Whole blood perfusion experiments Human and mouse blood samples were perfused through a parallel-plate transparent flow chamber containing a coverslip, coated with either plaque material or collagen, as described [5]. PPACK-anticoagulated blood was used for perfusion experiments without coagulation, respectively; ADP (10 mM) was co-perfused, as indicated [18]. Coagulation was introduced by co-perfusion of human or mouse citrate-anticoagulated blood with 0.1 volume of CaCl2 (75 mM), MgCl2 (37.5 mM) and tissue factor (20 pM) in Hepes buffer pH 7.45 (NaCl 110 mM, glucose 10 mM, Hepes 5 mM, KCl 2.7 mM and 0.1% bovine serum albumin). Wall shear rate was 300–1000 s21, as indicated. After blood perfusion, thrombi on coverslips were rinsed with Hepes buffer supplemented with 2 mM CaCl2, 2 mM MgCl2 and heparin (1 U/ml). Recording of real-time videos and capturing of phase-contrast images was with a non-confocal microscopic system [13]. Videos were analyzed offline for embolizing events (single or clustered platelets) per aggregate [18]. Randomly captured images were analyzed for surface area coverage, area distribution of individual segmented features or integrated fluorescence intensity with Image-Pro (ImagePro, Silver Spring MD, USA) or Metamorph software (MDS Analytical Technologies, Downingtown, PA, USA) [27]. In specific experiments, OG488-fibrinogen (25 mg/ml) was added to human or (P2Y12-deficient) mouse blood and, after perfusion over collagen or plaque material, fluorescence accumu- lation was detected by confocal laser scanning microscopy. Confocal images were recorded off-line with a BioRad 2100 multiphoton system at fixed settings of argon laser power, scanning rate and photomultiplier gain [15]. Coating of plaque material Human atherosclerotic plaques were collected at autopsy from a carotid artery and used in compliance with institutional guidelines (Department of Pathology, Maastricht University). Permission was obtained from the local Medical Ethics Committee. Murine atherosclerotic plaques were collected from the aortic arches of 6 Apoe2/2 mice, fed with a Western-type diet with 0.15% cholesterol for 18–20 weeks. After resection, atherosclerotic specimens were frozen into liquid nitrogen and stored at 280uC. Thawed human and mouse atherosclerotic tissues were homogenized in phosphate- buffered saline. Plaque homogenates were pooled at 165 mg wet tissue weight/ml, as described [5]. Coverslips were coated with 2 plaque material at a density of 170 mg wet weight/cm . Cone and plate(let) experiments An Impact-R cone and plate(let) analyzer (CPA, DiaMed, Cressier, Switzerland) was used to evaluate platelet adhesion and aggregation on a surface under defined flow conditions [28]. Citrate-anticoagu- lated blood (130 ml) was placed in a polystyrene well and subjected to a shear of 500–5000 s21 for 2 min. The platelet aggregates were washed and stained by May-Gru¨nwald stain, according to the manufacturer’s instructions (DiaMed). Platelet deposition was evaluated by an image analyzer system connected to the microscope, 2 measuring the average size of the aggregates in mm . Thrombin generation experiments Thrombin generation was measured in citrate-anticoagulated human or mouse PRP (16108 platelets/ml), as before [29]. Briefly, PRP was preincubated with inhibitors, and then treated with ADP (20 mM) or vehicle for 10 min. Samples (4 volumes) were pipetted into a polystyrene 96-wells plate (Immulon 2HB, Dynex Technol- ogies, Chantilly, VA, USA), already containing 1 volume of buffer A (20 mM Hepes, 140 mM NaCl and 0.5% bovine serum albumin) P2Y12 in Atherothrombosis and tissue factor (6 pM). Coagulation was started by adding 1 volume of buffer B (2.5 mM Z-GGR-AMC, 20 mM Hepes, 140 mM NaCl, 100 mM CaCl2 and 6% bovine serum albumin). Samples were run at least in duplicate. First-derivative curves of accumulation of fluorescence in human plasma were converted into nM thrombin using a human calibrator. Statistical analysis Data are presented as means 6 SE. Groups were compared using the non-parametric Mann-Whitney U test (one-tailed) using the statistical package for social sciences (SPSS 15.0, Chicago, IL, USA). Size distribution of platelet aggregates was evaluated by x2 analysis [27]. A p-value below 0.05 was considered significant. Results Inhibition of mouse P2Y12 receptors causes unstable thrombus formation after rupture of an atherosclerotic plaque To study the role of platelet P2Y12 in atherothrombosis, we used a recently established mouse model of acute plaque rupture [26]. Mice deficient in ApoE were fed a cholesterol-enriched diet for 18–20 weeks. Plaque-containing carotid arteries were dissected free from surrounding tissue, and CFSE-labeled Apoe2/2 platelets were injected to enable measurement of fluorescence by intravital microscopy. In control animals infused with vehicle solution, targeted treatment with an ultrasound probe resulted in acute rupture and subsequent non-occlusive thrombus formation (Fig. 1A). As described, this thrombotic process relies on GPVI- induced platelet activation as well as on thrombin generation and coagulation [26]. Mice were infused with effective concentrations of one of the reversible P2Y12 antagonists, ticagrelor or cangrelor. In blood samples taken from some of these mice, it was checked that the ticagrelor or cangrelor interventions abolished ADP- and collagen-induced platelet aggregation (data not shown). In other mice, the infusion of either P2Y12 antagonist greatly suppressed platelet accumulation induced by carotid plaque rupture, in that only loose thrombi appeared, which consisted of single-layered fluorescent platelets, in contrast to the compact bright fluorescent thrombi in vehicle-treated mice (Fig. 1B). The reduced thrombus formation was also apparent from time-courses of integrated fluorescence intensity after plaque rupture (Fig. 1C). Similarly as described [26], in vehicle-treated mice, plaque rupture caused a biphasic pattern of rapid increase and slower decline in fluorescence accumulation. Only few single platelets were seen to leave the thrombus in the declining phase. Since fluorescence bleaching was relatively low, the declining phase had a different cause, likely contraction of the platelet-fibrin thrombus. Off-line quantification of images showed that P2Y12 inhibition with either antagonist reduced the size of thrombi compared to the vehicle control (Fig. 1D). Video analysis further revealed that the thrombi formed in the presence of P2Y12 inhibitor were relatively unstable. With ticagrelor or cangrelor present, 4–5 fold more emboli were shed from thrombi in comparison to control mice (Fig. 1E). Together, these data pointed to a marked inhibiting effect of the reversible P2Y12 antagonists on in vivo thrombus formation following plaque rupture. Absence of functional P2Y12 receptors suppresses thrombus formation and thrombin generation, but increases disaggregation To study the mechanism by which P2Y12 contributes to thrombus stabilization, we performed flow chamber experiments PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10130