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Clopidogrel reduces platelet–leucocyte aggregation, monocyte activation and RANTES secretion in type 2 diabetes mellitus
  1. S A Harding1,
  2. J Sarma2,
  3. J N Din2,
  4. P M Maciocia2,
  5. D E Newby2,
  6. K A A Fox2
  1. 1Department of Cardiology, Wellington Hospital, Wellington, New Zealand
  2. 2Centre for Cardiovascular Sciences, University of Edinburgh, Edinburgh, UK
  1. Correspondence to:
    Dr Scott Harding
    Department of Cardiology, Wellington Hospital, Private Bag 7902, Wellington, New Zealand; scott.harding{at}

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Patients with diabetes mellitus have an increased risk of developing atherosclerosis and its sequelae. Atherosclerosis is an inflammatory disease involving multiple interactions between platelets, leucocytes and endothelial cells.1 Clopidogrel, a specific antagonist of the ADP P2Y12 receptor, inhibits both platelet activation and aggregation induced by ADP.2 Although clopidogrel has well-documented antithrombotic actions, its potential anti-inflammatory effects have been little investigated. We examined whether specific platelet inhibition with clopidogrel would reduce systemic inflammatory markers and specifically platelet, monocyte and endothelial activation in patients with type 2 diabetes mellitus.


We enrolled 20 patients with type 2 diabetes mellitus without clinical evidence of cardiovascular disease, malignancy, chronic inflammatory disorders, intercurrent illness, renal or hepatic insufficiency or contraindications to clopidogrel who had not taken antiplatelet agents within the preceding two weeks. Ethical approval was obtained from the local ethics committee and all participants provided written informed consent. They were treated with clopidogrel 75 mg daily for 28 days. Fasting peripheral venous blood samples were obtained at baseline and at 28 days.

Plasma concentrations of soluble CD40 ligand (sCD40L) (Bender MedSystems) and sE-selectin (R&D Systems) were determined by enzyme-linked immunosorbent assays. Plasma concentrations of the chemokine regulated on activation normal T cell expressed presumed secreted (RANTES, CCL5) were assessed with the human chemokine flow cytometric bead array (BD Biosciences).

To evaluate CD40L and P-selectin on platelets, whole blood was diluted 1:10 with phosphate-buffered saline (PBS) and incubated with anti-CD42a:fluorescein isothiocyanate (FITC) (Serotec), anti-P-selectin:phycoerythrin (PE) (DakoCytomation), anti-CD40L:PE (DakoCytomation) and appropriate isotype controls for 20 min before the cells were fixed with 1% paraformaldehyde. To evaluate CD40 and CD11b on monocytes, blood was diluted 1:2 with PBS and incubated with the following monoclonal antibodies: anti-CD14:FITC (Serotec), anti-CD40:PE (Serotec), anti-CD11b:PE (Serotec) and appropriate isotype-matched controls for 20 min. Thereafter, samples were fixed and the red cells lysed by the addition of FACS-Lyse (Becton Dickinson). To determine platelet–monocyte and platelet–neutrophil binding, whole blood was diluted 1:2 with PBS and incubated with anti-CD14:PE (DakoCytomation) and anti-CD42a:FITC or isotype-matched controls for 20 min at room temperature, before FACS-Lyse was added. Platelet–monocyte and platelet–neutrophil aggregates were defined as monocytes or neutrophils positive for CD42a. At least 2500 cells were measured by flow cytometry (EPICS XL2; Beckman-Coulter). Samples were analysed with EXPO 32 software (Cytometry Systems).

Continuous variables are reported as mean (SD). Data were statistically analysed with a paired t test for normally distributed variables or the Wilcoxon matched pairs test for non-parametric variables. GraphPad Prism (GraphPad Software, San Diego, California, USA) was used for all statistical analyses. Significance was taken at p < 0.05.


Participants (51 (7) years) had a diagnosis of diabetes for 6 (4) years with mean haemoglobin A1c of 7.9 (1.4)% indicating moderate glycaemic control. Baseline and four-week fasting plasma glucose concentrations, haemoglobin A1c and fasting lipid profiles were similar (data not shown, NS).

