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.

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.

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.

Direct Real Time Visualization of Platelet Calclium Signaling In Vivo: Role of Platelet Activation and Thrombus Formation in a Living Mouse.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 325-325 ◽  
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Intracellular Calcium ◽  
Platelet Activation ◽  
Vessel Wall ◽  
High Speed ◽  
Critical Role ◽  
Calcium Mobilization ◽  
Recipient Mouse ◽  
Intracellular Calcium Mobilization ◽  
Platelet Accumulation

Abstract Intracellular calcium mobilization plays a critical role in platelet signaling. Upon platelet activation, an intracellular calcium mobilization leads to the activation of various intracellular and membrane proteins, including integrins involved in both platelet shape change and aggregation. The goal of the present study was to monitor platelet calcium mobilization in vivo in an intact animal and to determine which intracellular pathways are dominant in platelet accumulation into the developing thrombus. Platelets were isolated from mice, washed, loaded with a calcium-sensitive fluorochrome, Fura2-AM and then infused into a recipient mouse. We studied Fura2-AM loaded platelet incorporation during arterial thrombus development following laser injury of the vessel wall in the cremaster microcirculation of living mice using high speed intravital widefield digital microscopy. Fura-2 loaded platelets were monitored by excitation at 380 nm; this fluorescence reports the basal calcium levels in platelets. Calcium mobilization was monitored by excitation at 340 nm where the fluorescence intensity reflects Fura2-calcium complex formation. We observed that platelets bind to the growing thrombus independent of calcium mobilization. However, the stable incorporation of platelets into the thrombus correlated with a significant intracellular calcium increase. Once the thrombus reached maximal size at about 100 seconds, the calcium mobilization also reached maximal intensity. Subsequently, platelets that did not mobilize calcium dissociated from the thrombus. We confirmed these observations by using platelets treated with the calcium chelators, BAPTA-AM or EGTA-AM. We observed a significant inhibition of platelet accumulation into the thrombus, indicating that the intracellular calcium increase is necessary in vivo for the stable accumulation of the platelets into the thrombus. We also evaluated the involvement in vivo of two platelet agonists, ADP and thromboxane A2 (TxA2), on calcium mobilization and platelet incorporation into thrombi. When platelets were treated with aspirin or with the P2Y1 antagonist A3P5P (adenosine 3′-phosphate-5′-phosphate), we observed a partial decrease in both calcium mobilization and platelet accumulation into the thrombus. These results indicate that TxA2 and ADP via the P2Y1 receptor are involved in vivo in platelet activation upon vessel wall injury in this thrombosis model. When platelets were treated with both compounds, we completely inhibited the calcium increase and the incorporation of platelets into the thrombus. Altogether, our results directly show, for the first time in vivo, the importance of the calcium mobilization on platelet accumulation into the developing thrombus. The platelet agonists TxA2 and ADP both play an important and complementary role on platelet activation by acting on the mobilization of the intracellular calcium.

Negative Regulation of Gq-Mediated Pathways in Platelets by G12/13 Pathways through Fyn Kinase

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2854-2854
Author(s):  
Soochong Kim ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Calcium ◽  
Signaling Pathways ◽  
Platelet Function ◽  
Shape Change ◽  
Wild Type Mouse ◽  
Calcium Mobilization ◽  
Negative Regulator ◽  
Platelet Response ◽  
Calcium Response

