P2 receptors, platelet function and pharmacological implications

2008 ◽  
Vol 99 (03) ◽  
pp. 466-472 ◽  
Author(s):  
Christian Gachet
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Knockout Mice ◽  
P2 Receptors ◽  
The Other ◽  
Antithrombotic Drugs ◽  
Adp Receptors ◽  
Inflammatory Processes ◽  
G Protein Coupled

SummaryADP and ATP play a crucial role in platelet activation and their receptors are potential targets for antithrombotic drugs. The ATP-gated cation channel P2X1 and the two G protein-coupled ADP receptors, P2Y1 and P2Y12, selectively contribute to platelet aggregation and formation of a thrombus.Owing to its central role in the growth and stabilization of a thrombus, the P2Y12 receptor is an established target of antithrombotic drugs like the thienopyridines clopidogrel or prasugrel, or competitive antag-onists such as cangrelor or AZD6140.The optimal inhibition of this receptor to reach clinical efficacy while preserving patients from unacceptable bleeding is a matter of debate. On the other hand, studies in P2Y1 and P2X1 knockout mice and using selective P2Y1 and P2X1 antagonists have shown that these receptors are also attractive targets for new antithrombotic compounds. Finally, the regulation by the P2 receptors of the platelet involvement in inflammatory processes is also briefly discussed.

Evidence for Two Distinct G-protein-coupled ADP Receptors Mediating Platelet Activation

1999 ◽  
Vol 81 (01) ◽  
pp. 111-117 ◽  
Author(s):  
Laurent Gousset ◽  
Vinay Bhaskar ◽  
Diana Vincent ◽  
Albert Tai ◽  
Elwood Reynolds ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Calcium ◽  
G Protein ◽  
Platelet Activation ◽  
Shape Change ◽  
Calcium Mobilization ◽  
Intracellular Calcium Mobilization ◽  
Adp Receptors ◽  
Adp Receptor ◽  
G Protein Coupled

SummaryThe identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTP γS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2Y1receptor. By contrast, the P2Y1receptor antagonist adeno-sine 3’,5’-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Giproteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1and coupled to Gq.

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

2020 ◽  
Vol 21 (11) ◽  
pp. 3932 ◽  
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Kyung-Mee Park ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Functional Role ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
G Protein Coupled ◽  
Gpcr Desensitization ◽  
Stimulation Of

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.

scholarly journals Positron Emission Tomography in the Inflamed Cerebellum: Addressing Novel Targets among G Protein-Coupled Receptors and Immune Receptors

Pharmaceutics ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 925
Author(s):  
Margit Pissarek
Keyword(s):  
G Protein ◽  
Cannabinoid Receptors ◽  
G Protein Coupled Receptors ◽  
Translocator Protein ◽  
Brain Diseases ◽  
Present Contribution ◽  
Binding Behavior ◽  
Positron Emission ◽  
Inflammatory Processes ◽  
G Protein Coupled

Inflammatory processes preceding clinical manifestation of brain diseases are moving increasingly into the focus of positron emission tomographic (PET) investigations. A key role in inflammation and as a target of PET imaging efforts is attributed to microglia. Cerebellar microglia, with a predominant ameboid and activated subtype, is of special interest also regarding improved and changing knowledge on functional involvement of the cerebellum in mental activities in addition to its regulatory role in motor function. The present contribution considers small molecule ligands as potential PET tools for the visualization of several receptors recognized to be overexpressed in microglia and which can potentially serve as indicators of inflammatory processes in the cerebellum. The sphingosine 1 phosphate receptor 1 (S1P1), neuropeptide Y receptor 2 (NPY2) and purinoceptor Y12 (P2Y12) cannabinoid receptors and the chemokine receptor CX3CR1 as G-protein-coupled receptors and the ionotropic purinoceptor P2X7 provide structures with rather classical binding behavior, while the immune receptor for advanced glycation end products (RAGE) and the triggering receptor expressed on myeloid cells 2 (TREM2) might depend for instance on further accessory proteins. Improvement in differentiation between microglial functional subtypes in comparison to the presently used 18 kDa translocator protein ligands as well as of the knowledge on the role of polymorphisms are special challenges in such developments.

