Role of P2Y12 Receptor in Thrombosis

2016 ◽  
pp. 307-324 ◽  
Author(s):  
Yaqi Zhang ◽  
Si Zhang ◽  
Zhongren Ding
Keyword(s):  
P2y12 Receptor

scholarly journals Central role of the P2Y12 receptor in platelet activation

2004 ◽  
Vol 113 (3) ◽  
pp. 340-345 ◽  
Author(s):  
Robert T. Dorsam ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Activation ◽  
P2y12 Receptor

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

2010 ◽  
Vol 429 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Analia Garcia ◽  
Soochong Kim ◽  
Kamala Bhavaraju ◽  
Simone M. Schoenwaelder ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Induce Platelet Aggregation ◽  
Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

scholarly journals THE ROLE OF PLATELET REACTIVITY UNDER NOVEL P2Y12 RECEPTOR INHIBITORS IN PREDICTING 1-YEAR BLEEDING EVENTS

2016 ◽  
Vol 67 (13) ◽  
pp. 2140
Author(s):  
Ioanna Xanthopoulou ◽  
Niki Vlassopoulou ◽  
Ilianna Bei ◽  
Ioanna Pentara ◽  
Angelos Perperis ◽  
...  
Keyword(s):  
Platelet Reactivity ◽  
P2y12 Receptor ◽  
Bleeding Events

scholarly journals Role of the P2Y12 Receptor in the Modulation of Murine Dendritic Cell Function by ADP

2010 ◽  
Vol 185 (10) ◽  
pp. 5900-5906 ◽  
Author(s):  
Abduelhakem Ben Addi ◽  
Dorothée Cammarata ◽  
Pamela B. Conley ◽  
Jean-Marie Boeynaems ◽  
Bernard Robaye
Keyword(s):  
Dendritic Cell ◽  
Cell Function ◽  
P2y12 Receptor ◽  
Dendritic Cell Function

scholarly journals A Potential Protective Role of Platelets during Septic Shock Does Not Depend on Their Purinergic Receptors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2537-2537 ◽  
Author(s):  
Beatrice Hechler ◽  
Chloé Zimmermann ◽  
Yannick Rabouel ◽  
Stéphanie Magnenat ◽  
Mélanie Burban ◽  
...  
Keyword(s):  
Septic Shock ◽  
Platelet Count ◽  
Purinergic Receptors ◽  
Plasma Concentrations ◽  
Protective Role ◽  
P2y12 Receptor ◽  
Severe Thrombocytopenia ◽  
P2y1 Receptor ◽  
Tnf Α

