Kuijpers1* Abstract Stabilizing Role of Platelet P2Y12 Receptors in Shear- Dependent Thrombus Formation on Ruptured Plaques Reyhan Nergiz-Unal1, Judith M. E. M. Cosemans1, Marion A. H. Feijge1, Paola E. J. van der Meijden 1 , Robert F. Storey2, J. J. J. van Giezen 3 , Mirjam G. A. oude Egbrink4, Johan W. M. Heemskerk1, Marijke J. E. 1 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands, 2 Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom, 3 AstraZeneca R&D Mo¨ lndal, Mo¨ lndal, Sweden, 4 Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands Background: In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y12 receptors. However, such models do not predict involvement of P2Y12 in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y12 in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis. Methodology: Plaques in the carotid arteries from Apoe2/2 mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y12 blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated. Principal Findings: Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y12 blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y12 reduced the thrombi and increased embolization events. These P2Y12 effects were present at shear rates .500 s21, and they persisted in the presence of coagulation. P2Y12-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding. Conclusions/Significance: Platelet P2Y12 receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y12 function is restricted to high shear flow conditions, and is preserved in the presence of coagulation. Citation: Nergiz-Unal R, Cosemans JMEM, Feijge MAH, van der Meijden PEJ, Storey RF, et al. (2010) Stabilizing Role of Platelet P2Y12 Receptors in Shear- Dependent Thrombus Formation on Ruptured Plaques. PLoS ONE 5(4): e10130. doi:10.1371/journal.pone.0010130 Editor: Yu Wang, Hong Kong University, Hong Kong Received October 6, 2009; Accepted March 12, 2010; Published April 12, 2010 Copyright: 2010 Nergiz-Unal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Partial financial support for the conductance of this study was provided by AstraZeneca. Support is acknowledged from the Hacettepe University (Ankara, Turkey), the Netherlands Organization for Scientific Research (11.400.0076) and the Netherlands Heart Foundation (2005B079). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: J.J.J. van Giezen is an employee of AstraZeneca. Partial financial support for the conductance of this study was provided by AstraZeneca who own the patent for ticagrelor. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: Marijke.Kuijpers@bioch.unimaas.nl Introduction Rupture of an atherosclerotic plaque and occlusive arterial thrombus formation is the major cause of acute cardiovascular incidents and deaths in the Western countries [1]. The development of a thrombus is a complex and dynamic process, involving platelet aggregation, generation of thrombin and formation of a network of fibrin, to strengthen the platelet aggregate and stabilize the thrombus [2,3]. In vivo mouse models of experimental thrombosis have indicated a major role of platelet receptors in the process of thrombus formation and stabilization [4]. However, the studies have been carried out by damaging healthy mouse arteries in an artificial way, e.g. by laser-induced tissue ablation, free radical-generating agents or mechanical disruption. Thrombus formation in diseased, atherosclerotic arteries following plaque rupture has hardly been investigated. Ruptured atherosclerotic plaques expose several platelet-adhe- sive and -activating components, such as collagen types I and III, von Willebrand factor (VWF), lysophosphatidic acid, thrombos- pondin, fibronectin, vitronectin, fibrin/fibrinogen and oxidized low density lipoprotein [5,6,7,8,9,10]. In addition, plaques contain tissue factor which, upon de-encryption, activates the extrinsic coagulation system [5], while the intrinsic system of factor XII activation is triggered via collagen [11]. To which extent each of these plaque components contribute to thrombus formation is unclear, though the platelet-activating roles of VWF and collagen are well-described [1]. Collagen-bound VWF mediates the initial tethering and transient adhesion of platelets via glycoprotein PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10130