GRK2 Regulates ADP Signaling in Platelets Via P2Y ₁ and P2Y ₁₂

Abstract

Venous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA ₂, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y ₁ and P2Y ₁₂. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation.

Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 ^-/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA ₂, or convulxin. Underlying these changes in GRK2 ^-/- platelets is an increase in Ca ²⁺ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I ₂. Furthermore, platelet aggregation can be restored in GRK2 ^-/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y ₁₂ signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 ^-/- mice treated with the P2Y ₁₂ antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 ^-/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation.

Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y ₁ and P2Y ₁₂-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases.