RESUMO
AIM OF THE STUDY: To present three pediatric cases of auditory brainstem implantation (ABI) and review literature data concerning this topic. PATIENTS: The first two children had a neurofibromatosis type II with bilateral sensorineural deafness; in both cases, the implant was inserted during the surgical removal of a vestibular schwannoma; the third patient had profound deafness due to bilateral cochlear nerve insufficiency associated with inner ear malformation. RESULTS: Two postoperative complications were observed: patient 1 had a persistent fever which required the replacement of the fat graft used to seal the translabyrinthine approach; patient 3 had a CSF leakage requiring additional surgery and lumbar external drainage. In our three patients, the numbers of active electrodes were 6/22 (Cochlear ABI 24M ABI), 11/12 (Medel Opus II ABI) and 11/12 (implant Medel), respectively. Due to additional major surgical procedures and to disappointing functional results of the ABI, patient 1 stopped wearing her implant 18 months after implantation. Nine months after surgery, patient 2 achieved open-set speech recognition and was very satisfied with the implant. Six months after implantation, patient 3 (cochlear nerve deficiency), who was 3.5 years-old at the time, clearly reacted to some environmental sounds but was not yet able to achieve speech recognition. CONCLUSIONS: ABI has now entered the list of treatments that can be proposed in pediatric profound sensorineural deafness. Its major risks of complications are CSF leakage and non-auditory side effects. Its outcomes are worse and less predictable than cochlear implants. Thus, its indications must remain restricted to cases meeting the following conditions: absence of alternative option to restore hearing, patients and parents high level of motivation and realistic expectations.
Assuntos
Implantes Auditivos de Tronco Encefálico , Tronco Encefálico/cirurgia , Neurofibromatose 2/cirurgia , Neuroma Acústico/cirurgia , Tronco Encefálico/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Neurofibromatose 2/diagnóstico , Neuroma Acústico/diagnóstico , Resultado do TratamentoAssuntos
Citoesqueleto de Actina/metabolismo , Plaquetas/metabolismo , Microscopia Confocal , Microtúbulos/metabolismo , Trombina/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Plaquetas/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina D/farmacologia , Camundongos , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Transdução de Sinais , Tiazolidinas/farmacologiaRESUMO
Targeted therapy is certainly considered the future of cancer treatment. Several new molecules targeting critical intracellular signaling actors, particularly kinases, are arriving in clinics and many other are under development. However, proteins targeted by these drugs are common to many cell types and are particularly implicated in the highly dynamic processes of platelet activation. Therefore, the effects of targeted drugs, including kinase inhibitors, on platelet activation have to be considered in clinical practice. Moreover, their analysis also represents an opportunity to increase our knowledge in platelet biology and physiology and to develop novel antiplatelet strategies. In this review we briefly describe the major platelet signaling pathways that may be affected by these new drugs and discuss some clinical implications of their use.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Terapia de Alvo Molecular , Ativação Plaquetária/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Dasatinibe , Humanos , Neoplasias/tratamento farmacológico , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologiaRESUMO
BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare but severe complication of heparin therapy in which immunoglobulin G (IgG) antibodies against the platelet factor 4-heparin complex activate platelets through the FcγRIIA receptor. Clustering of FcγRIIA initiates signaling cascades involving tyrosine kinases including the spleen tyrosine kinase (Syk). Moreover, besides the critical role of platelets, the expression of tissue factor (TF) by human monocytes triggered by HIT antibodies has been shown to contribute to the hypercoagulability and the thrombotic complications in HIT patients. OBJECTIVES: We investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B-cell related hematological malignancies, on FcγRIIA-mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies-induced expression of TF and procoagulant activity of monocytic cells. RESULTS: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA-induced LAT phosphorylation and activation of phosphoinositide 3-kinase, Akt, phospholipase Cγ2 and p38 MAP-kinase. As a consequence, FcγRIIA-induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. CONCLUSION: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies-mediated platelet activation and monocyte procoagulant activity.
Assuntos
Heparina/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Monócitos/metabolismo , Oxazinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Tromboplastina/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quinase SykAssuntos
Plaquetas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ticlopidina/análogos & derivados , Difosfato de Adenosina/farmacologia , Idoso , Clopidogrel , Feminino , Humanos , Masculino , Métodos , Pessoa de Meia-Idade , Fosforilação , Ticlopidina/administração & dosagem , Ticlopidina/farmacologiaRESUMO
The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.
Assuntos
Plaquetas/metabolismo , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Purinérgicos P2/fisiologia , Actinas/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , GTP Fosfo-Hidrolases/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Quinases Ativadas por p21RESUMO
Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.
Assuntos
Monofosfato de Adenosina/análogos & derivados , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Monofosfato de Adenosina/farmacologia , Animais , Apirase/farmacologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de VarreduraRESUMO
Over the last few years, many reports have extended our knowledge of the inositol lipid metabolism and brought out some exciting information about the location, the variety and the role of phosphoinositides (PIs). Besides the so-called "canonical PI pathway" leading to the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), the precursor of the intracellular second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG), many other metabolic pathways have been identified to produce seven different polyphosphoinositides. Several of these quantitatively minor lipid molecules appear to be specifically involved in the control of cellular events, such as the spatial and temporal organisation of key signalling pathways, the rearrangement of the actin cytoskeleton or the intracellular vesicle trafficking. This is consistent with the fact that many of the enzymes, such as kinases and phosphatases, involved in the tight control of the intracellular level of polyphosphoinositides, are regulated and/or relocated through cell surface receptors for extracellular ligands. The remarkable feature of PIs, which can be rapidly synthesised and degraded in discrete membrane domains or even subnuclear structures, places them as ideal regulators and integrators of very dynamic mechanisms of cell regulation. In this review, we will summarise recent studies on the potential location, the metabolic pathways and the role of the different PIs. Some aspects of the temporal synthesis of D3 PIs will also be discussed.
