RESUMO
Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.
Assuntos
Receptores de Tromboxano A2 e Prostaglandina H2 , Trombose , Plaquetas , Humanos , Agregação Plaquetária , Proteínas Proto-Oncogênicas c-pim-1/genética , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Trombose/tratamento farmacológicoRESUMO
The principal demonstrated role of the nonvisual arrestins in vivo is to limit G protein-coupled receptor (GPCR) signaling. Nonetheless, a direct demonstration of this fundamental ability in platelets remains lacking, despite the prominent role played by GPCRs in platelet activation. This paper describes the basic characterization of the activatory responses of platelets from mice lacking arrestin-3 (arr3-/-), revealing pleiotropic roles dependent on GPCR ligand. Functionally, arrestin-3 acts as a brake on platelet aggregation regardless of ligand tested. Downstream of P2Y receptors, arr3-/- mice show increased secretion and integrin activation mirrored by enhanced intracellular calcium signaling and global PKC-dependent phosphorylation. Furthermore, P2Y12 receptor (P2Y12R) activity as assessed by ADP-mediated reduction of VASP phosphorylation is enhanced in arr3-/-mice. Downstream of PAR receptors there are similar increases in secretion and integrin activation in arr3-/- mice, together with enhanced PKC activity. Last, in arr3-/- mice the TP receptor displays unaltered PKC activity but markedly reduced calcium responses, which together with the kinetics of the aggregation response suggested a unique positive regulatory role for arrestin-3 in TP signaling. Overall, this paper reveals pleiotropic roles for arrestin-3 dependent on GPCR ligand describing for the first time a negative regulatory function for arrestin-3 in platelets.
Assuntos
Arrestinas/metabolismo , Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Plaquetas/citologia , Humanos , CamundongosRESUMO
Ticagrelor is a potent antagonist of the P2Y12 receptor (P2Y12R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca2+ release in washed platelets vs other P2Y12R antagonists. This additional effect of ticagrelor beyond P2Y12R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of Gs-coupled adenosine A2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y12R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y12R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y12R, limiting basal Gi-coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y12R that contribute to its effective inhibition of platelet activation.
Assuntos
Adenosina/análogos & derivados , Plaquetas/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Ativação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y12/metabolismo , Adenosina/farmacologia , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Feminino , Humanos , Masculino , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , TicagrelorRESUMO
Platelet activity is regulated by a number of surface expressed G protein-coupled receptors (GPCRs) including the α isoform of the thromboxane receptor (TPα receptor). With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare GPCR variants including in the TBXA2R gene, which encodes the TPα receptor. The study of patients with naturally occurring variants within TBXA2R associated with bleeding and abnormal TPα receptor function has provided a powerful insight in defining the critical role of TPα in thrombus formation. This review will highlight how the identification of these function-disrupting variants of the platelet TPα has contributed important structure-function information about these GPCRs. Further we discuss the potential implications these findings have for understanding the molecular basis of mild platelet based bleeding disorders.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Hemorragia/sangue , Hemorragia/genética , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Alelos , Genótipo , Hemorragia/diagnóstico , Humanos , FenótipoRESUMO
OBJECTIVE: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS: We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. CONCLUSIONS: Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists.
Assuntos
Plaquetas/metabolismo , Ativação Plaquetária , Receptores de Trombina/metabolismo , Idoso , Plaquetas/efeitos dos fármacos , Estudos de Casos e Controles , Simulação por Computador , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Inglaterra , Feminino , Genótipo , Glicosilação , Células HEK293 , Humanos , Ligação de Hidrogênio , Lactonas/farmacologia , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Peptídeos/farmacologia , Fenótipo , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Transporte Proteico , Piridinas/farmacologia , Receptor PAR-1/efeitos dos fármacos , Receptor PAR-1/metabolismo , Receptores de Trombina/química , Receptores de Trombina/efeitos dos fármacos , Receptores de Trombina/genética , Relação Estrutura-Atividade , Trombina/farmacologia , TransfecçãoRESUMO
Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.
Assuntos
Transtornos Plaquetários/diagnóstico , Monitoramento de Medicamentos/métodos , Hemorragia/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Ativação Plaquetária/fisiologia , Inibidores da Agregação Plaquetária/farmacologia , Adulto , Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Feminino , Estudos de Associação Genética , Voluntários Saudáveis , Hemorragia/sangue , Hemorragia/fisiopatologia , Humanos , Masculino , Ativação Plaquetária/efeitos dos fármacos , Valor Preditivo dos Testes , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Sensibilidade e Especificidade , Adulto JovemRESUMO
Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.
