Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function

Ramachandran, Rithwik; Mihara, Koichiro; Thibeault, Pierre E.; Vanderboor, Christina; Petri, Bjoern; Saifeddine, Mahmoud; Bouvier, Michel; Hollenberg, Morley D.

doi:10.1124/mol.116.106526

Cited by 23 publications

(40 citation statements)

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“…In keeping with emerging literature for other GPCRs, we now know that activated PAR4 can directly couple to multiple G-protein-signalling pathways including Gaq/11 and the Ga12/13 pathway (Woulfe, 2005;Kim et al, 2006) but is thought not to engage Gai dependent signalling pathways (Kim et al, 2006). PAR4 can also recruit and signal through b-arrestins (Li et al, 2011;Ramachandran et al, 2017). In recent work, we identified a Cterminal motif in PAR4 that was critical for PAR4 signalling through the Gaq/11 calcium signalling pathway and for recruiting b-arrestin-1/-2 (Ramachandran et al, 2017).…”

Section: Introductionmentioning

confidence: 64%

“…The mutant receptor with an 8-amino acid C-terminal deletion (dRS-PAR4) failed to internalize following activation with the PAR4 agonists thrombin or AYPGKF-NH2 suggesting a role for b-arrestins in PAR4 trafficking. A pepducin targeting this C-terminal motif was also effective in attenuating PAR4-dependent platelet aggregation and thrombosis in vivo (Ramachandran et al, 2017). These recent findings point to the exciting possibility that it might be possible to therapeutically target PAR4 signalling in a pathway-specific manner.…”

Section: Introductionmentioning

confidence: 90%

“…Cells stably expressing PAR4-YFP or dRS-PAR4-YFP were maintained in the above media supplemented with 600 µg/mL of geneticin (Gibco). dRS-PAR4-YFP expressing HEK-293 cells has been previously described (Ramachandran 2017). PAR1-KO-PAR4-YFP-HEK-293 (Mihara et al, 2016) and b-arrestin-1/2-KO HEK-293 cells (Thibeault et al, 2019) have been previously characterized.…”

Section: Cell Culture and Transfectionsmentioning

confidence: 99%

“…PAR4 can also recruit and signal through b-arrestins (Li et al, 2011;Ramachandran et al, 2017). In recent work, we identified a Cterminal motif in PAR4 that was critical for PAR4 signalling through the Gaq/11 calcium signalling pathway and for recruiting b-arrestin-1/-2 (Ramachandran et al, 2017). The mutant receptor with an 8-amino acid C-terminal deletion (dRS-PAR4) failed to internalize following activation with the PAR4 agonists thrombin or AYPGKF-NH2 suggesting a role for b-arrestins in PAR4 trafficking.…”

Section: Introductionmentioning

confidence: 99%

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Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA andβ-Arrestin

et al. 2020

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Proteinase-Activated Receptors (PARs) are a four-member family of G-protein coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types including platelets, vascular smooth muscle cells and neuronal cells. In studying PAR4 signalling and trafficking, we observed dynamic changes in the cell membrane with spherical membrane protrusions that resemble plasma membrane blebbing. Since non-apoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release we sought to elucidate the signalling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/Cas9-mediated gene-editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gaq/11-and Gai-signalling pathways and is dependent on signalling via the b-arrestin-1/-2 and RhoA signalling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. Significance Statement We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and b-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signalling in PAR4 since membrane blebbing was dependent on some, but not all, signalling pathways activated by PAR4.

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Section: Introductionmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 90%

Section: Cell Culture and Transfectionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA andβ-Arrestin

et al. 2020

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“…1). Although both PAR1 and P2Y12 are GPCR, they signal through different intracellular messenger pathways (Jin et al, 1998;Boeynaems et al, 2005;Ramachandran et al, 2017;Sanchez Centellas et al, 2017). Convulxin signals through glycoprotein IV (Marlas et al, 1983;Niedergang et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Acetylcholine Inhibits Platelet Activation

Bennett

Ture

Schmidt

et al. 2019

J Pharmacol Exp Ther

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Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but fewer endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO), have been identified. Acetylcholinesterase inhibitors, such as donepezil, can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets. We measured the effect of acetylcholine or analogs of acetylcholine on human platelet activation ex vivo. Acetylcholine and analogs of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and glycoprotein IIb IIIa conformational changes. Conversely, we found that antagonists of the acetylcholine receptor, such as pancuronium, enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase, such as donepezil, also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Our data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for antithrombotic therapies.

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Single Amino Acid Replacement in G‐7039 Leads to a 70‐fold Increase in Binding toward GHS‐R1a

et al. 2019

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The growth hormone secretagogue receptor type 1a (GHS‐R1a) is a class A rhodopsin‐like G protein coupled receptor (GPCR) that is expressed in a variety of human tissues and is differentially expressed in benign and malignant prostate cancer. Previously, the peptidomimetic [1‐Nal4,Lys5(4‐fluorobenzoyl)]G‐7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC50=69 nm), required a lengthy four‐step radiosynthesis, and had a cLogP above 8. To address these challenges, we now report on changes targeted at the 4th position of G‐7039. A 2‐fluoropropionic acid (2‐FPA) group was added on to Lys5 to determine the potential binding affinity of the [18F]‐2‐FP radiolabeled analogue, which could be prepared by simplified radiochemistry. Lead candidate [Tyr4,Lys5(2‐fluoropropionyl)]G‐7039 exhibited an IC50 of 0.28 nm and low picomolar activity toward GHS‐R1a. Molecular docking revealed a molecular basis for this picomolar affinity.

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Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function

Cited by 23 publications

References 50 publications

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA andβ-Arrestin

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA andβ-Arrestin

Acetylcholine Inhibits Platelet Activation

Single Amino Acid Replacement in G‐7039 Leads to a 70‐fold Increase in Binding toward GHS‐R1a

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