Assuntos
Fosfatidilinositóis/metabolismo , Transdução de Sinais , Animais , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Humanos , Metabolismo dos Lipídeos , Modelos Biológicos , Modelos Químicos , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
FcgammaRIIA, the only Fcgamma receptor present in platelets, is involved in heparin-associated thrombocytopenia (HIT). Recently, adenosine diphosphate (ADP) has been shown to play a major role in platelet activation and aggregation induced by FcgammaRIIA cross-linking or by sera from HIT patients. Herein, we investigated the mechanism of action of ADP as a cofactor in FcgammaRIIA-dependent platelet activation, which is classically known to involve tyrosine kinases. We first got pharmacologic evidence that the ADP receptor coupled to Gi was required for HIT sera or FcgammaRIIA clustering-induced platelet secretion and aggregation. Interestingly, the signaling from this ADP receptor could be replaced by triggering another Gi-coupled receptor, the alpha(2A)-adrenergic receptor. ADP scavengers did not significantly affect the tyrosine phosphorylation cascade initiated by FcgammaRIIA cross-linking. Conversely, the Gi-dependent signaling pathway, initiated either by ADP or epinephrine, was required for FcgammaRIIA-mediated phospholipase C activation and calcium mobilization. Indeed, concomitant signaling from Gi and FcgammaRIIA itself was necessary for an efficient synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger playing a critical role in the process of phospholipase Cgamma2 activation. Altogether, our data demonstrate that converging signaling pathways from Gi and tyrosine kinases are required for platelet secretion and aggregation induced by FcgammaRIIA.
Assuntos
Antígenos CD/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de IgG/fisiologia , Difosfato de Adenosina/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Anticoagulantes/efeitos adversos , Antígenos CD/farmacologia , Plaquetas/enzimologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Epinefrina/farmacologia , Proteínas de Ligação ao GTP/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Heparina/efeitos adversos , Humanos , Isoenzimas/metabolismo , Fosfatos de Fosfatidilinositol/biossíntese , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos alfa 2/fisiologia , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/efeitos dos fármacos , Trombocitopenia/induzido quimicamente , Trombocitopenia/patologia , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismoRESUMO
Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.
Assuntos
Plaquetas/fisiologia , Colágeno/farmacologia , Fosfatos de Inositol/sangue , Integrinas/sangue , Fosfatidilinositol 3-Quinases/sangue , Transdução de Sinais , Trombopoetina/farmacologia , Androstadienos/farmacologia , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Cinética , Ácidos Fosfatídicos/sangue , Fosfatidilinositóis/sangue , Agregação Plaquetária , Receptores de Colágeno , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombopoetina/fisiologia , WortmaninaRESUMO
Entry of the bacterial pathogen Listeria monocytogenes into non-phagocytic mammalian cells is mainly mediated by the InlB protein. Here we show that in the human epithelial cell line HEp-2, the invasion protein InlB activates sequentially a p85beta-p110 class I(A) PI 3-kinase and the phospholipase C-gamma1 (PLC-gamma1) without detectable tyrosine phosphorylation of PLC-gamma1. Purified InlB stimulates association of PLC-gamma1 with one or more tyrosine-phosphorylated proteins, followed by a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels and a release of intracellular Ca2+ in a PI 3-kinase-dependent manner. Infection of HEp-2 cells with wild-type L. monocytogenes bacteria also induces association of PLC-gamma1 with phosphotyrosyl proteins. This interaction is undetectable upon infection with a deltainlB mutant revealing an InlB specific signal. Interestingly, pharmacological or genetic inactivation of PLC-gamma1 does not significantly affect InlB-mediated bacterial uptake, suggesting that InlB-mediated PLC-gamma1 activation and calcium mobilization are involved in post-internalization steps.
Assuntos
Isoenzimas/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Proteínas de Membrana/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas de Bactérias , Cálcio/metabolismo , Ativação Enzimática , Células Epiteliais/microbiologia , Humanos , Listeria monocytogenes/fisiologia , Proteínas de Membrana/metabolismo , Fosfolipase C gama , Fosforilação , Células Tumorais Cultivadas , Tirosina/metabolismo , VirulênciaRESUMO
The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.
Assuntos
Plaquetas/enzimologia , Proteínas de Transporte , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Tirosina Quinase da Agamaglobulinemia , Androstadienos/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/fisiologia , Cálcio/metabolismo , Cromonas/farmacologia , Humanos , Integrinas/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/enzimologia , Megacariócitos/metabolismo , Camundongos , Morfolinas/farmacologia , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas/antagonistas & inibidores , Receptores de Colágeno , WortmaninaRESUMO
Flavonoids provide a large number of interesting natural compounds that are consumed daily and exhibit more or less potent and selective effects on some signaling enzymes as well as on the growth and proliferation of certain malignant cells in vitro. Among the identified signal transducers, phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) are now considered key players in many cellular responses including cell multiplication, apoptosis, and transformation. Despite their lack of strict specificity, some flavonoids provide valuable bases for the design of analogues that could be used to specifically block particular isoforms of PI 3-kinase or PKC and their downstream-dependent cellular responses.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Animais , Divisão Celular/efeitos dos fármacos , Humanos , Células Tumorais CultivadasRESUMO
Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.