Assuntos
Exoma/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Trombocitopenia/genética , Plaquetas/patologia , Predisposição Genética para Doença , Humanos , Mutação de Sentido Incorreto , Contagem de PlaquetasRESUMO
P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient.
Assuntos
Endossomos/metabolismo , Regulação da Expressão Gênica , Receptores Purinérgicos P2Y12/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Difosfato de Adenosina/metabolismo , Transporte Biológico , Plaquetas/metabolismo , Linhagem Celular , Endocitose , Células HEK293 , Humanos , Ligantes , Proteínas Mutantes/metabolismo , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.
Assuntos
Coagulação Sanguínea , Plaquetas/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Vesículas Secretórias/metabolismo , Trombose/enzimologia , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/patologia , Proteínas de Transporte/farmacologia , Feminino , GTP Fosfo-Hidrolases/genética , Hemostáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Vesículas Secretórias/genética , Trombina/metabolismo , Trombina/farmacologia , Trombose/genética , Trombose/patologia , Proteínas rho de Ligação ao GTPRESUMO
BACKGROUND: Optimal treatment of acute ST-elevation myocardial infarction (STEMI) involves rapid diagnosis, and transfer to a cardiac centre capable of percutaneous coronary intervention (PCI) for immediate mechanical revascularisation. Successful treatment requires rapid return of perfusion to the myocardium achieved by thromboaspiration, passivation of the culprit lesion with stent scaffolding and systemic inhibition of thrombosis and platelet activation. A delicate balance exists between thrombosis and bleeding and consequently anti-thrombotic and antiplatelet treatment regimens continue to evolve. The desire to achieve reperfusion as soon as possible, in the setting of high platelet reactivity, requires potent and fast-acting anti-thrombotic/anti-platelet therapies. The associated bleeding risk may be minimised by use of short-acting anti-thrombotic intravenous agents. However, effective oral platelet inhibition is required to prevent recurrent thrombosis. The interaction between baseline platelet reactivity, timing of revascularisation and effective inhibition of thrombosis is yet to be formally investigated. METHODS/DESIGN: We present a protocol for a prospective observational study in patients presenting with acute STEMI treated with primary PCI (PPCI) and receiving bolus/infusion bivalirudin and prasugrel therapy. The objective of this study is to describe variation in platelet reactivity, as measured by the multiplate platelet function analyser, at presentation, the end of the PPCI procedure and 1, 2, & 24 hours post-procedure. We intend to assess the prevalence of high residual platelet reactivity within 24 hours of PPCI in acute STEMI patients receiving prasugrel and bivalirudin. Additionally, we will investigate the association between high platelet reactivity before and after PPCI and the door-to-procedure completion time.This is a single centre study with a target sample size of 108 participants. DISCUSSION: The baseline platelet reactivity on presentation with a STEMI may impact on the effect of acute anti-thrombotic and anti-platelet therapy and expose patients to a heightened risk of bleeding or ongoing thrombosis. This study will define the baseline variation in platelet reactivity in a population of patients experiencing acute STEMI and assess the pharmacodynamic response to combined treatment with bivalirudin and prasugrel. The data obtained from this trial will be hypothesis generating for future trials testing alternative pharmacotherapies in the acute phase of treatment for STEMI. TRIAL REGISTRATION: This study has approval from Wiltshire research ethics committee (10/H0106/87) and is registered with current controlled trials (http://www.controlled-trials.com/ISRCTN82257414).
Assuntos
Plaquetas/efeitos dos fármacos , Monitoramento de Medicamentos/métodos , Infarto do Miocárdio/terapia , Fragmentos de Peptídeos/uso terapêutico , Intervenção Coronária Percutânea , Piperazinas/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária , Sistemas Automatizados de Assistência Junto ao Leito , Projetos de Pesquisa , Tiofenos/uso terapêutico , Plaquetas/metabolismo , Protocolos Clínicos , Trombose Coronária/sangue , Trombose Coronária/etiologia , Trombose Coronária/prevenção & controle , Quimioterapia Combinada , Inglaterra , Hemorragia/induzido quimicamente , Hirudinas/efeitos adversos , Humanos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico , Fragmentos de Peptídeos/efeitos adversos , Intervenção Coronária Percutânea/efeitos adversos , Piperazinas/efeitos adversos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/efeitos adversos , Cloridrato de Prasugrel , Valor Preditivo dos Testes , Estudos Prospectivos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Tiofenos/efeitos adversos , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND AND PURPOSE: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. EXPERIMENTAL APPROACH: Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. KEY RESULTS: Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. CONCLUSION AND IMPLICATIONS: Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.
