Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

Youngheun Jee,Seung-Hun Lee,Sanggu Kim,Preeti Kumari Chaudhary,Kyung-Mee Park,Soochong Kim

doi:10.3390/ijms21113932

Abstract

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Highlights

Platelet aggregation is important to maintain hemostasis and thrombosis
We found that the dose response curves were left-shifted, and there was little difference between WT and GRK6−/− platelet in response to high dose of agonists
GPCR kinases (GRKs) were known to be involved in the desensitization of G protein-coupled receptors (GPCRs) in other cell types, the functional role of these signaling molecules and their mechanisms in regulating platelet physiological events have not been fully understood

Summary

Introduction

Platelet aggregation is important to maintain hemostasis and thrombosis. Various agonists including thrombin, adenosine diphosphate (ADP), and thromboxane, mediate their cellular effects through the G protein-coupled receptors (GPCRs) to induce platelet activation, and these GPCRs have been the most common target for anti-thrombotic drug development. Thromboxane A2 (TxA2) exerts its actions via the G protein-coupled thromboxane A2 receptor (TP receptor) and requires co-activation of Gq and Gi pathways to cause platelet aggregation [2,3,4]. It has been shown to activate Gi pathways through the P2Y12 receptor activation by the secreted ADP. Thrombin and thrombin receptor-activating peptides mediate their effect via the G protein-coupled protease-activated receptors (PARs), by direct coupling to Gq and G12/13. PARs cause Gi stimulation indirectly through the P2Y12 receptor activation by the secreted ADP [5]. It has been shown that co-stimulation of serotonin and epinephrine, which acts via the Gq-coupled 5HT2A receptor and the Gz-coupled α2A adrenergic receptor, respectively, induces platelet aggregation [6]. Collagen and collagen-related peptide (CRP) activate platelets by stimulating tyrosine phosphorylation of the Fc receptor (FcR) γ-chain that contains an immuno-receptor tyrosine-based activation motif (ITAM) through the Ig receptor, glycoprotein (GP)VI [7]

Methods

Results

Conclusion