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.

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.

PAR1-Induced Human Platelet Shape Change, Aggregation and Secretion Requires Gα13 Switch Region I Signaling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1522-1522
Author(s):  
Jin-Sheng Huang ◽  
Lanlan Dong ◽  
Guy C. Le Breton
Keyword(s):  
G Protein ◽  
Platelet Activation ◽  
Platelet Function ◽  
Human Platelet ◽  
Shape Change ◽  
Human Platelets ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Rhoa Activation ◽  
Platelet Shape

Abstract While it is known that platelets possess multiple G protein signaling pathways that contribute to the different platelet functional responses, the relative participation of these individual pathways in platelet shape change, aggregation and secretion is not well characterized. To a large extent this is due to the lack of suitable reagents which selectively interfere with specific G protein signaling events, and which can be applied to the study of intact human platelets. With the exception of pepducins, which modulate receptor-G protein coupling (Kuliopulos, A. and Covic, L. Life Sciences 74, 255–262, 2003), the field has for the most part been limited to agents which interfere with different downstream kinases or other downstream effectors. However, the G protein pathways share many of these downstream targets, and consequently, it has been difficult to assign a specific platelet function to a certain G protein. In order to address this issue, it was reasoned that more direct information about specific G protein involvement in human platelet activation might be obtained by interfering with the initial G protein signal transduction events, rather than by interfering with the secondary downstream consequences of this transduction process. Based on this consideration, the present study used a specific Gα13 switch region I (SRI) peptide to investigate the involvement of Gα13 signaling in protease-activated receptor 1 (PAR1)-mediated human platelet function. Specifically, a myristoylated peptide representing the Gα13 SRI (Myr-G13SRIpep) was synthesized and evaluated for its effects on PAR1 activation. Initial studies using dot blot and mass spectrum analysis demonstrated that Myr-G13SRIpep, and its random sequence control (Myr-G13SRIRandom-pep), were equally taken up by intact human platelets. Radioligand binding experiments revealed that Myr-G13SRIpep did not interfere with PAR1-ligand interaction. Subsequent experiments demonstrated that G13SRIpep specifically bound to platelet p115Rho guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation. These results suggest a direct interaction of Gα13 SRI with p115RhoGEF, and indicate a possible mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies revealed that Myr-G13SRIpep inhibited PAR1-stimulated platelet shape change, aggregation and dense granule secretion in a dose-dependent manner. On the other hand, Myr-G13SRIpep did not inhibit platelet activation induced by ADP, A23187 or PAR4 activating peptide (AYPGKF). Taken together, these findings demonstrate that the inhibitory effects of Myr-G13SRIpep are limited to PAR1 signaling mechanisms and are not due to nonspecific effects on platelet function. These results also suggest a significant role for Gα13 SRI signaling in the process of PAR1-mediated human platelet activation. In additional studies it was found that Myr-G13SRIpep also inhibited low-dose thrombin-induced aggregation and PAR1-induced intraplatelet calcium mobilization. Collectively, these results provide evidence that: 1. interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2. signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes; 3. Gα13 SRI signaling is involved in low-dose thrombin-induced platelet aggregation as well as PAR1-mediated calcium mobilization; and 4. permeable peptides representing SRI of Gα-subunits should be a useful approach for studying individual G protein signaling pathways in intact cells.

Thrombopoietin Activates Human Platelets and Induces Tyrosine Phosphorylation of p80/85 Cortactin

1998 ◽  
Vol 79 (01) ◽  
pp. 195-201 ◽  
Author(s):  
Guillaume Huret ◽  
Jean-Pierre Loza ◽  
Raphaël Adda ◽  
Josiane Melle ◽  
Jacques Maclouf ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Actin Polymerization ◽  
Shape Change ◽  
Human Platelets ◽  
Cortical Actin ◽  
Fibrinogen Binding ◽  
Serotonin Secretion ◽  
Acid Pathway

SummaryThe mechanism of human platelet activation by thrombopoietin (TPO) was investigated in vitro. We found that rHuTPO stimulated thromboxane A2 formation and serotonin secretion, despite the absence of shape change and aggregation. Blockade of the arachidonic acid pathway did not inhibit rHuTPO-induced platelet secretion. rHuTPO stimulated the tyrosine phosphorylation of 64, 80/85, 95, 130 and 140 kDa proteins, but phosphoproteins of 100-105 and 125 kDa obtained when platelets aggregated in the presence of thrombin were absent. rHuTPO stimulated and potentiated the thrombin-induced tyrosine phosphorylation of a 80 kDa protein identified as the cortical actin-associated protein, p80/85 cortactin. When platelets were aggregated in the presence of rHuTPO and fibrinogen, cortactin phosphorylation was enhanced as compared to rHuTPO alone. Treatment with RGDS or cytochalasin D respectively reduced or abolished cortactin tyrosine phosphorylation. This confirms the existence of fibrinogen binding-dependent and independent pools of phosphorylated cortactin, both requiring intact actin polymerization. Cytoskeleton-binding proteins may be implicated in in vitro platelet activation by rHuTPO.