Clopidogrel treatment reduced platelet surface P-selectin (5.6 (2.8)% v 3.7 (1.8)%, p  =  0.002) but not CD40L expression (3.3 (0.7)% v 3.4 (1.0)%, p  =  0.7). Monocyte surface expression of CD40 (40.7 (5.9)% v 35.0 (6.5)%, p  =  0.007) and CD11b (74.8 (7.5) v 60.0 (5.9) mean fluorescence intensity, p  =  0.02) were reduced after clopidogrel treatment. Clopidogrel treatment reduced both platelet–monocyte (23.2 (7.6)% v 17.8 (7.4)%, p  =  0.01) and platelet–neutrophil (7.7 (2.9)% v 6.0 (2.3)%, p  =  0.04) binding (fig 1).

Figure 1

 (A) Platelet–monocyte aggregates, (B) platelet–neutrophil aggregates, (C) monocyte surface expression of CD40 and (D) monocyte surface expression of CD11b before and after four weeks’ treatment with clopidogrel in patients with type 2 diabetes. MFI, mean fluorescence intensity.

Plasma concentrations of the platelet-derived chemokine RANTES (3722 (1123.0) pg/ml v 1476 (1229) pg/ml, p < 0.0001) were greatly reduced. However, concentrations of sCD40L (0.24 (0.42) ng/l v 0.23 (0.32) ng/l, p  =  0.24) and sE-selectin (67.8 940.3) ng/ml v 70.0 (40.5) ng/ml, p  =  0.44) were unchanged after clopidogrel treatment.


Over the past decade it has become apparent that complex signalling occurs between platelets, leucocytes and endothelial cells, and that these interactions have proinflammatory and proatherosclerotic consequences.1,3 Xiao and Théroux4 have previously reported that clopidogrel attenuated the excess platelet–monocyte and platelet–neutrophil conjugates in patients with acute coronary syndromes. Consistent with these findings, our results show that clopidogrel treatment in patients with diabetes mellitus also reduces platelet–leucocyte conjugates despite considerably lower levels of platelet activation. In addition, we have shown that clopidogrel was associated with less monocyte surface expression of CD40 and CD11b indicating reduced monocyte activation. Reduction of platelet–leucocyte interactions and monocyte activation may well contribute to the clinical benefits of clopidogrel.

RANTES mediates monocyte and lymphocyte recruitment to sites of vascular injury and has a key role in the progression of atherosclerosis. RANTES may be secreted by several cell types including activated platelets and leucocytes.3 In our study clopidogrel reduced both platelet and monocyte activation and it is therefore not surprising that clopidogrel also greatly reduced plasma RANTES concentrations. This reduced RANTES secretion provides another potential anti-inflammatory mechanism that may contribute to the clinical benefits of clopidogrel.

CD40L has been shown to mediate a broad range of proinflammatory and prothrombotic responses. In contrast to P-selectin and RANTES, neither platelet surface expression of CD40L nor sCD40L was reduced by clopidogrel. Variable effects of clopidogrel on sCD40L have previously been reported. Xiao and Théroux4 reported a 27% reduction in plasma concentrations of sCD40L in patients with acute coronary syndromes, whereas Quinn et al5 found that clopidogrel did not affect pre- or post-procedure sCD40L concentrations in patients undergoing percutaneous coronary intervention. In the present study, baseline platelet activation and sCD40L levels were substantially lower than in the acute coronary syndrome population studied by Xiao and Théroux,4 in which the sCD40L reductions were greatest with baseline concentrations > 0.5 ng/ml. In our study only one participant had an sCD40L concentration > 0.5 ng/ml and in this patient clopidogrel caused a 23% reduction. These findings suggest that clopidogrel may not have the same effect on CD40L in patients with lower baseline levels of platelet activation and sCD40L.

In conclusion, clopidogrel treatment in patients with type 2 diabetes mellitus not only reduces platelet activation but also reduces platelet–leucocyte interactions, monocyte activation and plasma concentrations of the chemokine RANTES. These findings provide further support for the hypothesis that clopidogrel has important anti-inflammatory actions that may contribute to its clinical benefits.



  • Grant support: Drs Harding and Din were supported by grants from the British Heart Foundation (PG/2001/068; PG/03/009)

  • Competing interests: Professor Fox has received grant support for the Global Registry of Acute Coronary Events (GRACE) from Sanofi-Aventis. All other authors have no conflict of interest to declare

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