Regular Article Platelet activation in patients with peripheral vascular disease: Reproducibility and comparability of platelet markers ☆ A. Burdess a, ⁎, A.E. Michelsen b , F. Brosstad b , K.A.A. Fox c , D.E. Newby c , A.F. Nimmo d a The University of Edinburgh, Centre for Cardiovascular Sciences, UK b Research Institute for Internal Medicine, University of Oslo, Norway c The University of Edinburgh, Centre for Cardiovascular Sciences, UK d The University of Edinburgh, Department of Anaesthesiology, UK abstract article info Article history: Received 16 November 2010 Received in revised form 9 August 2011 Accepted 15 August 2011 Available online 19 September 2011 Keywords: Peripheral arterial disease Platelet-monocyte aggregation Reproducibility Background: Many markers of platelet activation have been described but their reproducibility and compara- bility in patient populations are poorly defined. Objectives: We sought to compare markers of platelet and monocyte activation with platelet-monocyte ag- gregates, a proposed gold standard of in vivo platelet activation, and assess their reproducibility in patients with peripheral arterial disease: a population with substantial platelet activation, inflammation and risk of thrombotic events. Patients/Methods: Thirty patients with peripheral vascular disease attended on two occasions to permit within-day and between-day comparisons. In vivo platelet and monocyte activation were determined by flow-cytometric quantification of platelet-monocyte aggregation, platelet surface expression of P-selectin and CD40L, platelet-derived microparticles, and monocyte surface expression of CD40 and CD11b. Plasma concentrations of platelet-derived microparticles, soluble P-selectin and CD40L were measured by enzyme- linked immunosorbant assays. Results: Platelet-monocyte aggregation (36.7±7.86%), and platelet surface expression of P-selectin (5.8± 1.65%) and CD40L (3.3± 1.45%) demonstrated comparable within-day (mean difference ± co-efficient of repro- ducibility; 0.9±15.4%, 0.21±1.65% and 0.2±2.8% respectively) and between-day reproducibility (2.0±12.4%, 0.10±2.25% and 0.9±6.4% respectively). Platelet-monocyte aggregates correlated well with other platelet (r= 0.30-0.50, P b 0.02) and monocyte (r=0.27-0.47, P b 0.03) activation markers. Flow cytometric and assay quantified platelet-derived microparticles showed poorer reproducibility (co-efficient of reproducibility N 40). Conclusions: In patients with peripheral arterial disease, measurements of platelet-monocyte aggregates have good reproducibility and consistently reflect other markers of platelet and monocyte activation. © 2011 Elsevier Ltd. All rights reserved. Introduction Atherothrombosis is the leading cause of mortality in the western world. Platelets play a major role in the inflammatory and thrombotic progression of atherosclerosis [1,2]. Indeed, increased platelet activity is present in patients at increased risk of atherothrombotic events and predicts adverse cardiovascular events [3–8]. The measurement of platelet activity is therefore crucial to our understanding of the patho- physiology of atherothrombosis, the prediction of adverse cardiovascu- lar events, and the development of novel therapeutic interventions. Despite the development of several techniques, there is still no generally accepted ideal measure of platelet activation. A variety of methods exist, including platelet aggregometry, point of care devices, flow cytometric assessment of platelet surface antigens, and plasma markers of platelet activation: all have advantages and disadvantages [9]. Historically considered the gold standard, platelet aggregometry requires the preparation of platelet rich plasma and a high sample volume. Centrifugation and washing procedures may produce cell loss and artefactually activate platelets. Many of the point-of-care systems assess ex vivo platelet aggregation to various exogenous ago- nists. Although more labour intensive, flow cytometry is emerging as the new sensitive gold standard with measurement of surface expres- sion of platelet antigens providing an assessment of in vivo platelet activation. It requires only a small sample volume, is performed on whole blood, and allows analysis of platelets in their physiological milieu. One of the most commonly studied markers of platelet activa- tion is the α-granule membrane protein, P-selectin, that is present only on the surface of activated degranulated platelets. However, Thrombosis Research 129 (2012) 50–55 ☆ This work was supported by grants from the British Heart Foundation (FS/05/038); European Society of Vascular Surgery Research Awards and Royal College of Surgeons of Edinburgh Research Grants. ⁎ Corresponding author at: Centre for Cardiovascular Sciences, Chancellor's Building, Royal Infirmary of Edinburgh, Little France, Edinburgh, EH16 4SB, UK. Tel.: + 44 131 242 6432, +44 7813602353(Mobile); fax: +44 131 242 6422. E-mail address: anne.burdess@ed.ac.uk (A. Burdess). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.08.015 Contents lists available at SciVerse ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres