Abstract
Arrestins in concert with GPCR kinases (GRKs) function in G protein-coupled receptor (GPCR) desensitization in various cells. Therefore, we characterized the functional differences of arrestin3 versus arrestin2 in the regulation of GPCR signaling and its desensitization in platelets using mice lacking arrestin3 and arrestin2. In contrast to arrestin2, platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in arrestin3-deficient platelets compared to wild-type (WT) platelets, while non-GPCR agonist CRP-induced platelet aggregation and secretion were not affected. Surprisingly, in contrast to GRK6, platelet aggregation induced by the co-stimulation of serotonin and epinephrine was significantly potentiated in arrestin3-deficient platelets, suggesting the central role of arrestin3 in general GPCR desensitization in platelets. In addition, the second challenge of ADP and AYPGKF restored platelet aggregation in arrestin3-deficient platelets but failed to do so in WT and arrestin2-deficient platelets, confirming that arrestin3 contributes to GPCR desensitization. Furthermore, ADP- and AYPGKF-induced Akt and ERK phosphorylation were significantly increased in arrestin3-deficient platelets. Finally, we found that arrestin3 is critical for thrombus formation in vivo. In conclusion, arrestin3, not arrestin2, plays a central role in the regulation of platelet functional responses and thrombus formation through general GPCR desensitization in platelets.
Highlights
Platelets have been established as a critical player in hemostasis and thrombosis through their activation via different signaling pathways both in humans and animals.Initially, platelets are activated by a contact-dependent pathway via collagen-induced glycoprotein VI (GPVI) signaling or vWF-induced GPIb-IX-V signaling upon the exposure of the sub-endothelial matrix [1,2]
We further show that arrestin3 regulates as Gq -coupled P2Y1 (ADP) and PAR4 receptor desensitization resulting in the regulation of Gq - and Gi -mediated signaling in platelets
Similar to arrestin3, arrestin2 had no effect on CRP-induced platelet aggregation and dense granule secretion. These results suggest that arrestin2, unlike arrestin3, does not play any role in PAR-induced G protein-coupled receptor (GPCR) desensitization in platelets
Summary
Platelets have been established as a critical player in hemostasis and thrombosis through their activation via different signaling pathways both in humans and animals. Several mechanisms prevent the hyperactivation of GPCR signaling, among which the involvement of arrestins along with GRKs has recently been shown as the most important mechanism for turning off the growing number of GPCR-mediated signaling transduction pathways in various cells These are the only two protein families besides heterotrimeric G proteins that have shown the capacity to interplay with the activated conformation of 7-transmembrane (7-TM) receptors, and they are considered as the key modulators of GPCR phosphorylation, desensitization, intracellular trafficking, and re-sensitization [5,6,7]. Despite the importance of arrestins in GPCR-mediated signaling, the functional role of arrestins in platelet activation, as well as their underlying mechanism, has yet to be established. Arrestin is critical for the regulation of platelet function via general GPCR desensitization
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