RESUMO
A metabolically protected analog of substance P, [pGlu5-MePhe8-MeGly9]SP(5-11) (DiMe-C7), was approximately equipotent with substance P in causing increased locomotor activity after microinfusion into the ventral tegmental area of rat brain, but the effects of DiMe-C7 on behavior were considerably prolonged. There was little metabolic degradation of tritiated DiMe-C7 for up to 1 hour after infusion, whereas tritiated substance P was completely degraded within 10 minutes.
Assuntos
Encéfalo/metabolismo , Fragmentos de Peptídeos , Substância P/análogos & derivados , Animais , Comportamento Animal/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Substância P/metabolismo , Substância P/farmacologiaRESUMO
Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.
Assuntos
Colágeno/metabolismo , Ativação Plaquetária , Proteínas Tirosina Quinases/fisiologia , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/sangue , Plaquetas/metabolismo , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Fosfolipase C gama , Fosforilação , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismoRESUMO
The adapter protein SLP-76 is expressed in T lymphocytes and hematopoietic cells of the myeloid lineage, and is known to be a substrate of the protein tyrosine kinases that are activated after ligation of the T-cell antigen receptor. Transient overexpression of SLP-76 in a T-cell line potentiates transcriptional activation after T-cell receptor ligation, while loss of SLP-76 expression abrogates several T-cell receptor-dependent signaling pathways. Mutant mice that lack SLP-76 manifest a severe block at an early stage of thymocyte development, implicating SLP-76 in signaling events that promote thymocyte maturation. While it is clear that SLP-76 plays a key role in development and activation of T lymphocytes, relatively little is understood regarding its role in transducing signals initiated after receptor ligation in other hematopoietic cell types. In this report, we describe fetal hemorrhage and perinatal mortality in SLP-76-deficient mice. Although megakaryocyte and platelet development proceeds normally in the absence of SLP-76, collagen-induced platelet aggregation and granule release is markedly impaired. Furthermore, treatment of SLP-76-deficient platelets with collagen fails to elicit tyrosine phosphorylation of phospholipase C-gamma2 (PLC-gamma2), suggesting that SLP-76 functions upstream of PLC-gamma2 activation. These data provide one potential mechanism for the fetal hemorrhage observed in SLP-76-deficient mice and reveal that SLP-76 expression is required for optimal receptor-mediated signal transduction in platelets as well as T lymphocytes.
Assuntos
Plaquetas/metabolismo , Feto/anormalidades , Hemorragia/patologia , Fosfoproteínas/deficiência , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Plaquetas/citologia , Células da Medula Óssea/citologia , Colágeno/farmacologia , Precursores Enzimáticos/metabolismo , Histocitoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Fosfolipase C gama , Fosforilação , Fosfotirosina/análise , Agregação Plaquetária/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Quinase Syk , Fosfolipases Tipo C/metabolismoRESUMO
BACKGROUND: We previously demonstrated that Dok2 is rapidly phosphorylated on tyrosine residues in platelets in response to thrombin, the immunoreceptor tyrosine-based activation motif-coupled collagen receptor glycoprotein (GP) VI, and by integrin alphaIIbbeta3. OBJECTIVES AND METHODS: In this study we further delineate the regulation of phosphorylation of Dok2 and compare this to the related adapter Dok1. RESULTS: We demonstrate expression of Dok1 in platelets and the unexpected observation that the adapter protein undergoes tyrosine phosphorylation in response to thrombin but not to GPVI or integrin alphaIIbbeta3. Furthermore, Dok1 phosphorylation is transient, peaking at 30 s and returning to basal by 5 min, whereas Dok2 phosphorylation is delayed but sustained. Dok2 phosphorylation, but not that of Dok1, is inhibited by Src kinase inhibitors and by chelation of intracellular calcium. Further, phosphorylation of Dok2 by thrombin and integrin alphaIIbbeta3 in mouse platelets is independent of Syk and phospholipase Cgamma2. Additionally, Dok2 coimmunoprecipitates with integrin alphaIIbbeta3 downstream of Src kinases. CONCLUSIONS: These results demonstrate differential modes of regulation of Dok1 and Dok2 in platelets. Further, they raise the interesting possibility that Dok2 plays an important role in integrin outside-in signaling through a physical and functional interaction with integrin alphaIIbbeta3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Animais , Plaquetas , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Transdução de SinaisRESUMO
BACKGROUND: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRgamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). OBJECTIVE: To determine why GPVI-FcRgamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. METHODS AND RESULTS: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcRgamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. CONCLUSION: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcRgamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.
