The membrane expression of P-selectin, but not monocyte-platelet aggregates, is influenced by variability in response to aspirin in patients with coronary artery disease

Although anti-platelet therapy is the mainstay of cardiovascular disease, the fact that thrombosis still occurs despite the use of 75 mg/d aspirin implies that in some subjects (perhaps 10– 20%) this agent is sub-effective. This variability in response to aspirin has led to the concept of a ‘suboptimal response to aspirin’, also described as ‘high on-treatment platelet reactivity’ or ‘aspirin resistance’ [1]. This suboptimal response in patients undergoing percutaneous coronary intervention has been associated with increased risk of thrombosis and increased major adverse clinical events in long-term followup [2]. However, research into aspirin variability (and so the concept of resistance) is confounded by variation in the daily dose of aspirin taken by patients, and the lack of a clear consensus of a definition, the latter partly due to numerous laboratory methods that include aggregometry (the most common method)[3]. Quantification of monocyte-platelet aggregates (MPAs) is an additional method that may prove useful in thrombosis research [4]. Increased MPAs are present in a variety of cardiovascular conditions, and although several workers compared MPAs with soluble and membrane expression of P-selectin [5], none have adequately addressed their possible link with aspirin resistance, pertinent as monocytes are sensitive to this drug [6]. We therefore hypothesised that variability in the response to aspirin would influence soluble and membrane expression of P-selectin, and MPAs. We tested this hypothesis in patients with existing coronary artery disease (CAD), and therefore on 75 mg aspirin daily. We recruited 50 patients (12 women, 11 diabetics) with proven stable CAD. Twenty-seven patients had a history of angina, 15 a previous myocardial infarction, 21 had coronary artery bypass grafting, 11 a stroke/transient ischaemic attack, 8 peripheral artery disease and 18 had percutaneous coronary intervention. No patient was within three months of their index event. Drugs prescribed to the CAD patients were a statin to 45 (so that total cholesterol was mean 4.2 standard deviation (SD) 0.9 mmol/l), an angiotensin converting enzyme inhibitor or angiotensin receptor blocker to 40, a beta blocker to 30, a calcium channel blocker to 14, a diuretic to 15, a nitrate to 11 (giving SBP/DBP 138 [23] and 74 [12] mmHg) and insulin to 2 (giving HbA1c 6.3 [0.6]%). Exclusion criteria were current use of oral or parenteral anticoagulation or other anti-platelet drugs, bleeding abnormalities and/or significant hepatic, neoplastic, renal, connective tissue disease or inflammatory disease. The project had the approval of the Local Research Ethics Committee and written informed consent was obtained from each subject. Venous blood was taken into 3.2% sodium citrate for light transmission aggregometry (LTA) on 270 ml aliquots of platelet rich plasma (1000 rpm, 10 minutes) with a 4-channel PAP aggregometer (Alpha Labs, Basingstoke, UK) according to standard protocols using 30 mL of arachidonic acid (Sigma Aldrich, UK, 0.5 mg/ml). Percentage of light transmission was collected at three minutes after the addition of the agonist. Thromboxane B2 was assessed in serum, and soluble P-selectin was measured in citrated plasma by ELISA (R&D Systems, Abingdon, UK). MPAs and platelet P-selectin expression were measured by flow cytometry (FACScalibor, Becton Dickinson, Oxford, UK). Briefly, the platelet cloud was identified in forward and side scatter, gated and platelet identity confirmed by CD42aPerCP (all mAbs from Becton Dickinson, Oxford, UK). To quantify activated platelets, P-selectin was expressed as the percentage of platelets expressing the molecule above isotype control, and by the mean fluorescence intensity (MFI) at rest and after stimulation with arachidonic acid at 125mg/ml for five minutes. MPAs were defined in a separate forward and side scatter gate by events co-staining with CD42a-PerCP and with CD14-PE [7]. Data distributed normally are presented as mean and SD and analysed by student’s t-test. Data distributed