Lipid Receptor GPR31 (G-Protein–Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis

Objective: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein–coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4–mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop–derived pepducin, GPR310 (G-protein–coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection ( P =0.0018) against ferric chloride–induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE–mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y 12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. Conclusions: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi–signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

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Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo

10.1101/2021.11.19.469234 ◽

2021 ◽

Author(s):

Amro Elgheznawy ◽

Patricia Oeftering ◽

Maximilian Englert ◽

Friederike Kaiser ◽

Charly Kusch ◽

...

Keyword(s):

G Protein ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Platelet Response ◽

G Protein Coupled Receptor ◽

Icp Ms ◽

And Function ◽

G Protein Coupled

Zinc (Zn2+) is considered as an important mediator for thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these well-known Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

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G-Protein-Coupled Receptor-Mediated MAPK and PI3-Kinase Signaling Is Maintained in Chinese Hamster Ovary Cells after γ-Irradiation

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111425859 ◽

2011 ◽

Vol 17 (3) ◽

pp. 361-369 ◽

Cited By ~ 4

Author(s):

Ronald I. W. Osmond ◽

M. Henry Martin-Harris ◽

Michael F. Crouch ◽

Janet Park ◽

Eric Morreale ◽

...

Keyword(s):

G Protein ◽

Cell Lines ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Calcium Flux ◽

Kinase Signaling ◽

G Protein Coupled Receptor ◽

Screening Programs ◽

Cellular Models ◽

G Protein Coupled

To expedite G-protein-coupled receptor (GPCR) drug screening studies, cell lines amenable to transfection (e.g. CHO cells) have been widely used as cellular models. These cells can be frozen in a ready-to-use format, allowing screening of a single batch of cells and validation of the cellular material prior to the screening run. A common method used to deliver frozen cells to screening programs is to γ-irradiate the cells, abrogating cell division after thawing and ensuring consistency in the number of cells analyzed per well. With the recognition that signaling proteins such as ERK and Akt are important markers of GPCR activation, along with the availability of suitable assays for their measurement, these outputs have become important for GPCR screening programs. Here we show that several γ-irradiated and frozen CHO-K1 cell lines expressing transfected GPCRs, initially optimized for performing cAMP or AequoScreen calcium flux assays, can be used for the measurement of GPCR-mediated ERK and Akt phosphorylation. Furthermore, CHO-K1 cells transfected with NOP or GAL1 receptors show pharmacology for a number of agonists and antagonists that is consistent with non-irradiated cultured lines. These data indicate that γ-irradiated CHO-K1 cells can be reliably used for the measurement of GPCR-mediated kinase signaling outputs.

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Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C

Blood ◽

10.1182/blood.v100.10.3561 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3561-3569 ◽

Cited By ~ 39

Author(s):

Andrew C. Pearce ◽

Jonathan I. Wilde ◽

Gina M. Doody ◽

Denise Best ◽

Osamu Inoue ◽

...

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

G Protein ◽

Cell Types ◽

Human Platelets ◽

Glycoprotein Vi ◽

Activation Motif ◽

G Protein Coupled ◽

Collagen Receptor

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

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The P2Y12 Antagonists, 2-Methylthioadenosine 5′-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels

Journal of Biological Chemistry ◽

10.1074/jbc.m809780200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16108-16117 ◽

Cited By ~ 42

Author(s):

Subhashini Srinivasan ◽

Fozia Mir ◽

Jin-Sheng Huang ◽

Fadi T. Khasawneh ◽

Stephen C.-T. Lam ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Molecular Mechanisms ◽

Human Platelet ◽

Receptor Activation ◽

Human Platelets ◽

Triethylammonium Salt ◽

P2y12 Antagonists ◽

Integral Role ◽

G Protein Coupled

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors.

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Targeting Orphan G Protein-Coupled Receptor 17 with T0 Ligand Impairs Glioblastoma Growth

Cancers ◽

10.3390/cancers13153773 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3773

Author(s):

Phuong Doan ◽

Phung Nguyen ◽

Akshaya Murugesan ◽

Kumar Subramanian ◽

Saravanan Konda Konda Mani ◽

...

Keyword(s):

Cell Cycle ◽

Signaling Pathways ◽

G Protein ◽

Tumor Regression ◽

Xenograft Model ◽

Calcium Flux ◽

G Protein Coupled Receptor ◽

Wild Mice ◽

Endogenous Expression ◽

G Protein Coupled

Glioblastoma, an invasive high-grade brain cancer, exhibits numerous treatment challenges. Amongst the current therapies, targeting functional receptors and active signaling pathways were found to be a potential approach for treating GBM. We exploited the role of endogenous expression of GPR17, a G protein-coupled receptor (GPCR), with agonist GA-T0 in the survival and treatment of GBM. RNA sequencing was performed to understand the association of GPR17 expression with LGG and GBM. RT-PCR and immunoblotting were performed to confirm the endogenous expression of GPR17 mRNA and its encoded protein. Biological functions of GPR17 in the GBM cells was assessed by in vitro analysis. HPLC and histopathology in wild mice and an acute-toxicity analysis in a patient-derived xenograft model were performed to understand the clinical implication of GA-T0 targeting GPR17. We observed the upregulation of GPR17 in association with improved survival of LGG and GBM, confirming it as a predictive biomarker. GA-T0-stimulated GPR17 leads to the inhibition of cyclic AMP and calcium flux. GPR17 signaling activation enhances cytotoxicity against GBM cells and, in patient tissue-derived mesenchymal subtype GBM cells, induces apoptosis and prevents proliferation by stoppage of the cell cycle at the G1 phase. Modulation of the key genes involved in DNA damage, cell cycle arrest, and in several signaling pathways, including MAPK/ERK, PI3K–Akt, STAT, and NF-κB, prevents tumor regression. In vivo activation of GPR17 by GA-T0 reduces the tumor volume, uncovering the potential of GA-T0–GPR17 as a targeted therapy for GBM treatment. Conclusion: Our analysis suggests that GA-T0 targeting the GPR17 receptor presents a novel therapy for treating glioblastoma.

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