Assuntos
Células Sanguíneas/metabolismo , Plaquetas/metabolismo , Colágeno/fisiologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de IgG/metabolismo , Receptores Imunológicos/fisiologia , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Glicoproteínas da Membrana de Plaquetas/genética , Ratos , Receptores Imunológicos/genética , Transdução GenéticaRESUMO
BACKGROUND: The small GTPase Rac1 plays a critical role in lamellipodia assembly in platelets on matrix proteins in the absence or presence of G protein-coupled receptor (GPCR) agonists. Rac mediates actin assembly via Scar/WAVE, a family of scaffolding proteins that direct actin reorganization by relaying signals from Rac to the Arp2/3 complex. OBJECTIVE: To evaluate the role of Scar/WAVE-1 in mediating platelet activation and cytoskeletal reorganization. METHODS AND RESULTS: Using specific antibodies, we demonstrate that murine platelets, like human platelets, express Scar/WAVE-1 and Scar/WAVE-2. Lamellipodia formation in Scar/WAVE-1(-/-) platelets is markedly inhibited on immobilized collagen-related peptide (CRP) and on laminin, both of which signal through the collagen receptor GPVI. In contrast, lamellipodia formation on collagen, which requires release of the GPCR agonists ADP and thromboxane A(2), is not altered. Immobilized fibrinogen supports limited formation of lamellipodia in murine platelets, which is not altered in Scar/WAVE-1(-/-) platelets. As with Rac1(-/-) platelets, Scar/WAVE-1(-/-) platelets exhibit a marked inhibition of aggregation in response to CRP, whereas the response to the GPCR agonist thrombin is not altered. Platelet aggregation on immobilized collagen under shear, which is dependent on signaling by matrix and GPCR agonists, was unaltered in the absence of Scar/WAVE-1. CONCLUSION: This study demonstrates a major role for Scar/WAVE-1 in mediating platelet cytoskeletal reorganization and aggregate formation downstream of activation by GPVI but not by GPCR agonists.
Assuntos
Plaquetas/metabolismo , Citoesqueleto/metabolismo , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/deficiência , Difosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Fibrinogênio/metabolismo , Hemorreologia , Humanos , Técnicas In Vitro , Laminina/metabolismo , Camundongos , Camundongos Knockout , Selectina-P/metabolismo , Peptídeos/metabolismo , Pseudópodes/metabolismo , Estresse Mecânico , Trombina/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
BACKGROUND: MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cell's contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation. OBJECTIVE: To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin). RESULTS: Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model. CONCLUSIONS: MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.
Assuntos
Miosina não Muscular Tipo IIA/biossíntese , Miosina não Muscular Tipo IIA/fisiologia , Actinas/metabolismo , Amidas/farmacologia , Plaquetas/citologia , Plaquetas/metabolismo , Movimento Celular , Colágeno/química , Colágeno/metabolismo , Citoesqueleto/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Modelos Biológicos , Cadeias Leves de Miosina/química , Fosforilação , Agregação Plaquetária , Piridinas/farmacologia , Trombose/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Plaquetas/fisiologia , Proteínas de Transporte/metabolismo , Integrinas/metabolismo , Isoenzimas/metabolismo , Proteínas de Membrana , Fosfoproteínas/metabolismo , Ativação Plaquetária/fisiologia , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismo , Animais , Plaquetas/metabolismo , Ativação Enzimática , Humanos , Camundongos , Fosfolipase C gama , Fosforilação , Receptores de ColágenoRESUMO
Essentials There is a clinical need for new technologies to measure platelet function in whole blood. Mild bleeding disorders were evaluated using multiple electrode aggregometry (MEA). MEA is insensitive at detecting patients with mild platelet function and secretion defects. More studies are required to investigate MEA in patients with a defined set of platelet disorders. SUMMARY: Background Multiple electrode aggregometry (MEA) measures changes in electrical impedance caused by platelet aggregation in whole blood. This approach is faster, more convenient and offers the advantage over light transmission aggregometry (LTA) of assessing platelet function in whole blood and reducing preanalytical errors associated with preparation of platelet-rich plasma (PRP). Several studies indicate the utility of this method in assessing platelet inhibition in individuals taking antiplatelet agents (e.g. aspirin and clopidogrel). Objective Our current study sought to evaluate the ability of MEA in diagnosing patients with mild bleeding disorders by comparison with light transmission lumi-aggregometry (lumi-LTA). Methods Forty healthy subjects and 109 patients with a clinical diagnosis of a mild bleeding disorder were recruited into the UK Genotyping and Phenotyping of Platelets study (GAPP, ISRCTN 77951167). MEA was performed on whole blood using one or two concentrations of ADP, PAR-1 peptide, arachidonic acid and collagen. Lumi-LTA was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Results Of 109 patients tested, 54 (49%) patients gave abnormal responses by lumi-LTA to one or more agonists. In contrast, only 16 (15%) patients were shown to have abnormal responses to one or more agonists by MEA. Conclusions In this study we showed that MEA is less sensitive in identifying patients with abnormal platelet function relative to lumi-LTA.
