Thromboxane A2 inhibition of SKCa after NO synthase block in rat middle cerebral artery

NO/prostanoid independent, EDHF-mediated hyperpolarization and dilation in rat middle cerebral arteries is mediated solely by endothelial cell IK(Ca). However, when the NO-pathway is also active, both SK(Ca) and IK(Ca) contribute to EDHF responses. As the SK(Ca) component can be inhibited by stimulation of thromboxane A(2) (TxA(2)) TP receptors and NO has the potential ability to inhibit thromboxane synthesis, we investigated whether TxA(2) might explain loss of functional input from SK(Ca) during NOS inhibition in cerebral arteries. EXPERIMENTAL APPROACH: Rat middle cerebral arteries were mounted in a wire myograph. Endothelium-dependent responses to the PAR2 agonist, SLIGRL were assessed as simultaneous changes in smooth muscle membrane potential and tension. KEY RESULTS: Responses were obtained in the presence of L-NAME as appropriate. Inhibition of TP receptors with either ICI 192,605 or SQ 29,548, did not affect EDHF mediated hyperpolarization and relaxation, but in their presence neither TRAM-34 nor apamin (to block IK(Ca) and SK(Ca) respectively) individually affected the EDHF response. However, in combination they virtually abolished it. Similar effects were obtained in the presence of the thromboxane synthase inhibitor, furegrelate, which additionally revealed an iberiotoxin-sensitive residual EDHF hyperpolarization and relaxation in the combined presence of TRAM-34 and apamin. CONCLUSIONS AND IMPLICATIONS: In the rat middle cerebral artery, inhibition of NOS leads to a loss of the SK(Ca) component of EDHF responses. Either antagonism of TP receptors or block of thromboxane synthase restores an input through SK(Ca). These data indicate that NO normally enables SK(Ca) activity in rat middle cerebral arteries.

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell-dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell-DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4(+) T cells. During inflammation, weak tetramer-binding TP-deficient CD4(+) T cells were preferentially expanded compared with TP-proficient CD4(+) T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4(+) T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4(+) T cells and with augmented accumulation of follicular helper T cells (TFH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4(+) T cell-DC interactions by TXA2-TP signaling improves the overall quality of adaptive immune responses.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs)

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets. We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -‘good’ outcome no relapse > 3 years; ‘poor’ outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in ‘good’ outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression. We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI2/TXA2 ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. © 2010 Elsevier B.V. All rights reserved.

Human group II 14 kDa phospholipase A2 activates human platelets

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

COX-derived prostanoid pathways in gastrointestinal cancer development and progression : novel targets for prevention and intervention

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies. COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE 2, which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA 2 in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD 2 and its metabolite 15d-PGJ2, PGF 1α and PGI 2. Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity.A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field. © 2011 Elsevier B.V.

Cyclooxygenase-2-linked attenuation of hypoxia-induced pulmonary hypertension and intravascular thrombosis

Exogenous prostacyclin is effective in reducing pulmonary vascular resistance in some forms of human pulmonary hypertension (PH). To explore whether endogenous prostaglandins played a similar role in pulmonary hypertension, we examined the effect of deleting cyclooxygenase (COX)-gene isoforms in a chronic hypoxia model of PH. Pulmonary hypertension, examined by direct measurement of right ventricular end systolic pressure (RVESP), right ventricular hypertrophy (n = 8), and hematocrit (n = 3), was induced by 3 weeks of hypobarichypoxia in wild-type and COX-knockout (KO) mice. RVESP was increased in wild-type hypoxic mice compared with normoxic controls (24.4 ± 1.4 versus 13.8 ± 1.9 mm Hg; n = 8; p < 0.05). COX-2 KO mice showed a greater increase in RVESP following hypoxia (36.8 ± 2.7 mm Hg; p < 0.05). Urinary thromboxane (TX)B2 excretion increased following hypoxia (44.6 ± 11.1 versus 14.7 ± 1.8 ng/ml; n = 6; p < 0.05), an effect that was exacerbated by COX-2 gene disruption (54.5 ± 10.8 ng/ml; n = 6). In contrast, the increase in 6-keto-prostacyclin1α excretion following hypoxia was reduced by COX-2 gene disruption (29 ± 3 versus 52 ± 4.6 ng/ml; p < 0.01). Tail cut bleed times were lower following hypoxia, and there was evidence of intravascular thrombosis in lung vessels that was exacerbated by disruption of COX-2 and reduced by deletion of COX-1. The TXA2/endoperoxide receptor antagonist ifetroban (50 mg/kg/day) offset the effect of deleting the COX-2 gene, attenuating the hypoxia-induced rise in RVESP and intravascular thrombosis. COX-2 gene deletion exacerbates pulmonary hypertension, enhances sensitivity to TXA2, and induces intravascular thrombosis in response to hypoxia. The data provide evidence that endogenous prostaglandins modulate the pulmonary response to hypoxia. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics.

Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

Atividade pró-trombótica da toxina ExoU de Pseudomonas aeruginosa detectada em modelos experimentais in vivo e in vitro

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes.

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin a(11b)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca2+ with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca2+ release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLC gamma 2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity. (c) 2005 Elsevier Inc. All rights reserved.

Les prostanoïdes contrôlent la circulation placentaire : implication dans la prééclampsie

Au cours de la grossesse, une perfusion placentaire adéquate est indispensable au bon développement du fœtus. Dans certaines maladies comme la prééclampsie, celle-ci est altérée, compromettant ainsi la vie du fœtus, mais aussi celle de sa mère. Le retrait du placenta mène à la disparition des symptômes de la prééclampsie, suggérant un rôle central de ce dernier dans la maladie. Le placenta étant dépourvu d’innervation autonome, le tonus vasculaire placentaire doit être sous le contrôle de facteurs humoraux et tissulaires. Les vaisseaux placentaires sont très réactifs aux prostanoïdes. Le rapport thromboxane A2 (TXA2)/prostacycline (PGI2) est fortement augmenté dans les placentas de grossesses avec prééclampsie. De plus, le taux d’isoprostane, marqueur du stress oxydatif, est accru dans les placentas de femmes avec prééclampsie. Finalement, la prééclampsie s’accompagne d’un stress oxydatif placentaire marqué. Les espèces réactives de l’oxygène sont connues d’une part, pour oxyder l’acide arachidonique (AA), formant ainsi des isoprostanes et d’autre part, pour augmenter la production de TXA2 dans différents tissus, suite à l’activation des cyclooxygénases (COXs). Nous proposons que : 1. les prostanoïdes sont parmi les molécules endogènes qui contrôlent le tonus vasculaire placentaire. 2. la maladie modifie la réponse aux isoprostanes dans les vaisseaux placentaires. 3. l’induction d’un stress oxydatif placentaire entraîne une réponse vasoactive par activation de la voie du métabolisme de l’AA. Nous avons tout d’abord montré, dans des placentas obtenus de grossesses normotensives, que l’U-46619, un mimétique de la TXA2, de même que l’isoprostane, 8-iso-prostaglandine E2 (8-isoPGE2), ont augmenté fortement la pression de perfusion dans les cotylédons perfusés in vitro et la tension dans les anneaux d’artères chorioniques suspendus dans des bains à organe isolé. En revanche, dans les artères chorioniques de placentas obtenus de grossesses avec prééclampsie, ces réponses étaient modifiées puisque la réponse maximale à l’U-46619 était augmentée et celle à la 8-isoPGE2 diminuée. D’autre part, nous avons montré que les réponses maximales aux deux prostanoïdes étaient augmentées dans les vaisseaux placentaires de grossesse normale ou avec prééclampsie issus d’une délivrance prématurée par rapport à ceux d’une délivrance à terme. Ceci suggère une évolution de la réactivité des artères placentaires au cours du 3e trimestre de grossesse. En outre, les vaisseaux placentaires ont répondu aux prostanoïdes de façon semblable qu’ils aient été issus d’un accouchement vaginal ou d’une césarienne élective. Ceci indique que les prostanoïdes placentaires n’interviennent pas dans le processus de délivrance. D’un autre côté, l’utilisation de bloqueurs spécifiques des récepteurs TP à la TXA2, le SQ29,548 et l’ICI192,605, et des récepteurs EP à la prostaglandine E2, l’AH6809, nous ont permis de mettre en évidence le fait que l’U-46619 et la 8-isoPGE2 pouvaient agir de façon non-sélective sur l’un ou l’autre des récepteurs. Ces résultats supportent donc nos 2 premières hypothèses : les prostanoïdes font partie des molécules endogènes qui peuvent contrôler le tonus vasculaire placentaire et la prééclampsie modifie la réponse aux isoprostanes dans les artères chorioniques d’une manière compatible avec l’augmentation de la production de ces substances qui elle, est probablement le résultat du stress oxydatif. En revanche, en ce qui concerne les substances capables de jouer la contrepartie vasodilatatrice, l’utilisation d’un inhibiteur des synthases de monoxyde d’azote, le L-NAME, et celle d’inhibiteurs des COXs, l’ibuprofène, l’indométacine et le N-2PIA, ne nous a pas permis de mettre en évidence un quelconque rôle du monoxyde d’azote ou des prostanoïdes vasodilatatrices à ce niveau. Finalement, nous avons montré que l’induction d’un stress oxydatif dans les cotylédons perfusés in vitro et les artères chorioniques entraînait une vasoconstriction marquée. Celle-ci semble résulter de l’action des prostanoïdes puisqu’un blocage des récepteurs TP ou des COXs diminuait significativement la réponse maximale au peroxyde d’hydrogène. Les prostanoïdes impliquées dans la réponse au stress oxydatif proviendraient essentiellement d’une activation des COXs puisque l’étude ne nous permet pas de conclure à une quelconque implication des isoprostanes dans cette réponse. Ces observations confirment donc notre hypothèse que, dans le placenta, le stress oxydatif possède des propriétés vasoactives par activation du métabolisme de l’AA. En résumé, les résultats obtenus dans les placentas de grossesses normotensives et avec prééclampsie suggèrent que les prostanoïdes sont des molécules d’importance dans la régulation du tonus vasculaire placentaire. Le fait que la prééclampsie modifie la réponse aux prostanoïdes pourrait expliquer pourquoi la perfusion placentaire est altérée chez ces patientes. En outre, il apparaît évident qu’il existe un lien étroit entre le stress oxydatif et la voie de synthèse des prostanoïdes placentaires. Cependant d’autres études sont nécessaires pour mieux comprendre la nature de ce lien, qui pourrait, d’une certaine façon, jouer un rôle important dans le développement de la prééclampsie.