Role of G protein–gated inwardly rectifying potassium channels in P2Y12 receptor–mediated platelet functional responses

Blood ◽  
2004 ◽  
Vol 104 (5) ◽  
pp. 1335-1343 ◽  
Author(s):  
Haripriya Shankar ◽  
Swaminathan Murugappan ◽  
Soochong Kim ◽  
Jianguo Jin ◽  
Zhongren Ding ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Shape Change ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Girk Channels ◽  
Girk Channel ◽  
Inwardly Rectifying

Abstract The role of the Gi-coupled platelet P2Y12 receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to αIIbβ3 activation in human platelets has not been delineated. As the P2Y12 receptor has been shown to activate G protein–gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y12 receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(±)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)–, 2-methylthioADP (2-MeSADP)–, U46619-, and low-dose thrombin–mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y12-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced Gq-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y12 receptor–selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y12 receptor. We conclude that GIRK channels are important functional effectors of the P2Y12 receptor in human platelets.

Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo

Blood ◽  
2010 ◽  
Vol 115 (10) ◽  
pp. 2008-2013 ◽  
Author(s):  
Valérie Martin ◽  
Julie Guillermet-Guibert ◽  
Gaétan Chicanne ◽  
Cendrine Cabou ◽  
Martine Jandrot-Perrus ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Activation ◽  
Critical Role ◽  
G Protein Coupled Receptors ◽  
In Vivo Studies ◽  
Null Mouse ◽  
Functional Responses ◽  
Activation Motif ◽  
G Protein Coupled

Abstract During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110β null mouse model and demonstrated a critical role of PI3Kβ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glyco-protein VI-Fc receptor γ-chain complex, and its contribution in response to G-protein–coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110β null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein–coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3Kβ is also acting downstream of αIIbβ3. In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl3 injury of the carotid, suggesting that PI3Kβ is a potential target for antithrombotic drugs.

scholarly journals Decoding Gq Signaling in Platelets

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 866-866
Author(s):  
Xi Chen ◽  
Shuchi Gupta ◽  
Matthew Cooper ◽  
Daniel Dehelian ◽  
Lawrence F. Brass ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Shape Change ◽  
Rgs Proteins ◽  
Functional Responses ◽  
Binding Interface ◽  
Computational Alanine Scanning ◽  
Binding Interfaces

Abstract Most platelet agonists work through G protein coupled receptors (GPCRs), activating pathways that involve members of the Gq, Gi, and G12 families of heterotrimeric G proteins. Gq is critical for most functional responses during platelet activation. A defect in Gq expression in patients leads to impaired platelet secretion and aggregation, which is associated with bleeding diathesis. We have previously shown that the Regulators of G protein Signaling (RGS) proteins function as negative regulators for platelet activation by limiting G protein-dependent signaling, but much remains to be learned about the mechanisms by which G proteins can be regulated during platelet activation. Here we took advantage of an RGS-insensitive Gq(G188S) mutant mouse line to decipher the regulatory complex of Gq-dependent signaling in platelets. The G188S mutation disrupts RGS18 and Gq interaction in a way similar to what we have observed in an analogous Gi2(G184S) mutation. However, in contrast to increased platelet activation in Gi2(G184S) expressing platelets, G188S expressing platelets have decreased activation in response to thrombin, TxA2 and ADP, but not to collagen. The hemostatic thrombi formed in G188S mice are significantly reduced with constant embolization as compared with WT controls. Underlying these changes is a decrease in Gq-dependent signaling events, whereas shape change, which is G13-mediated, is unaffected. Our data further reveal that PLCβ3 and GRK2 complex with activated Gq, but not to Gi2. The G188S mutation in Gq not only prevents the binding of RGS proteins to Gq but also impairs the Gq/PLCβ3 interaction, though it does not affect the GRK2 binding to Gq. Instead, there an increase of GRK2 binding to Gq. Finally, the structural analysis shows that PLCβ3 shares a large overlapping binding area with RGS18 to Gq. Computational alanine scanning to predict binding interfaces indicates that the G188S mutation resides in a region important for the binding of both PLC and RGS. The PLC binding interface also overlaps with that of GRK2, which suggests that when PLC is in complex with Gq, it precludes the binding of both RGS proteins and GRK2. In the G188S mutant, however, PLC binding is reduced, which may reduce competition and allow more GRK2 to bind. The binding interfaces of RGS and GRK2 are not predicted to be overlapping, raising the possibility that the two proteins could bind simultaneously to activated WT Gq. Collectively, these observations 1) indicate that the feedback mechanism of Gq inactivation in platelets is different from that of Gi2, 2) reveal, for the first time, that a Gq/PLCβ/RGS/GRK2 signaling node is present in platelets and plays an important role in Gq-dependent signaling during platelet activation, and 3) show that the G188S mutation affect Gq-mediated signaling by disrupting Gq/PLCβ/RGS interaction. Disclosures No relevant conflicts of interest to declare.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

2021 ◽  
pp. 247255522097979
Author(s):  
Kyung-Soon Lee ◽  
Edelmar Navaluna ◽  
Nicole M. Marsh ◽  
Eric M. Janezic ◽  
Chris Hague
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Near Infrared ◽  
Human Cells ◽  
Receptor Subtypes ◽  
G Protein Coupled Receptor ◽  
Functional Responses ◽  
Epitope Tag ◽  
Nir Imaging ◽  
G Protein Coupled

We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives ( t1/2) using LICOR NIR imaging–polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

scholarly journals Adhesion G Protein–Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms

2015 ◽  
Vol 88 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Kelly R. Monk ◽  
Jörg Hamann ◽  
Tobias Langenhan ◽  
Saskia Nijmeijer ◽  
Torsten Schöneberg ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
In Vitro Pharmacology ◽  
G Protein Coupled
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