(GP)Ib-V-IX. Stable platelet adhesion to VWF is achieved via integrin aIIbb3 and adhesion to collagen via the platelet receptors, GPVI and integrin a2b1 [2,12,13]. Recent in vitro studies suggest that these receptors also mediate platelet adhesion to collagen in damaged plaques [5,6]. Subsequent activation responses of adhered platelets include mobilization of cytosolic Ca2+, secretion of autocoids like ADP, activation of integrin aIIbb3 (fibrinogen receptor) and formation of pseudopods, all of which events help to recruit circulating platelets into a thrombus [2,14]. Part of the activated platelets become procoagulant by Ca2+-dependent exposure of phosphatidylserine on their surface, which is required for local thrombin generation [15]. The autocrine agent ADP activates platelets via two receptors, P2Y1 and P2Y12; the first of which mediates shape change and initiates platelet aggregation, while the latter is required for complete aggregation [16]. The importance of P2Y12 was revealed by the observation that the damage of arteries in P2Y12-deficient mice resulted in markedly delayed and unstable thrombus formation [17]. Mechanistically, we and others have shown that continuous signaling via P2Y12 is required to prevent platelet disaggregation and to maintain aIIbb3 in its active conformation [18,19]. The P2Y12 receptor also has a role in platelet procoagulant activity by potentiating tissue factor-induced throm- bin generation via sustained Ca2+ mobilization [20,21]. In the present paper, we used a recently developed mouse model of thrombus formation on acutely ruptured plaques to study the role of P2Y12 receptors in atherothrombosis. We investigated effects of the reversible P2Y12 antagonists, ticagrelor and cangrelor, not only using this in vivo model, but also in perfusion studies of whole (P2Y12-deficient) mouse or human blood perfused over collagen-containing plaque material. The results point to a P2Y12-dependent stabilization of thrombus formation at high shear, that is maintained under conditions of coagulation. This observation may have important implications for antithrombotic treatment in patients. Materials and Methods Materials Fibrillar Horm collagen (type I) was purchased from Nycomed Pharma (Munich, Germany). Native fibrillar type I collagen was prepared from bovine tendon with minimal protease treatment, as described [22]. Fibrinogen labeled with Oregon Green (OG) 488 was from Invitrogen (Carlsbad, CA, USA). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Molecular Probes (Leiden, the Netherlands). Rat-anti-mouse CD62 labeled with FITC (Wug.E9) and rat-anti-mouse GPIIbIIIa (JON/A) labeled with PE were from Emfret Analytics (Wu¨ rzburg, Germany). Cangrelor (AR-C69931MX) and ticagrelor (AZD6140) were kindly provided by AstraZeneca (Mo¨ lndahl, Sweden). Ketamine and xylazine were from Eurovet (Bladel, the Netherlands). Sources of other materials are described elsewhere [23]. Animals Wild type C57BL/6 mice (12 weeks old) and Apoe2/2 mice on C57BL/6 background (4 weeks old) were obtained from Charles River (Maastricht, The Netherlands). The Apoe2/2 mice were fed a Western-type diet with 0.15% cholesterol for 18–20 weeks, i.e. until plaques had developed in the carotid arteries near the bifurcation. Mice homozygously deficient in P2Y12 receptors [24] and corresponding wild types (C57BL/6 background) were bred and housed, as described previously [25]. Animal experiments were approved by the research ethics committees of the universities of Maastricht (The Netherlands) and Sheffield (UK). P2Y12 in Atherothrombosis In vivo plaque rupture and measurement of thrombus formation Acute rupture of plaques in the carotid arteries was provoked by targeted ultrasound treatment, as described before [26]. Briefly, Apoe2/2 mice fed with cholesterol-containing diet were anesthe- tized by subcutaneous injection of ketamine and xylazine (0.1 mg/ g and 0.02 mg/g body weight, respectively). The carotid arteries were carefully dissected free from surrounding tissue, and the anesthetized animals were injected intravenously with 10% CFSE- labeled platelets, which were obtained from a donor mouse of the same genotype. The mice were then injected with vehicle solution, ticagrelor or cangrelor, as described below. Body temperature was held at 37uC during all procedures. Using brightfield illumination and intravital microscopy, a plaque was selected in one of the carotid arteries near the bifurcation. The tip (0.5 mm diameter) of a titanium ultrasound probe was then placed at the plaque shoulder region. Rupture of the plaque was induced by ultrasound application during 10 s at 6 kHz, using a VibraCell VCX130 processor (Sonics, Newtown, CT, USA). Thrombus formation was recorded in real time by capturing 12-bit fluorescence images at 33 Hz during at least 10 min, using a back-thinned electron multiplier C9100-12 EM- CCD camera (Hamamatsu, Japan) at fixed gain settings. Local rupture of the plaques was verified by two-photon laser scanning microscopy and histological staining of sections of the carotid arteries [26]. Time-series of fluorescence images were analyzed using Wasabi (Hamamatsu) software. Per region of interest (ROI), correspond- ing to the site of thrombus formation, total pixel intensity was calculated and corrected for background intensity. To quantify thrombus size, digital images taken at specific time points were processed using ImagePro software (Media Cybernetics, Silver- spring, MD, USA). Within the carotid artery, two similar ROIs were defined, one representing the thrombus area and an adjacent ROI representing the background. A threshold level was set by eliminating all pixels with intensity lower than 99.0% of the pixels of the background ROI. Intensities (gray levels) of all pixels in the thrombus ROI were then integrated. No image processing was applied. In vivo administration of P2Y12 antagonist and blood collection Anesthetized mice were injected intravenously with vehicle solution (5% mannitol in saline), ticagrelor (AZD6140) or cangrelor (AR-C69931MX). Ticagrelor was infused at a dose of 420 mg/kg for 1 min, followed by 60 mg/kg/min for 15 min; cangrelor was infused at 3 mg/kg/min for 15 min, and infusions were continued during the experiment. Mice for control experiments were bled retro-orbitally under anesthetics after 15 min of infusion of vehicle solution, ticagrelor or cangrelor. Mouse blood was collected into 40 mM PPACK and 5 U/ml heparin or trisodium citrate, as indicated [13]. Platelet-rich plasma (PRP) was isolated and used for platelet activation analysis. Human blood taken by venipuncture, was collected into 40 mM PPACK or 12.9 mM trisodium citrate, as indicated. Flow-cytometric evaluation of platelets with active aIIbb3 or P- selectin exposure was performed by activation with ADP (40 mM) or convulxin (100 ng/ml) in 20x diluted whole mouse blood, followed by labeling with JON/A mAb or anti-CD62 mAb, respectively. Detection of fluorescence was with a FACScan flow cytometer, equipped with an argon laser (Becton-Dickinson, Franklin Lakes, NJ). The percentage of positive cells was analyzed using WinMDI 2.8 software (http://facs.scripps.edu). PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10130