Assuntos
Síndrome Coronariana Aguda , Difosfatos , Humanos , Ticagrelor/farmacologia , Ticagrelor/metabolismo , Ticagrelor/uso terapêutico , Difosfatos/metabolismo , Difosfatos/farmacologia , Difosfatos/uso terapêutico , Adenosina/farmacologia , Agonismo Inverso de Drogas , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Difosfato de Adenosina/farmacologia , Difosfato de Adenosina/metabolismo , Plaquetas , Síndrome Coronariana Aguda/tratamento farmacológico , Síndrome Coronariana Aguda/complicações , Receptores Purinérgicos P2Y12/metabolismoRESUMO
BACKGROUND AND PURPOSE: Thromboxane A2 (TXA2) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. EXPERIMENTAL APPROACH: Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. KEY RESULTS: BRET studies revealed that F-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. CONCLUSION AND IMPLICATIONS: F-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.
RESUMO
We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.
Assuntos
Arrestina/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica/fisiologia , Receptores Purinérgicos P2Y12/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Arrestina/genética , Western Blotting , Linhagem Celular , Células Cultivadas , Endocitose , Humanos , Imunoprecipitação , Domínios PDZ/genética , Domínios PDZ/fisiologia , Fosfoproteínas/genética , Ligação Proteica/genética , RNA Interferente Pequeno , Receptores Purinérgicos P2Y12/genética , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit ß-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR â A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of ß-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.
Assuntos
Membrana Celular/metabolismo , Multimerização Proteica/fisiologia , Receptor PAR-2/metabolismo , Receptores de Trombina/metabolismo , Transdução de Sinais/fisiologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Membrana Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/fisiologia , Receptor PAR-2/genética , Receptores de Trombina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.
Assuntos
Plaquetas/metabolismo , Receptores Purinérgicos P2Y12/fisiologia , Sequência de Aminoácidos , Endocitose , Humanos , Dados de Sequência Molecular , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/efeitos dos fármacosRESUMO
The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.
Assuntos
Plaquetas/fisiologia , Domínios PDZ/fisiologia , Ativação Plaquetária/fisiologia , Transporte Proteico/fisiologia , Receptores Purinérgicos P2Y12/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Arrestina/metabolismo , Células CHO , Linhagem Celular Transformada , Clatrina/metabolismo , Cricetinae , Hemostasia/fisiologia , Humanos , Mutagênese/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genéticaRESUMO
BACKGROUND: Patients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. OBJECTIVES: To characterize the mechanism of altered platelet function in COVID-19 patients. METHODS: The platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. RESULTS: COVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbß3 activation and P-selectin expression. Agonist-stimulated integrin αIIbß3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. CONCLUSIONS: Overall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil-associated platelets. Platelet-driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.
Assuntos
COVID-19 , Trombose , Humanos , Proteoma/metabolismo , COVID-19/complicações , Proteômica , Fosfatidilserinas/metabolismo , Inflamação/metabolismo , Trombose/etiologia , Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ativação Plaquetária , Selectinas/metabolismoRESUMO
The activity and traffic of G-protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y(1) and P2Y(12) responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y(1) receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y(12) receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y(1) receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y(1) receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y(1) receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y(1) receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.
Assuntos
Receptores Purinérgicos P2/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Plaquetas/fisiologia , Humanos , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Nexinas de Classificação , Células Tumorais CultivadasRESUMO
Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the µ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.
Assuntos
Analgésicos Opioides/farmacologia , Oligopeptídeos/fisiologia , Receptores Opioides mu/agonistas , Receptores Opioides mu/fisiologia , Analgésicos Opioides/metabolismo , Animais , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Células HEK293 , Humanos , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos WistarRESUMO
As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field.