Abstract Platelets contain high levels of Src family kinases (SFKs) suggesting an important role for these enzymes in platelet function. In this study, we have investigated the regulation of platelet function by SFKs downstream of G12/13 pathways. Calcium-independent platelet shape change induced by selective G12/13 stimulation with YFLLRNP was potentiated with a small mobilization of intracellular calcium in the presence of SFK inhibitors PP1 or PP2, which was abolished by the chelation of intracellular calcium, suggesting that SFKs downstream of G12/13 negatively regulate calcium mobilization in platelets. In addition, PP1 or PP2 caused a leftward shift of human platelet aggregation, secretion, and calcium response induced by low concentrations of agonists that activate platelets through G12/13 signaling such as PAR1-activating peptide SFLLRN and PAR4-activating peptide AYPGKF. However, 2-MeSADP-induced calcium response and platelet aggregation were not affected by the presence of PP1 or PP2, suggesting that SFKs downstream of G12/13, but not Gq/Gi, pathways are involved in this platelet response. Moreover, platelet aggregation and secretion caused by combined stimulation of G12/13 and Gi/Gz (YFLLRNP + 2-MeSADP with P2Y1 antagonist/epinephrine) were also potentiated in the presence of PP1 or PP2 confirming the contribution of SFKs downstream of G12/13 as a negative regulator to platelet activation. Potentiation of platelet aggregation in the presence of SFK inhibitors was not affected in the presence of GF109203X, while PP2 failed to potentiate platelet aggregation in the presence of 5,5′-dimethyl-BAPTA indicating that potentiation of cytosolic calcium may have an important role in this enhanced platelet responses by SFK inhibition. Moreover, PP1 or PP2 failed to potentiate platelet responses in the presence of Gq selective inhibitor YM-254890 or in Gq-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of Gq signaling pathways. Importantly, AYPGKF-induced platelet aggregation, secretion, and calcium response were potentiated in Fyn-, but not in Lyn-, deficient platelets compared to the wild-type mouse platelets, whereas 2-MeSADP-induced platelet response was not affected in these platelets. We conclude that SFKs activated downstream of G12/13, but not Gq/Gi, pathways negatively regulate platelet responses by inhibiting intracellular calcium mobilization in platelets through Gq signaling pathways. Specifically, we define that Fyn plays a major role in this negatively regulatory pathway.

Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 770-770 ◽  
Author(s):  
Yingying Mao ◽  
Todd M Getz ◽  
Jianguo Jin ◽  
Satya P. Kunapuli
Keyword(s):  
Intracellular Calcium ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Shape Change ◽  
Coagulation Factor ◽  
Calcium Mobilization ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Factor Xiia

Abstract Abstract 770 Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt. Disclosures: No relevant conflicts of interest to declare.

Fibrinogen Binding Is Independent of an Increase in Intracellular Calcium Concentration in Thrombin Degranulated Platelets

1995 ◽  
Vol 73 (02) ◽  
pp. 304-308 ◽  
Author(s):  
Fabio M Pulcinelli ◽  
James L Daniel ◽  
Silvia Riondino ◽  
Pier Paolo Gazzaniga ◽  
Leon Salganicoff
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Calcium ◽  
Binding Sites ◽  
Calcium Concentration ◽  
Calcium Release ◽  
Shape Change ◽  
Cytosolic Calcium ◽  
Calcium Mobilization ◽  
Induce Platelet Aggregation ◽  
Fibrinogen Binding

SummaryIn a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C(PLC) activation and calcium mobilization in TDP activated by ADP, epinephrine and U46619.ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

Effects of Enhanced P2X1 Receptor Ca2+ Influx on Functional Responses in Human Platelets

2002 ◽  
Vol 88 (09) ◽  
pp. 495-502 ◽  
Author(s):  
Michael Rolf ◽  
Martyn Mahaut-Smith
Keyword(s):  
Morphological Change ◽  
G Protein ◽  
Platelet Activation ◽  
Shape Change ◽  
Human Platelets ◽  
Functional Responses ◽  
P2x1 Receptor ◽  
G Protein Coupled

SummaryG-protein-coupled P2Y1 and P2Y12 receptors play key roles in platelet activation, however the importance of ionotropic P2X1 receptors remains unclear. Platelet P2X1 responses are highly labile in vitro, but were greatly enhanced by increasing [Ca2+]o in the range 1–10 mM. The P2X1 agonist α,β-MeATP stimulated a shape change which saturated at peak [Ca2+]i of ≥ 400 nM, without evidence for aggregation. The maximal P2X1-evoked transmission decrease was 82% of that obtained via P2Y1 receptors. α., β-MeATP caused a disc to sphere transformation in virtually all platelets, but lacked the long processes produced by ADP. Following block of P2Y1 receptors with A3P5PS, co-stimulation with α., β-MeATP and ADP failed to induce aggregation despite the generation of peak [Ca2+]i responses similar to those stimulated via P2Y1 receptors. Therefore early, transient Ca2+ influx via P2X1 receptors can contribute to platelet activation by stimulating a significant morphological change, but does not readily synergise with P2Y12 receptors to support aggregation.

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