P2Y12 in Atherothrombosis before ultrasound treatment. (A) Brightfield image of a carotid plaque (p) with ultrasound probe (up). Further, raw fluorescence images (left) and threshold masked images (right) of CFSE-labeled platelets before and after ultrasound treatment. Dotted area indicates location of carotid artery (bars, 100 mm). Time stamps point to images at baseline (210 s) or after plaque rupture (20 s). (B) Representative threshold masked images of thrombi on ruptured plaques of mice infused with vehicle, ticagrelor or cangrelor (bar, 100 mm). Note the microthrombi with P2Y12 antagonist formed within 1 min of ultrasound treatment. (C) Time-courses of integrated CFSE fluorescence intensity above background (arbitrary units, AU) of representative thrombi formed. (D) Quantification of thrombus size at various time points after ultrasound treatment. Data are integrated fluorescence intensities from threshold masked images. (E) Number of fluorescent emboli shed during 3 min after plaque rupture. Data are means 6 SE (n = 3–8), *p,0.05 and #p,0.1 compared to vehicle. doi:10.1371/journal.pone.0010130.g001 over native type I collagen with blood from Apoe2/2 mice which were infused with ticagrelor. Perfusion of blood from ticagrelor- treated mice resulted in diminished thrombus formation on collagen (Fig. 2A). The treatment significantly reduced platelet deposition, while it increased the disaggregation events (Fig. 2B, C). Flow cytometry using whole blood from mice infused with ticagrelor demonstrated near complete inhibition of aIIbb3 activation in response to ADP and of P-selectin expression in response to low convulxin (Fig. 2D). To confirm the role of P2Y12 in these platelet responses, experiments were performed with blood from P2Y12-deficient mice. Perfusion of blood from these mice also resulted in diminished thrombus formation (Fig. 3A), accompanied by increased disaggregation events (Fig. 3B). Furthermore, flow cytometry using whole blood from P2Y12-deficient mice demon- strated complete inhibition of aIIbb3 activation in response to ADP and of P-selectin expression in response to low convulxin (Fig. 3C). Ticagrelor was without further effect on these responses of P2Y12- deficient platelets (not shown). Since enhancement of platelet-dependent thrombin generation is an established outcome of P2Y12 signaling [20], effects of P2Y12 antagonism on thrombin generation were investigated in mouse and human PRP. Dose-response experiments demonstrated that 20 mM ticagrelor or cangrelor were maximally effective (data not shown). At this concentration, either antagonist partly reduced the stimulating effect of ADP on thrombin peak levels in mouse and human PRP (Fig. 4A, C). Since in particular the rate of thrombin generation is an important parameter in flow-dependent thrombus formation, we quantified the slope of the thrombin generation curves for effects of P2Y12 inhibition. both ticagrelor and cangrelor markedly suppressed this slope in mouse and human PRP (Fig. 4B, D). We reasoned that this contribution of P2Y12 may play a role in thrombus stabilization by increasing the availability of thrombin to activate platelets and form fibrin fibers. Inhibition of P2Y12 impairs thrombus formation and stabilization at high but not low shear rate The clinical efficacy of P2Y12 antagonists may depend on the local shear rate, which can vary considerably at arterial sites of vulnerable atherosclerotic plaques. To investigate this, human blood was perfused at low and high shear rate over a collagen- containing surface under physiological conditions allowing coagulation. Under control conditions in the absence of P2Y12 inhibitors, stable thrombi containing platelets and fibrin were formed at shear rates of 300 to 1000 s21 (Fig. 5A). Fibrin fibers were most clearly visible between adjacent thrombi. In the presence of ticagrelor or cangrelor, much smaller thrombi were formed. Especially at 1000 s21, fibrin fiber formation and the average thrombus size were drastically reduced with ticagrelor (Fig. 5B). Analysis of the size distribution patterns on coverslip showed that ticagrelor suppressed the formation of larger thrombi (.400 platelets) only at this higher shear rate (Fig. 5C). Interestingly, this was accompanied by a twofold increase in disaggregation events, again only at 1000 s21 (Fig. 5D). Similar effects of P2Y12 inhibition on thrombus stabilization at high shear were seen in flow studies without coagulation (not shown). Together, these experiments indicated that, under conditions of high shear and coagulation, P2Y12 antagonism suppressed the formation of stable, fibrin-contain- ing thrombi. To confirm the shear-dependent effect of P2Y12 inhibition, additional whole blood experiments were performed with a cone and plate(let) analyzer. This test evaluates platelet adhesion and aggregation to a surface under defined shear conditions [28]. At a low shear rate of 500 s21, the size of the platelet aggregates was not influenced by ticagrelor or cangrelor (Fig. 6A, B). However, at 5000 s21, the larger size aggregates became greatly sensitive to P2Y12 inhibition. This experiment hence supports the conclusion that P2Y12 contributes to thrombus formation especially at high shear rate. Inhibition or deficiency of P2Y12 suppresses thrombus formation on plaques under high shear flow To approach the clinical situation, plaque tissue was isolated from large atherosclerotic vessels obtained by autopsy. Cell-free homogenates were prepared from pools of four plaques [5], and these were used as thrombogenic surface for flow chamber studies. Human blood, supplemented with OG488-labeled fibrinogen, was perfused over the plaque homogenate, again under conditions of high shear and coagulation. This resulted in the assembly of large, fluorescent thrombi, which were interconnected by fluorescent fibrin fibers (Fig. 7A). Preincuba- tion of the blood with ticagrelor or cangrelor substantially reduced thrombus size as well as fibrin fiber formation (Fig. 7B, C). That thrombus size was reduced with either P2Y12 antagonist was further confirmed by morphometric analysis of the platelet aggregates (Fig. 7D). This was in agreement with analysis of the disaggregation events during blood flow, pointing to a marked destabilization of the thrombi formed in the presence of ticagrelor or cangrelor (Fig. 7E). To confirm these results based on pharmacological inhibition of P2Y12, flow experiments were performed using blood from P2Y12-deficient mice. Blood was perfused over homogenized plaque material, which was isolated from atherosclerotic aortic arches of cholesterol-fed Apoe2/2 mice. Perfusion of P2Y12- deficient blood over this material resulted in greatly reduced thrombus formation, leaving only a monolayer of platelets at the plaque surface (Fig. 8A). Deficiency in P2Y12 also suppressed the deposition of OG488-fibrinogen. Image analysis pointed to a significant reduction in both overall thrombus size (Fig. 8B) and label accumulation (Fig. 8C). Morphometric analysis showed the formation of more smaller aggregates with P2Y12- deficient blood (Fig. 8D). These thrombi were instable, as appeared from analysis of the platelet disaggregation events during flow (Fig. 8E). Altogether these results indicate that ADP, via P2Y12, stabilizes thrombi formed under flow on ruptured plaques in the mouse carotid artery in vivo, and on human and mouse plaque material in PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10130