P2Y12 in Atherothrombosis Figure 4. Procoagulant role of P2Y12 receptors in platelet-dependent thrombin generation. Mouse (A, B) or human (C, D) PRP was preincubated with vehicle, ticagrelor (20 mM) or cangrelor (20 mM), and stimulated with ADP (20 mM). Thrombin generation was measured after triggering with tissue factor (1 pM) and CaCl2 (16.7 mM). (A, C) Representative thrombin generation curves of PRP stimulated with ADP alone, or ADP in combination with P2Y12 inhibitor. (B, D) Quantitative effect of ticagrelor or cangrelor on ADP-stimulated rate of thrombin formation. Data are means 6 SE (n = 3–6), *p,0.05 compared to ADP + vehicle. doi:10.1371/journal.pone.0010130.g004 material, including tissue factor, oxidized low density lipopro- teins and lysophosphatidate [8,10,31]. A key role of platelet P2Y12 receptors in thrombus stabilization was confirmed by in vitro flow studies using either mouse or human blood. Markedly, when coagulation was introduced, blockage or deficiency of P2Y12 suppressed the formation of larger sized platelet aggregates as well as the formation of fibrin fibers, in particular at high shear rate. Furthermore, inhibition or absence of P2Y12 destabilized the thrombi formed on immobi- lized collagens or plaque material. Also cone-and-plate(let) experiments, where platelets adhered to a surface, showed an enhancing effect of P2Y12 on thrombus formation at high but not low shear rate. In earlier work, we and others have shown that autocrine, platelet-derived ADP contributes to thrombus growth and stability on collagen-containing surfaces [18,32,33]. The present data confirm this and indicate that, in addition, the thrombus- stabilizing role of P2Y12 is maintained in the presence of coagulation. Mechanistically, P2Y12 can contribute to the formation of stable thrombi via two different processes: (i) continuous signaling to phosphoinositide 3-kinases b and c with as a result sustained aIIbb3 activation [18]; (ii) enhancement of thrombin/fibrin generation due to increased Gi-mediated Ca2+ mobilization and platelet procoagulant activity [20,21,34]. The observations that ticagrelor and cangrelor retards ADP-dependent thrombin generation and fibrin formation relates to the latter function. In conjunction with the present results, others have reported that P2Y12 is implicated in shear-induced platelet aggregation via activation of Syk kinase and phosphoinositide 3- kinase [35]. Interestingly, another study describes no effect of cangrelor of collagen-dependent thrombus formation [36]. However, this study focused short-term processes (,1.5 min), during which thrombi are still in the growing phase and the contribution of adhesive receptors such as GP-Ib-V-IX is relatively high. Our time-dependent analysis however indicates that a role of P2Y12 becomes more prominent at later stages. The P2Y12-directed prodrug clopidogrel is increasingly used to prevent secondary ischemic events in patients with myocardial infarction or stroke [37,38]. Recently, clinical studies performed with the new, irreversible P2Y12 inhibitor prasugrel [39,40] and PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10130