Arrestin-2 Differentially Regulates PAR4 and P2Y12 Receptor Signaling in Platelets

Arrestins play important roles in the function of G Protein-Coupled Receptors (GPCRs) in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as molecular scaffolds to recruit phosphatidyl inositol-3 kinases (PI3Ks) to GPCRs and promote PI3K-dependent signaling. Due to the multifunctional role of arrestins, we sought to determine whether arrestins regulate Akt activation in platelets and thrombosis in living animals. Co-immunoprecipitation experiments indicate that arrestin-2 associates with PAR4 in thrombin-treated platelets and P2Y12 in ADP-treated platelets, but neither receptor in resting cells. Interestingly, association of arrestin-2 with PAR4 was also stimulated by ADP and PAR4-induced association of arrestin with PAR4 was inhibited by P2Y12 antagonists or apyrase. To determine the functional role of arrestin-2 in platelets, ADP- and thrombin receptor-stimulated Akt phosphorylation was compared in platelets from arrestin-2 knock-out versus WT mice. Akt phosphorylation stimulated by 0.8 mM AYPGKF PAR4 agonist peptide was reduced by an average of 77% in arrestin-2 knock-out platelets compared to WT controls (significantly different, p=0.007, n=3 in each group), but ADP-stimulated Akt phosphorylation was unaffected (p=.38, n=3 each). PAR4-stimulated fibrinogen binding was also reduced in arrestin2−/− platelets (by 58.5% in 1 mM AYPGKF-stimulated platelets compared to WT controls), whereas ADP-stimulated fibrinogen binding was not. Finally, arrestin2 knock-out mice were less sensitive to ferric chloride-induced thrombosis than WT mice: 55% of WT mice (n-=9) formed occlusive thrombi after 2min15sec exposure of the carotid artery to 10% ferric chloride, whereas only 11% of WT mice (n=9) formed occlusive thrombi under the same conditions. In conclusion, arrestin-2 associates with both PAR4 and P2Y12 receptors, but differentially regulates their signaling to Akt and fibrinogen binding and appears to play a net positive role in regulating thrombosis in vivo.