Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y1 Receptor Are Crucial for Gq Coupling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1566-1566
Author(s):  
Zhongren Ding ◽  
Florin Tuluc ◽  
Kavita R. Bandivadekar ◽  
Lili Zhang ◽  
Jianguo Jin ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Human Platelet ◽  
Inositol Phosphate ◽  
Shape Change ◽  
P2y Receptors ◽  
Human Platelets ◽  
Carboxyl Terminus ◽  
P2y1 Receptor ◽  
Functional Responses ◽  
C Terminus

Abstract ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y1 and P2Y12 receptors. The Gq-activating P2Y1 receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y1 receptor in Gq activation. Human P2Y1 receptors, either wild type (P2Y1-WT) or a mutant in which the C-terminus was truncated (P2Y1-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y1-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y1-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y1 receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y1-WT- and P2Y1-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y1-ΔR340-L373 and P2Y1-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y1 receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y1 receptor are essential for Gq coupling and subsequent platelet activation.

Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling

2005 ◽  
Vol 288 (3) ◽  
pp. C559-C567 ◽  
Author(s):  
Zhongren Ding ◽  
Florin Tuluc ◽  
Kavita R. Bandivadekar ◽  
Lili Zhang ◽  
Jianguo Jin ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Inositol Phosphate ◽  
Chinese Hamster ◽  
P2y Receptors ◽  
Similar Data ◽  
Amino Acid Residues ◽  
Functional Responses ◽  
Intracellular Ca ◽  
Arginine Residues ◽  
Stimulation Of

The P2Y1 ADP receptor activates Gq and causes increases in intracellular Ca2+ concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y1 receptor in Gq activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y1 receptor (P2Y1-WT cells), P2Y1-ΔR340-L373, or P2Y1-ΔD356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y1-ΔT330-L373, a mutant lacking the entire COOH terminus, completely lost their response to 2-MeSADP. Similar data were obtained by using these cell lines and measuring Ca2+ mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y1 receptor are essential for Gq coupling. Radioligand binding demonstrated that both the P2Y1-WT and P2Y1-ΔT330-L373-expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 COOH terminus failed to elicit Gq functional responses, indicating that the P2Y1 COOH terminus is essential but not sufficient for Gq activation. Finally, cells expressing a double-mutant P2Y1 receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the Gq-activating P2Y receptors completely lost their functional ability to activate Gq. We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y1 receptor are essential for Gq coupling.

Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

1984 ◽  
Vol 52 (03) ◽  
pp. 333-335 ◽  
Author(s):  
Vider M Steen ◽  
Holm Holmsen
Keyword(s):  
Inhibitory Action ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Early Step ◽  
Stimulus Response ◽  
Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

scholarly journals Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽  
1978 ◽  
Vol 51 (6) ◽  
pp. 1065-1071 ◽  
Author(s):  
S Levy-Toledano ◽  
G Tobelem ◽  
C Legrand ◽  
R Bredoux ◽  
L Degos ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Shape Change ◽  
Antigenic Site ◽  
Human Platelets ◽  
Clot Retraction ◽  
Igg Antibody ◽  
Normal Human ◽  
Induced Aggregation ◽  
Bovine Factor

Abstract In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

Partial Agonists as Probes of Human Platelet Stimulus-Response Coupling in the Response to ADP and Adrenaline

1979 ◽  
Author(s):  
M.C. Scrutton ◽  
J.A. Grant ◽  
C.M. Egan
Keyword(s):  
Cyclic Amp ◽  
Human Platelet ◽  
Shape Change ◽  
Human Platelets ◽  
Stimulus Response ◽  
Partial Agonists ◽  
Aggregation Response ◽  
A 23187 ◽  
Intracellular Ca ◽  
Adp Receptor