Rôle de la PKC delta dans la fonction plaquettaire en réponse à la thrombine via le récepteur GPIb alpha

La famille des protéines kinases C (PKC) est essentielle pour la fonction plaquettaire en réponse à la thrombine qui signale et active les plaquettes via les proteases activated receptors (PAR-1 et PAR-4) et le GPIbα. Ces derniers constituent les récepteurs de moyenne/faible et de hautes affinités pour la thrombine, respectivement. L’isoforme PKCδ régule positivement ou négativement la fonction des plaquettes tout dépendamment de la nature du stimulus. Cependant, son importance dans la fonction plaquettaire en réponse à la thrombine en aval de la GPIbα reste inconnue. L’objectif principal de ce projet de doctorat était de déterminer l'implication de l'axe thrombine/GPIbα/PKCδ dans la fonction plaquettaire et d’évaluer le rôle de cet axe dans la régulation de la thrombose. Dans les plaquettes humaines, le prétraitement avec l'inhibiteur spécifique de la PKCδ δ(V1-1)TAT, a significativement potentialisé l'activation et l’agrégation des plaquettes en réponse à de faibles concentrations de α-thrombine, mais pas en réponse à la γ-thrombine ou aux agonistes des PARs. Ce phénomène de potentialisation a été associé à une sécrétion accrue de granules, de génération de thromboxane A2 (TXA2) et une phosphorylation de la PKCδ sur la Tyr311, qui ont toutes été prévenues par l’inhibition spécifique du GPIbα à l’aide d’un anticorps monoclonal bloquant. En outre, l'inhibition de la p38 MAPK, ERK1/2 et le TXA2 a inversé ce processus de potentialisation. Les plaquettes murines déficientes en PKCδ étaient aussi plus réactives à la thrombine et ont montré une augmentation significative de l'agrégation, alors qu’une étude menée in vivo chez la souris PKCδ- /- a montré, suite à une stimulation par α-thrombine, une réaction thrombotique accrue caractérisée par une diminution significative du temps de saignement ainsi qu’une formation de thrombo-embolies pulmonaires. En bloquant le GPIbα, ces effets ont été renversés. Cette étude ouvre de nouvelles perspectives quant au rôle de la PKCδ dans les plaquettes en aval de GPIbα, où elle régule négativement la fonction plaquettaire en réponse à la thrombine. Ainsi, l'axe thrombine/GPIbα/PKCδ dans les plaquettes pourrait représenter un régulateur critique de la fonction plaquettaire et l'hémostase, et le dysfonctionnement de cette voie pourrait conduire à des événements thrombotiques.

