4 µg/mL), and arachidonic acid (AA; 1 mM).

 
Serum TxB2 levels were significantly higher in T2DM patients than in ND subjects (P = 0.001). Although serum TxB2 levels were markedly increased in T1DM, this difference was not significant (P = 0.32), and no significant difference was found between T1DM and T2DM patients (P = 0.29; Figure 2A). Similarly, collagen-induced TxB2 production was significantly higher in T2DM patients than in ND subjects (P = 0.001). Although collagen-induced TxB2 in T1DM were markedly increased compared with ND, this difference was not significant (P = 0.7), and no difference was found between T1DM and T2DM patients (P = 0.6; Figure 2B).


Figure 2
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  		Figure 2 Platelet and serum thromboxane B2 (TxB2) production in type 1 (T1DM), type 2 (T2DM) diabetic patients, and in non-diabetic (ND) subjects. Serum TxB2 concentration (expressed as picograms/millilitre) (A) and collagen-induced (4 µg/mL) TxB2 production (expressed as pg/108 cells) (B) in ND (dark grey columns), T1DM (white columns), and T2DM (light grey columns) patients under chronic treatment with aspirin.

 
In each group a statistically significant correlation was found between serum TxB2 levels and both collagen-induced PA (R = 0.796, P = 0.03 in T1DM, R = 0.654, P = 0.04 in T2DM and R = 0.776, P = 0.03 in ND) and collagen-induced TxB2 formation (R = 0.878, P = 0.005 in T1DM, R = 0.579, P = 0.04 in T2DM and R = 0.809, P = 0.04 in ND). No differences were found between patients under aspirin treatment for primary or secondary prevention in terms of PA (Mx%: 57.9 ± 12.4 vs. 56.5 ± 14.1 for collagen, P = 0.28; 16 ± 4 vs. 14 ± 1 for AA, P = 0.89; 41.7 ± 10.2 vs. 39.3 ± 13.3 for ADP, P = 0.59) and in terms of collagen-induced TxB2 production (113 ± 407 pg/108 cells in primary prevention vs. 705 ± 255 pg/108 cells in secondary prevention, P = 0.3).

We did not find any treatment-dependent differences in our patients, particularly between insulin and oral hypoglycaemic agents, in terms of PA (data not shown). In diabetic patients with fasting plasma glucose <126 mg/dL serum TxB2 production was 625 ± 182 pg/mL, while in those with fasting plasma glucose ≥126 mg/dL, serum TxB2 production was 1793 ± 203 pg/mL (P = 0.04). Weak but significant correlations were observed between TxB2 production and either plasma glucose (R = 0.25, P = 0.014) or HbAlc levels (R = 0.22, P = 0.026). Furthermore, by dividing patients into quartiles defined by the distribution of fasting plasma glucose (Figure 3A) or of HbA1c levels (Figure 3B), we observed significantly higher TxB2 production from bottom to top quartile (P for trend = 0.009 and 0.012, respectively). Serum high-sensitivity C-reactive protein levels were significantly higher in T2DM patients than in T1DM patients and in ND subjects (2.40 mg/L, range 0.15–18.8 mg/L vs. 0.15 mg/L, range 0.15–10.7 mg/L and 1.53 mg/L, range 0.15–12.4 mg/L, respectively, P = 0.01 for both). No correlation was found between serum high-sensitivity C-reactive protein levels and serum TxB2 production (R = – 0.3, P = 0.1). Furthermore, we did not find any correlation between serum TxB2 production and plasma salicylate levels (R = 0.14, P = 0.7).


Figure 3
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  		Figure 3 Association between serum thromboxane B2 (TxB2) production and glycaemic control. Comparison between quartiles of fasting glucose plasma levels (expressed in mg/dL) (A) or haemoglobin A1c (HbA1C, expressed as %) (B), and TxB2 production. P for trend = 0.009 and 0.012, respectively.

 
In vitro studies
In platelets from T1DM patients, in vitro incubation with 100 µM aspirin significantly reduced collagen-induced TxB2 production, compared with untreated platelets (44.6 ± 12.7 pg/108 cells vs. 845.4 ± 381.9 pg/108 cells, P = 0.003). The incubation with 100 µM aspirin and 10 µM NS-398 caused a further reduction of TxB2 production that was not significant, however, vs. aspirin alone [34.3 ± 10.1 pg/108 cells vs. 845.4 ± 381.9 pg/108 cells, and 34.3 ± 10.1 pg/108 cells vs. 44.6 ± 12.7 pg/108 cells; P = 0.0012 vs. non-treated and P = 0.71 vs. acetylsalicylic acid (ASA) alone, respectively]. Similarly, in platelets obtained from 50 T2DM patients, in vitro incubation with 100 µM aspirin significantly reduced collagen-induced TxB2 production, compared with untreated platelets (61.4 ± 11.5 pg/108 cells vs. 896.4 ± 232.3 pg/108 cells, P = 0.0014). The incubation with both 100 µM aspirin and 10 µM NS-398 caused a further reduction of TxB2 production that was not significant when compared with aspirin alone (48.1 ± 9.2 pg/108 cells vs. 896.4 ± 232.3 pg/108 cells, and 48.1 ± 9.2 pg/108 cells vs. 61.4 ± 11.5 pg/108 cells; P = 0.001 vs. non-treated and P = 0.26 vs. ASA, respectively; Figure 4).