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

2020 ◽  
Author(s):  
Layla Van Doren ◽  
Nga Nguyen ◽  
Christopher Garzia ◽  
Elizabeth Fletcher ◽  
Ryan Stevenson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
G Protein ◽  
Arterial Thrombosis ◽  
Platelet Reactivity ◽  
Human Platelets ◽  
Calcium Flux ◽  
G Protein Coupled Receptor ◽  
Carotid Artery Injury ◽  
G Protein Coupled

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.

scholarly journals The role of G protein-coupled receptor kinase 4 in cardiomyocyte injury after myocardial infarction

2020 ◽  
Author(s):  
Liangpeng Li ◽  
Wenbin Fu ◽  
Xue Gong ◽  
Zhi Chen ◽  
Luxun Tang ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Transgenic Mice ◽  
Cardiac Function ◽  
G Protein ◽  
Knockout Mice ◽  
Cardiac Dysfunction ◽  
Beclin 1 ◽  
Receptor Kinase ◽  
G Protein Coupled

Abstract Aims G protein-coupled receptor kinase 4 (GRK4) has been reported to play an important role in hypertension, but little is known about its role in cardiomyocytes and myocardial infarction (MI). The goal of present study is to explore the role of GRK4 in the pathogenesis and progression of MI. Methods and results We studied the expression and distribution pattern of GRK4 in mouse heart after MI. GRK4 A486V transgenic mice, inducible cardiomyocyte-specific GRK4 knockout mice, were generated and subjected to MI with their control mice. Cardiac infarction, cardiac function, cardiomyocyte apoptosis, autophagic activity, and HDAC4 phosphorylation were assessed. The mRNA and protein levels of GRK4 in the heart were increased after MI. Transgenic mice with the overexpression of human GRK4 wild type (WT) or human GRK4 A486V variant had increased cardiac infarction, exaggerated cardiac dysfunction and remodelling. In contrast, the MI-induced cardiac dysfunction and remodelling were ameliorated in cardiomyocyte-specific GRK4 knockout mice. GRK4 overexpression in cardiomyocytes aggravated apoptosis, repressed autophagy, and decreased beclin-1 expression, which were partially rescued by the autophagy agonist rapamycin. MI also induced the nuclear translocation of GRK4, which inhibited autophagy by increasing HDAC4 phosphorylation and decreasing its binding to the beclin-1 promoter. HDAC4 S632A mutation partially restored the GRK4-induced inhibition of autophagy. MI caused greater impairment of cardiac function in patients carrying the GRK4 A486V variant than in WT carriers. Conclusion GRK4 increases cardiomyocyte injury during MI by inhibiting autophagy and promoting cardiomyocyte apoptosis. These effects are mediated by the phosphorylation of HDAC4 and a decrease in beclin-1 expression.

Mode of action of P2Y12 antagonists as inhibitors of platelet function

2011 ◽  
Vol 105 (01) ◽  
pp. 96-106 ◽  
Author(s):  
Jackie Glenn ◽  
Ann White ◽  
Sue Fox ◽  
Hans van Giezen ◽  
Sven Nylander ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Function ◽  
Adenosine Diphosphate ◽  
P2y12 Receptor ◽  
Receptor Antagonists ◽  
Receptor Agonists ◽  
P2y12 Antagonists ◽  
Induced Aggregation ◽  
G Protein Coupled

SummaryP2Y12 receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y12 receptors. However, some P2Y12 receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y12 antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y12 receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A2A agonists, which act at Gs-coupled receptors. All three P2Y12 antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A2 mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y12 antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the Gs-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y12 antagonists acting through PAR1, TP, IP, EP4, A2A or EP3 receptors. All three P2Y12 antagonists enhanced VASPphosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y12 receptors and not through another G-protein-coupled receptor.

CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 326-326
Author(s):  
Wolfgang Bergmeier ◽  
Jill R. Crittenden ◽  
Crystal L. Piffath ◽  
Denisa D. Wagner ◽  
David E. Housman ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Knockout Mice ◽  
Thrombus Formation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Premature Stop Codon ◽  
Small Gtpase ◽  
Integrin Αiibβ3

Abstract Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.

A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1648-1648
Author(s):  
Ilaria Canobbio ◽  
Lucia Stefanini ◽  
Gianni F. Guidetti ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Lipid Rafts ◽  
Platelet Activation ◽  
Human Platelets ◽  
G Protein Coupled Receptors ◽  
Weak Stimulation ◽  
High Concentrations ◽  
G Protein Coupled

Abstract The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.

scholarly journals The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 4942-4954 ◽  
Author(s):  
Yotis A. Senis ◽  
Michael G. Tomlinson ◽  
Stuart Ellison ◽  
Alexandra Mazharian ◽  
Jenson Lim ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Activation ◽  
Drug Target ◽  
Tyrosine Phosphatase ◽  
Underlying Mechanism ◽  
G Protein Coupled Receptors ◽  
Bleeding Tendency ◽  
Platelet Surface ◽  
Surface Receptors ◽  
G Protein Coupled

Abstract Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase–linked and G protein–coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein–coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

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