Previous studies have identified 2', 3'-dialdehyde-ADF (oADP) and -dialcohol-ADP (or ABI as partial agonists at the ADP receptor, and Clonidine and methoxamine as partial agonists at the n-adrenoceptor.oADP and orADP cause shape change but not aggregation (Euxop, J, Biochem, 88, 543): Clonidine and methoxamine fail to cause aggregation but enhance the response to ADP and other agonists (Nature, in press). Preincubation of platelets with non-aggregating concentrations of the ionophore A-23187 induces a primai aggregation response to o ADP, and a primary aggregation + a secretion response to or ADH This response is specific to the partial agonists; is blocked by an ADP antagonist and by agents which raise cyclic AMP levels (e.g. PGE) or block intracellular Ca++ movemenl (e.g. tetracaine); and is not inhibited by specific chelation of extracellular Ca+. Under similar conditions A-23187 provokes a full aggregation response to Clonidine (which is blocked effectively by yohimbine but not by praiosin) and a primary aggrtion response to methoxamine. Preincubation of platelets with an adenylate cyclase inhibit; (SQ-22536) fails to provoke an aggregation response to oADP or orADP. These data suppt the concept that an increase in oytosolic [Ca2+], rather than a decrease in [cAMP], is key step in initiation of the response of human platelets to ADP and adrenaline.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

Mechanisms for Thrombopoietin-Induced Potentiation of Platelet Aggregation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3535-3535
Author(s):  
Paolo Lova ◽  
Francesca Campus ◽  
Alessandra Bertoni ◽  
Fabiola Sinigaglia ◽  
Cesare Balduini ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Shape Change ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Small Gtpase ◽  
P2y1 Receptor ◽  
Integrin Αiibβ3 ◽  
Granule Secretion ◽  
Induced Aggregation ◽  
Dependent Pathway

Abstract Thrombopoietin (TPO) is the major cytokine regulating megakaryocytes proliferation and thrombopoiesis. Binding of TPO to the c-Mpl receptor expressed on human platelets promotes tyrosine phosphorylation of a number of proteins and potentiates platelet aggregation induced by low concentrations of a variety of agonists including ADP. Nevertheless, TPO alone is not able to induce any platelet functional response, such as shape change, granule secretion, or aggregation. It is now clear that platelet aggregation induced by many different agonists results from the concomitant activation of both Gi-and Gq-dependent signaling pathways. In particular, activation of a Gi-dependent pathway is essential, albeit not sufficient, to elicit complete platelet aggregation. Recently, it has been proposed that the small GTPase Rap1B is an important element in the Gi-dependent pathway for platelet aggregation. ADP activates Gq and Gi-dependent pathways by binding to the P2Y1 and the P2Y12 receptors, respectively. In this study we investigated the contribution of TPO in platelet activation induced by ADP. We found that TPO could restore ADP-induced platelet aggregation when the Gq-coupled receptor was blocked, but failed to restore ADP-induced aggregation in absence of Gi stimulation. Similarly, TPO was unable to restore U46619-induced platelet aggregation, when the Gi-coupled P2Y12 receptor for ADP was blocked, but caused irreversible platelet aggregation when added in combination with epinephrine, which binds to a Gi-coupled receptor. TPO did not cause any detectable calcium movements or pleckstrin phosphorylation in human platelets. Moreover, TPO failed to restore ADP-induced cytosolic calcium incresases and pleckstrin phosphorylation when the Gq-coupled P2Y1 receptor was blocked, although aggregation was completely restored. Moreover, we found that TPO induced activation of Rap1B in platelets, and could completely restore ADP-induced Rap1B activation, which was partially reduced upon blockade of the P2Y1 receptor. Finally, ADP-induced binding of both fibrinogen and PAC-1 monoclonal antibody to integrin αIIbβ3 was prevented by the blockade of the Gq-coupled P2Y1 receptor, but completely restored by the simultaneous addition of TPO. These findings indicate that TPO can complement Gi-, but not Gq-dependent pathways for integrin αIIbβ3 activation and platelet aggregation, through a new mechanism that does not involve activation of phospholipase C, but may involve the small GTPase Rap1B.

scholarly journals Thrombopoietin potentiates activation of human platelets in association with JAK2 and TYK2 phosphorylation

1996 ◽  
Vol 316 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Belén RODRÍGUEZ-LIÑARES ◽  
Steve P. WATSON
Keyword(s):  
Shape Change ◽  
Physiological Role ◽  
Human Platelets ◽  
Dense Granule ◽  
Functional Responses ◽  
Dramatic Effect ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Dense Granule Secretion ◽  
Granule Secretion

