Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3K␣/␤ subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2؊/؊ mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.Arrestins are cytoplasmic proteins that were originally characterized by their ability to associate with agonist-activated G protein-coupled receptors (GPCRs), 2 mediating their internalization and desensitization (1). More recent studies suggest that arrestins play additional roles in GPCR signaling, by serving as scaffolds to recruit signaling complexes to the receptor, thereby facilitating activation of G protein-dependent and -independent pathways (2, 3). One such arrestinmediated pathway is the PI3K-dependent activation of the Ser-Thr kinase, Akt (4, 5). In fibroblasts, colorectal, and gastric carcinoma cells, arrestins have been found to play a critical role in localizing PI3K to GPCR complexes through an interaction with Src family kinases (SFKs) (6 -8). Perhaps most relevant for platelet agonists, thrombin-stimulated Akt phosphorylation involved activation of both G i and G q : G i -dependent signaling to Akt required ras activation, while G q -dependent Akt activation required arrestin-2 (9).Previous work from our laboratory and others has demonstrated that Akt-dependent pathways contribute to platelet activation by G protein-coupled receptors (10, 11). Yet, the mechanisms leading to Akt activation in platelets remain incompletely defined. Multiple laboratories have demonstrated that thrombin-dependent Akt phosphorylation in platelets is reduced by about 90% in the presence of inhibitors for the G i -coupled ADP receptor, P2Y12, and is blocked by inhibi...