Abstract Thrombocytopenia frequently occurs in septic shock patients and is associated with a worsened outcome. Experimental data indicate that platelets may have a protective role during sepsis. Our aims were to i) assess the potential protective role of platelets during septic shock and ii) evaluate the involvement of platelet P2Y1 and P2Y12 purinergic receptors. We used a model of polymicrobial-induced septic shock in mice by cecal ligation below the ileocecal valve and double puncture (CLP). The impact of moderate to severe thrombocytopenia was evaluated using mice deficient for the thrombopoietin receptor (Mpl-R-/-, 80% reduction in platelet count) and intravenous administration of a platelet specific rat anti-GPIbα monoclonal antibody (RAM.6, 2 mg/kg, 93% reduction in platelet count), respectively. To investigate the role of the P2Y12 receptor, WT mice were treated with clopidogrel, an irreversible inhibitor of the P2Y12 receptor, administered per os at 10 mg/kg, the day before and 2 h prior to surgery. Contribution of the P2Y1 receptor was evaluated using mice deficient for the P2Y1 receptor (P2Y1-/-). Mice underwent sham or CLP surgery (6 animals/group). Twenty hours post-surgery, blood and tissue samples were collected to evaluate critical parameters of septic shock. Mortality was recorded in a second group of animals. Twenty hours after CLP, mice with either normal or reduced platelet counts displayed signs of septic shock such as apathy, fur ruffling, conjunctivitis and diarrhea. Their mean arterial blood pressure (MAP) fell by 30% indicating systemic hypotension and organ failure characteristic of shock. Mpl-R-/- mice, displaying moderate thrombocytopenia, showed enhanced plasma concentrations of the pro-inflammatory cytokines TNF-α and IL-6, and of myeloperoxidase (MPO), indicating increased systemic inflammation relative to WT mice. These effects were more pronounced in RAM.6-treated mice displaying severe thrombocytopenia. Thrombin-antithrombin (TAT) complexes, reflecting in vivo thrombin generation, were enhanced in RAM.6-treated mice as compared to Mpl-R-/-mice or WT mice. Thrombocytopenia was also associated with increased plasma levels of aspartate aminotransferase (ASAT) relative to mice with normal platelet count, an effect also more pronounced in severely thrombocytopenic mice. These results indicate that depletion of platelets worsened organ injury during septic shock. Finally, severely thrombocytopenic mice succumbed more rapidly than control mice after CLP. Twenty hours after CLP, clopidogrel-treated mice and P2Y1-/- mice displayed signs of shock attested by a 30% decrease in MAP, similar to that observed in WT mice. Plasma concentrations of TNF-α, IL-6 and MPO were similarly increased in WT, clopidogrel-treated and P2Y1-/- mice. In addition, no differences in TAT levels were observed between the three groups of mice. Finally, the extent of liver damage, as evaluated by measurement of plasma ASAT levels, was similar between clopidogrel-treated mice, P2Y1-/-mice and WT mice. Overall, these results provide evidence for a beneficial role of platelets during experimental septic shock in mice. However, the platelet ADP receptors do not contribute to this effect, suggesting that anti-platelet therapy with P2Y12 receptor antagonists may not be beneficial in patients with septic shock. The mechanisms by which platelets modulate the host response to septic shock remain to be elucidated. Disclosures No relevant conflicts of interest to declare.

Abstract 599: Lighting Up P2Y12 Oligomers: Insights into the Role of Oligomerization in P2Y12 Function

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Aasma Khan ◽  
Jeffrey L Caplan ◽  
Donna Woulfe
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
G Protein ◽  
Time Lapse ◽  
Bimolecular Fluorescence Complementation ◽  
P2y12 Receptor ◽  
Live Cells ◽  
Bleeding Disorders ◽  
Mutant Forms

Introduction: Little is known about the role of P2Y12 oligomerization in receptor function and whether P2Y12 receptor mutations associated with human bleeding disorders may be explained by alterations in oligomerization. Objectives: 1) To determine whether P2Y12 homo- and hetero-oligomers are constitutive or dynamically regulated. 2) To explore whether P2Y12 mutants R256Q and R265W (previously detected in patients with abnormal bleeding, but with unaltered ADP binding) have different oligomerization affinities or kinetics and determine whether differences in P2Y12 oligomerization explain the functional defects. Methods: We employed a Venus-based Bimolecular Fluorescence Complementation (BiFC) approach in HEK293T cells transiently co-expressing P2Y12 or its mutant forms (R256Q or R265W) tagged with either the N-terminal (P2Y12-VN) or C terminal fragment (P2Y12-VC) of Venus, to characterize their homomeric interactions, in live cells using confocal microscopy and quantitative flow cytometry assays. Results: Agonist-independent formation of P2Y12 receptor homo-oligomers were detected on cell membranes. Time lapse imaging showed movement of P2Y12 receptor pairs from the endoplamic reticulum and Golgi network to the plasma membrane, suggesting that they are constitutive and required for export. Co-expression of P2Y12-VN with increasing amounts of P2Y12-VC demonstrated a dose-dependent increase in the fluorescence intensity of Venus, and reached saturation at a ratio of 1:3. Interestingly, the fluorescence intensities of homomeric P2Y12-R256Q-VN and R256Q-VC and, separately, P2Y12-R265W-VN and P2Y12R265W-VC were almost 4 times stronger than that of the wild type receptor as quantified in flow cytometry-based BiFC. Similar results were obtained in confocal microscopy. This suggests that these P2Y12 mutants form an increased number of dimers or oligomers with increased self-affinities. Conclusion: We demonstrate that P2Y12 forms constitutive homo-oligomers. Two mutations associated with bleeding disorders in patients have altered receptor-receptor interactions. Future investigation will explore the effect of mutations and receptor oligomers on G protein coupling and receptor: G protein stoichiometry.