Thrombopoietin (TPO), also known as the c-mpl ligand, stimulates rapid tyrosine phosphorylation of multiple proteins in human platelets including the Janus family kinases JAK2 and TYK2. On its own, TPO has no effect on platelet aggregation and dense-granule secretion but induces a general potentiation of these responses by other stimuli. The most dramatic effect is observed against threshold concentrations of agonists for aggregation. Shape change or weak reversible aggregation induced by low concentrations of thrombin, collagen and the thromboxane mimetic, U46619, are converted into irrreversible aggregation in the presence of TPO. A similar result is obtained in the presence of the ADP scavenger apyrase and cyclo-oxygenase inhibitor indomethacin. TPO also induces potentiation of dense-granule secretion measured through release of 5-hydroxy[3H]tryptamine. This effect is most striking against low concentrations of stimuli and is independent of aggregation as it is observed in the presence of chelation of extracellular Ca2+ with EGTA. TPO potentiates activation of phospholipase C and elevation of intracellular Ca2+, providing a molecular explanation for potentiation of functional responses. TPO may have an important physiological role in priming platelet activation in thrombocytopenia, an action that may help to compensate for the reduced platelet density.

scholarly journals Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽  
1978 ◽  
Vol 51 (6) ◽  
pp. 1065-1071
Author(s):  
S Levy-Toledano ◽  
G Tobelem ◽  
C Legrand ◽  
R Bredoux ◽  
L Degos ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Shape Change ◽  
Antigenic Site ◽  
Human Platelets ◽  
Clot Retraction ◽  
Igg Antibody ◽  
Normal Human ◽  
Induced Aggregation ◽  
Bovine Factor

In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

scholarly journals Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets

1993 ◽  
Vol 292 (3) ◽  
pp. 851-856 ◽  
Author(s):  
C Guinebault ◽  
B Payrastre ◽  
C Sultan ◽  
G Mauco ◽  
M Breton ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Human Platelet ◽  
Inositol Phosphate ◽  
Human Platelets ◽  
Significant Inhibition ◽  
Regulatory Subunit ◽  
Phosphoinositide Metabolism ◽  
Inositol Phosphate Production

In this study we have examined the implication of tyrosine kinase activities in aggregation, 5-hydroxytryptamine secretion and mainly phosphoinositide metabolism in response to human platelet stimulation by thrombin. Using the potent tyrosine kinase inhibitor tyrphostin AG-213, we have observed a significant inhibition of aggregation and 5-hydroxytryptamine release; however, this percentage inhibition was lower at high thrombin concentrations. On the other hand, tyrphostin treatment of metabolically 32P-labelled platelets significantly inhibited the thrombin-dependent accumulation of PtdIns(3,4)P2, which involves at least a PtdIns 3-kinase and/or a PtdIns3P 4-kinase, whereas the synthesis of phosphatidic acid (PtdOH), a good reflection of the phospholipase C (PLC) activation in platelets, was partially blocked. Inositol phosphate production was also inhibited by about 40% when tyrphostin-treated platelets were stimulated with thrombin. In addition, we show by Western-blot analysis that PLC gamma 1, as well as the regulatory subunit (p85) of the PtdIns 3-kinase, were present in the anti-phosphotyrosine immunoprecipitate isolated from thrombin-stimulated platelets. Furthermore, tyrphostin treatment clearly decreased the PLC gamma 1 and p85 contents in such an anti-phosphotyrosine immunoprecipitate. Our results provide the first evidence for a direct or indirect regulation of PtdIns(3,4)P2 accumulation and PLC gamma 1 activity by tyrosine phosphorylation during thrombin stimulation of human platelets.

Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y12 Receptor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3942-3942
Author(s):  
Zhongren Ding ◽  
Soochong Kim ◽  
Satya P. Kunapuli
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Human Platelet ◽  
P2y Receptors ◽  
Inverse Agonist ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Constitutive Activity ◽  
P2y12 Antagonists ◽  
Constitutively Active

Abstract Human platelets express two P2Y receptors: Gq-coupled P2Y1 and Gi-coupled P2Y12. Both P2Y1 and P2Y12 are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of human platelet ADP receptor we constructed a chimeric HA-tagged human P2Y12 receptor with its C-terminus replaced by the corresponding part of human P2Y1 receptor and stably expressed it in CHO-K1 cells. Interestingly, the chimeric P2Y12 mutant exhibited a high level of constitutive activity as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y12 mutant was abolished by pertussis toxin, a Gi inhibitor. The constitutively active P2Y12 mutant retained normal responses to 2-MeSADP, with an EC50 of 0.15 ± 0.04 nM. The constitutively active P2Y12 mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 as a potent P2Y12 inverse agonist whereas AR-C69931MX as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y12 receptor and the identification of potent inverse agonist.

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