Figure 4
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  		Figure 4 In vitro addition of aspirin and NS-398 reduces collagen-induced thromboxane B2 (TxB2) formation in diabetic patients. Effect of in vitro addition (10 min at 37°C) of 100 µM aspirin alone or in combination with NS-398 (10 µM) on collagen-induced (4 µg/mL) TxB2 production (expressed as pg/108 cells) in type 1 (T1DM) and type 2 (T2DM) diabetic patients.

 
COX-1 and COX-2 expression
COX-1 was highly expressed in all groups. Conversely, COX-2 was expressed in all diabetic patients analysed (n = 26 T1DM; n = 50 T2DM), but in only 23 out of 50 ND subjects (P = 0.03). However, it is worth noting that in all patients the amount of COX-2 was markedly lower than that of COX-1, in spite of the smaller amount of platelet proteins loaded for COX-1 assessment (1.5 x 107 platelets) with respect to those used for COX-2 detection (7.5 x 107 platelets). Figure 5 reports a representative western blot with samples from three T2DM and two ND subjects. The possibility of WBC-dependent COX-2 contamination in the platelet suspension was evaluated by analysing COX-2 expression in lysates of PBMC from the same patients, loaded up to a concentration five times higher (1.5 x 105 PBMC) than the upper cut-off of total WBC for patient enrolment (see Methods). COX-2 was undetectable in such PBMC lysates (data not shown), demonstrating that the COX-2 detected in platelet lysates from our patient populations was of intra-platelet origin only.


Figure 5
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  		Figure 5 Platelet COX-1 and COX-2 expression in diabetic patients and non-diabetic (ND) subjects. Immunodetection of COX-1 and COX-2 in lysated platelets from ND and T2DM (D), resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The amount of platelets loaded in the two settings was 7.5 x 107 platelets for COX-2 immunodetection and 1.5 x 107 platelets for COX-1 immunodetection. Lysates of PBMC (7.5 x 104) activated with lipopolysaccharide were used as positive controls (P) for COX-2 band identification.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Limitations of the study
 Conclusion
 Funding
 References
 
Our study demonstrates a reduced platelet sensitivity to the inhibitory action of aspirin assessed by higher PA and TxB2 production in both T1DM and T2DM patients, when compared with high-risk ND subjects. Thus, they provide an explanation for the lower sensitivity to the beneficial effects of aspirin in diabetic patients observed in clinical trials1–5 and might account for the higher platelet reactivity associated with enhanced risk for adverse cardiovascular events demonstrated in T2DM.26

Of note, the higher TxB2 production in our patients cannot be ascribed to lack of compliance to the antiplatelet treatment, as all our patients had detectable salicylate plasma levels.

In this study among diabetic patients, TxB2 production following platelet stimulation with collagen, ranged from 18 pg/108 cells to 7344 pg/108 cells, and correlated with serum levels of TxB2. More importantly, TxB2 production was much greater in diabetic patients than in ND. Incomplete inhibition of platelet COX-1 by oral aspirin in diabetic patients is supported, in our study, by the evidence that further in vitro addition of aspirin markedly reduced TxB2 production. This finding is in accordance with previous observations of our group demonstrating that among high-risk ND patients under chronic aspirin treatment, in vitro addition of aspirin reduced TxB2 formation in those with baseline TxB2 levels above the median value, while no additional inhibitory effect was obtained in those with baseline TxB2 levels below the median.22

Furthermore, in the present study all diabetic patients expressed platelet COX-2, although in aspirin-treated diabetic patients only a small proportion of TxB2 produced following platelet stimulation was COX-2-dependent. Indeed, in vitro platelet incubation with aspirin plus the COX-2 inhibitor NS-398 resulted in a further, albeit not significant, reduction in TxB2 production, when compared with aspirin alone. We can exclude that these findings are because of platelet contamination by PBMC, as no COX-2 was detectable in lysates of PBMC from the same patients.