Critical Role of Endogenous ADP Via P2Y12 Receptor in Maintenance of αIibβ3 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1655-1655
Author(s):  
Tsuyoshi Kamae ◽  
Masamichi Shiraga ◽  
Hirokazu Kashiwagi ◽  
Hisashi Kato ◽  
Seiji Tadokoro ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Thrombus Formation ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
P2y12 Receptor ◽  
Platelet Suspension ◽  
High Affinity ◽  
Static Conditions ◽  
Inside Out

Abstract Platelet αIIbβ3 (GPIIb-IIIa), a noncovalently associated heterodimer, is a prototypic integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor. αIIbβ3 plays a crucial role in platelet aggregation, a key event of hemostatic plug formation and pathologic thrombus formation. During thrombogenesis, the affinity of αIIbβ3 for macromolecular ligands is dynamically regulated. After exposure to subendothelial matrix, several mediators such as ADP and thromboxane A2, or shear stress, platelets becomes activated and inside-out signaling that induces a high-affinity state of αIIbβ3 for soluble ligands (αIIbβ3 activation) are generated. Previous studies revealed that activation of αIIbβ3 is a reversible process. When platelets are stimulated with weak agonists such as adenosine diphosphate (ADP) and epinephrine in the absence of fibrinogen, αIIbβ3 gradually looses its binding capacity. In contrast, thrombin can induce long-lasting αIIbβ3 activation in the absence of fibrinogen. Although much attention has been directed to the nature of inside-out signaling, the mechanisms by which αIIbβ3 keep in the high-affinity state still remains elusive. In this study, we have demonstrated a critical role of endogenous ADP via its P2Y12 receptor in the maintenance of αIIbβ3 activation. Washed platelets adjusted to 50 x 106/ml were stimulated with 0.2 U/ml thrombin or 5 mM U46619 under static conditions. After the 15-min stimulation, 1 mM AR-C69931MX (a P2Y12 antagonist), 1 mM A3P5P (a P2Y1 antagonist) or buffer alone was added to the suspensions for additional 5 min. The platelet suspensions were then incubated with FITC-PAC1 and PE-anti-CD62P for 30 min and analyzed in a flow cytometer. AR-C69931MX and A3P5P attenuated the number of activated αIIbβ3 about 95% and 45%, respectively on the already activated platelets with thrombin- or U46619 without inhibiting CD62P expression. In an another set of experiments, platelets stimulated with thrombin or U46619 for 15min were then diluted to 1:100 with buffer containing the same agonist concentration (0.5 x 106/ml). In these conditions the number of activated αIIbβ3 was also markedly decreased (~85% reduction). Furthermore, the reduction in activated αIIbβ3 by the dilution was reversed by the addition of exogenous ADP in a dose-dependent fashion. HPLC analysis revealed that the amounts of ADP released from thrombin- and U46619-stimulated platelets were 2.6 and 0.75 mmol/1011platelets, respectively, and these values were comparable with ADP doses required for sustained αIIbβ3 activation in the diluted platelet suspension. Thus, released endogenous ADP plays a critical role in the maintenance of αIIbβ3 activation, and certain platelet concentrations are needed for this action. We also examined Rap 1b activation during the maintenance of αIIbβ3 activation. Thrombin induced sustained Rap 1b activation in the absence of ligand. However, AR-C69931MX disrupted the sustained Rap 1b activation. Thus, there was a close relationship between the maintenance αIIbβ3 activation and Rap 1b activation. Our data provide that the continuous interaction between released ADP and P2Y12 receptor is critical for the maintenance of αIIbβ3 activation.

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