G-protein-coupled receptors signaling pathways in new antiplatelet drug development

Abstract

Platelet G-protein-coupled receptors influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce cardiovascular event occurrence in high-risk patients. The latter dual pathway blockade strategy is one of the greatest advances in the field of cardiovascular medicine. In addition to P2Y12, the platelet thrombin receptor, protease activated receptor-1, has also been recently targeted for inhibition. Blockade of protease activated receptor-1 has been associated with reduced thrombotic event occurrence when added to a strategy using P2Y12 and cyclooxygenase-1 inhibition. At this time, the relative contributions of these G-protein-coupled receptor signaling pathways to in vivo thrombosis remain incompletely defined. The observation of treatment failure in ≈10% of high-risk patients treated with aspirin and potent P2Y12 inhibitors provides the rationale for targeting novel pathways mediating platelet function. Targeting intracellular signaling downstream from G-protein-coupled receptor receptors with phosphotidylionisitol 3-kinase and Gq inhibitors are among the novel strategies under investigation to prevent arterial ischemic event occurrence. Greater understanding of the mechanisms of G-protein-coupled receptor-mediated signaling may allow the tailoring of antiplatelet therapy.

Keywords: GTP-binding proteins; blood platelet; coronary disease; purinerginc 2Y12 receptor agoists; receptors, thrombin.

Figure 1

G-protein–coupled receptor (GPCR) signaling in platelets. Binding of an agonist on the extracellular loop of the GPCR is associated with the exchange of GTP for GDP on the α subunit resulting in the dissociation of the α subunit from βγ subunit. Depending on the receptor type, the α subunit activates phospholipase C-β (PLC-β), Rho-GEF (guanine nucleotide exchange factor), or adenylyl cyclase, whereas the βγ subunit activates phosphotidylionisitol 3-kinase (PI3K) and PLC-β. Protease activated receptor (PAR) is activated by thrombin by creating a tethered ligand or through a noncanonical mechanism where cleavage by a proteinase occurs at a site different from the canonical cleavage site. The tethered ligand can also stimulate signaling through a G-protein–independent pathway involving β-arrestin–mediated signaling scaffold. β-arrestin is also involved in the internalization and desensitization of the PAR receptors.

Figure 2

An overview of signaling pathways of major G-protein–coupled receptors. Adenosine diphosphate activates platelets through P2Y₁ and P2Y₁₂ receptors. P2Y₁ is coupled to Gq, which activate phospholipase (PLC)-β through Gqα and its intracellular signaling leads to platelet shape change, granule secretion, and amplification of platelet activation through generation of thromboxane A2. P2Y₁₂ is coupled to Gi. The α subunit of Gi is associated with adenylyl cyclase and vasodilator stimulated phosphoprotein phosphorylase activity. The βγ subunit is associated with PI3-kinase that activates Akt, Rap1, ERK, and Src family kinases and G-protein–gated inwardly rectifying potassium channels (GIRK) in addition to PLC-β activity as described above. Activation of P2Y₁₂ leads to glycoprotein (GP) IIb/IIIa activation and stable platelet aggregation. Epinephrine is associated with Gz protein, which on activation inhibits adenylyl cyclase activity through α subunit. Prostaglandin I2 (PGI₂) receptor is associated with Gs protein, which on activation stimulates adenylyl cyclase activity through α subunit. DAG indicates diacylglycerol; ERK, extracellular-signal-regulated kinase; IP3, inositol 1,4,5-triphosphate; MLCK, myosin light chain kinase; PLA2, phospholipase A2; Pls, phospholipids; Tx A₂, thromboxane A₂; and VASP, vasodilator-stimulated phosphoprotein.

Figure 3

P2Y₁₂, protease activated receptor (PAR)-1 and PAR-4 signaling pathways in platelets. Thrombin binds to the hirudin-like sequence of the N-terminal exodomain of the PAR-1 and cleaves extracellular domain, thereby exposing tethered ligand with SFLLRN sequence. The tethered ligand rapidly undergoes conformational change and binds to ligand-binding site I located in the N-terminal exodomain of the receptor. Subsequent conformational change in the PAR-1 receptor leads the activation of G_12/13 and Gq that finally results in shape change, granule secretion and transient platelet aggregation through Rho-GEF (guanine nucleotide exchange factor) and phospholipase C-β (PLC-β) activities, respectively. Thrombin binds to the active site of the PAR-4 through a low-affinity, negatively charged cluster of amino acid residues and cleaves extracellular domain exposing tethered ligand with GYPGOV sequence. Similar to PAR-1, PAR-4 activation through tethered ligand leads to the activation of Rho-GEF and PLC-β that finally results in prolonged and irreversible platelet aggregation. PAR-1–mediated platelet aggregation may be transient and reversible unless strengthened by additional signaling from secreted ADP through P2Y₁₂ receptor and from PAR-4 receptor. DAG indicates diacylglycerol; IP3, inositol 1,4,5-triphosphate; PI3K, phosphotidylionisitol 3-kinase; Tx A₂, thromboxane A₂; and VASP, vasodilator-stimulated phosphoprotein.