Alteraciones respiratorias durante el sueño en niños con Síndrome Down a la altura de Bogotá

Objetivo: determinar la frecuencia de las diferentes alteraciones respiratorias durante el sueño (ARS) e hipertensión pulmonar (HTP) y establecer la saturación de oxígeno (SpO2) en vigilia, sueño y durante los eventos en niños con Síndrome Down (SD) a la altura de Bogotá (2640m) de acuerdo a grupos de edad e IMC. Métodos: estudio descriptivo de corte transversal, se incluyeron todos los niños con SD con sospecha de ARS remitidos a polisonograma (PSG) de octubre de 2011 a enero de 2013 a la Fundación Neumológica Colombina (FNC). Se dividieron en 3 grupos: apnea obstructiva, apnea obstructiva y central, sin apneas. Resultados: 74 niños, el 36,5% mujeres, edad media 4 años. 47,3% presento apnea obstructiva, más frecuente en >2 años, 35,1% apnea obstructiva y central, más frecuente en < 2 años y 17,6 % sin apnea. SpO2 promedio en apnea obstructiva 84,63%, apnea obstructiva y central: 81,8% y sin apnea: 86,85% (p 0,058). 23% presento obesidad, 16% con apnea obstructiva. 53 pacientes tenían ecocardiograma: 28% HTP, 53,3% tuvo apnea obstructiva y 26,7 apnea obstructiva y central, no diferencias significativas. SpO2 promedio en HTP 88,3% vigilia, 86,2% sueño REM, 85,7 % sueño no REM, no diferencia significativa comparada con pacientes sin HTP. Conclusiones: Las ARS son frecuentes en los niños con SD, La desaturación está presente en los niños con SD independiente del tipo de apnea. A todos los niños SD se les debe practicar un PSG en el primer año de vida.

A structural basis for the inhibition of collagen-stimulated platelet function by quercetin and structurally related flavonoids

Background and purpose: Molecular mechanisms underlying the links between dietary intake of flavonoids and reduced cardiovascular disease risk are only partially understood. Key events in the pathogenesis of cardiovascular disease, particularly thrombosis, are inhibited by these polyphenolic compounds via mechanisms such as inhibition of platelet activation and associated signal transduction, attenuation of generation of reactive oxygen species, enhancement of nitric oxide production and binding to thromboxane A2 receptors. In vivo, effects of flavonoids are mediated by their metabolites, but the effects and modes of action of these compounds are not well-characterized. A good understanding of flavonoid structure–activity relationships with regard to platelet function is also lacking. Experimental approach: Inhibitory potencies of structurally distinct flavonoids (quercetin, apigenin and catechin) and plasma metabolites (tamarixetin, quercetin-3′-sulphate and quercetin-3-glucuronide) for collagen-stimulated platelet aggregation and 5-hydroxytryptamine secretion were measured in human platelets. Tyrosine phosphorylation of total protein, Syk and PLCγ2 (immunoprecipitation and Western blot analyses), and Fyn kinase activity were also measured in platelets. Internalization of flavonoids and metabolites in a megakaryocytic cell line (MEG-01 cells) was studied by fluorescence confocal microscopy. Key results: The inhibitory mechanisms of these compounds included blocking Fyn kinase activity and the tyrosine phosphorylation of Syk and PLCγ2 following internalization. Principal functional groups attributed to potent inhibition were a planar, C-4 carbonyl substituted and C-3 hydroxylated C ring in addition to a B ring catechol moiety. Conclusions and implications: The structure–activity relationship for flavonoids on platelet function presented here may be exploited to design selective inhibitors of cell signalling.