Regardless of the mechanism, higher platelet TxB2 production in diabetic patients on aspirin may be clinically relevant, as TxB2 production must be reduced by at least 95% in order to achieve platelet inhibition.27

Taken together, our findings confirm and expand those of previous studies showing enhanced excretion of urinary 11-dehydro-TxB2 in diabetic patients, even when on aspirin.28,29

The reduced sensitivity to aspirin in T2DM might be related to hyperglycaemia or to associated inflammation.8 The demonstration in our study of a correlation between in vitro TxB2 production and systemic levels of fasting glucose or HbA1c suggests that hyperglycaemia per se might play an important role, beyond that played by inflammation. This notion is confirmed in our study by the demonstration that sensitivity to aspirin was equally reduced in T1DM and T2DM patients, while high-sensitivity C-reactive protein levels were higher in T2DM.

The molecular mechanisms by which hyperglycaemia might reduce the sensitivity to the antiplatelet action of aspirin are probably multiple and cannot be deduced from the results of our study.

The reduced inhibitory action of aspirin on platelet COX-1, observed in our study, might be ascribed either to glycation-induced conformational changes of platelet membranes with resulting impaired aspirin entrance10 and/or to a less-efficient acetylation previously demonstrated in platelets resuspended in high glucose medium.30

The presence of platelet COX-2 observed in all diabetic patients might contribute to the reduced sensitivity of platelets to aspirin. The persistence of COX-2 expression might be consequent either to glucose-induced megakariocyte COX-2 expression, similar to that observed in monocytes,31 and/or to the presence of a larger number of newborn platelets found in diabetic patients,9 since newborn platelets express COX-2.32


   Limitations of the study
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Limitations of the study
Conclusion
 Funding
 References
 
Our study has some limitations. First, the sample size of T1DM is small, therefore our population is large enough to detect differences between ND and T1DM in aggregometric tests, but might be too small to detect lack of statistical difference in TxB2 production between T1DM patients and ND. Secondly, in this study we did not evaluate clinical endpoints. Further larger studies are needed to determine the clinical implication of a reduced platelet sensitivity to aspirin in the diabetic population.


   Conclusion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Limitations of the study
 Conclusion
Funding
 References
 
This study shows for the first time that the reduced sensitivity of diabetic patients to the beneficial effects of aspirin on cardiovascular risk might be caused by both less-efficient inhibition of platelet COX-1 and enhanced COX-2 expression. Furthermore, the similar findings in T1DM and T2DM, as well as the association between TxA2 production and systemic levels of fasting glucose and HbA1c suggests that hyperglycaemia per se is likely to play an important role in determining the sensitivity of diabetic patients to the beneficial effects of aspirin.

It also remains to establish whether an optimal glycaemic control might restore the sensitivity of diabetic patients to the beneficial effects of aspirin and whether other drugs, able to specifically inhibit TxA2 receptors, might be more effective in diabetic patients alone or in addition to aspirin. All of these hypotheses need to be tested in further larger prospective studies.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Limitations of the study
 Conclusion
 Funding
References
 
This work was supported partially by a grant PRIN of the Italian Ministry of Education, University and Scientific Research (MIUR), partially by a grant ex 60% and partially by a grant ‘Monzino’ conv.101/04, all to F.M.P.

Conflict of interest: none declared.


   Footnotes
 
{dagger} The first two authors contributed equally to the study. Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Limitations of the study
 Conclusion
 Funding
 References
 

  1. Steering Committee of the Physicians' Health Study Research Group. Final report on the aspirin component of the ongoing Physicians' Health Study. N Engl J Med (1989) 321:129–135.[Abstract]
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  6. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, Ramírez C, Sabaté M, Jimenez-Quevedo P, Hernández R, Moreno R, Escaned J, Alfonso F, Bañuelos C, Costa MA, Bass TA, Macaya C. Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes (2005) 54:2430–2435.[Abstract/Free Full Text]
  7. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, Ramírez C, Sabaté M, Jimenez-Quevedo P, Hernández R, Moreno R, Escaned J, Alfonso F, Bañuelos C, Costa MA, Bass TA, Macaya C. Influence of aspirin resistance on platelet function profiles in patients on long-term aspirin and clopidogrel after percutaneous coronary intervention. Am J Cardiol (2006) 97:38–43.[CrossRef][Web of Science][Medline]
  8. Watala C, Golanski J, Pluta J, Boncler M, Rozalski M, Luzak B, Kropiwnicka A, Drzewoski J. Reduced sensitivity of platelets from type 2 diabetic patients to acetylsalicylic acid (aspirin)—its relation to metabolic control. Thromb Res (2004) 113:101–113.[CrossRef][Web of Science][Medline]
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