Assuntos
Transtornos da Coagulação Sanguínea/diagnóstico , Agregação Plaquetária , Testes de Função Plaquetária/métodos , Adolescente , Adulto , Idoso , Transtornos da Coagulação Sanguínea/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Impedância Elétrica , Eletrodos , Feminino , Humanos , Luz , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Testes de Função Plaquetária/instrumentação , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Reino Unido , Adulto JovemRESUMO
Essentials Dimeric high-affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets. Spatio-temporal organization of platelet GPVI-dimers was evaluated using advanced microscopy. Upon platelet adhesion to collagenous substrates, GPVI-dimers coalesce to form clusters. Clustering of GPVI-dimers may increase avidity and facilitate platelet activation SUMMARY: Background Platelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets. Objective To identify higher-order oligomerization (clustering) of pre-existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signaling. Methods GPVI was located by use of fluorophore-conjugated GPVI dimer-specific Fab (antigen-binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI-specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real-time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α2 ß1 , and phosphotyrosine. Results Upon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen-adhered platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI dimers or fibrinogen-induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α2 ß1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton. Conclusions Platelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI-associated signaling molecules, which may be crucial for the initiation and persistence of signaling.
Assuntos
Plaquetas/metabolismo , Colágeno/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Actinas/metabolismo , Vasos Sanguíneos/lesões , Adesão Celular , Citoesqueleto/metabolismo , Humanos , Microscopia Confocal , Ativação Plaquetária , Adesividade Plaquetária , Multimerização Proteica , Transdução de SinaisRESUMO
BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.
Assuntos
Plaquetas/fisiologia , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Pseudópodes/metabolismo , Fator de von Willebrand/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Benzodiazepinas/farmacologia , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Citoesqueleto , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Pseudópodes/efeitos dos fármacos , Ristocetina/farmacologia , Transdução de Sinais , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP , Quinases da Família src/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
The transmembrane adapter LAT (linker for activation of T cells) plays a central role in signalling by ITAM bearing receptors expressed on T cells, natural killer cells, mast cells and platelets. Receptor engagement leads to the phosphorylation of tyrosine residues present in the intracellular domain of LAT and formation of a multiprotein complex with other adapter molecules and enzymes including Grb2, Gads/SLP-76 and PLCgamma isoforms. These signalling events predominantly take place in glycolipid-enriched membrane domains. The constitutive presence of LAT in GEMs enables its function as the main scaffolding protein for the organization of GEM-localized signalling. The study of LAT-deficient mice and LAT-deficient cell lines further emphasizes the importance of LAT for these signalling cascades but also defines the existence of LAT-independent events downstream of the Syk-family kinase-ITAM complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Membrana , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Transdução de Sinais , Animais , Linfócitos B/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Linfócitos T/metabolismo , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismoRESUMO
Stimulation of HEL megakaryocytic cells by Fc gammaRIIA crosslinking is associated with tyrosine phosphorylation of syk and phospholipase C gamma2 (PLCgamma2) and is accompanied by formation of inositol phosphates and release of intracellular Ca2+. These responses are inhibited by the kinase inhibitors, staurosporine and ST271. In contrast, the G-protein receptor agonist, thrombin induces formation of inositol phosphates and release of intracellular calcium without an increase in tyrosine phosphorylation. The plant lectin wheat germ agglutinin (WGA) stimulates tyrosine phosphorylation of syk and PLCgamma2 but surprisingly does not stimulate formation of inositol phosphates and induce release of intracellular Ca2+. WGA also inhibited formation of inositol phosphates and release of intracellular Ca2+ by Fc gammaRIIA crosslinking and thrombin-stimulation. A similar inhibitory effect of WGA was observed against elevation of Ca2+ by the same two stimuli in MEG-01 megakaryotic cells. The results demonstrate a novel pathway of inhibition of PLC on crosslinking of cell surface proteins that is not present in platelets.
Assuntos
Fosfolipases Tipo C/antagonistas & inibidores , Aglutininas do Germe de Trigo/farmacologia , Antígenos CD , Cálcio/metabolismo , Linhagem Celular , Reagentes de Ligações Cruzadas , Humanos , Fosfatos de Inositol/metabolismo , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de IgG , Trombina , Células Tumorais CultivadasRESUMO
Ins(1,4,5)P3 receptors in adrenal cortical and cerebellar membranes can be distinguished by their affinities for Ins(1,4,5)P3 as well as the potencies with which heparin and Mg2+ inhibit binding. We have found that the differences in Ins(1,4,5)P3 affinity and heparin inhibition are maintained upon receptor solubilization and purification. In contrast to this, heparin-agarose affinity purification of solubilized cerebellar receptors reduces the potency of Mg2+ inhibition to that in adrenal cortex. These results suggest that Ins(1,4,5)P3 receptors in adrenal cortex are structurally distinct from those in cerebellum. Monoclonal antibodies raised against C- and N-terminal regions of mouse cerebellar Ins(1,4,5)P3 receptors recognize 250-300-kDa proteins in both rat cerebellum and bovine adrenal cortex.
Assuntos
Córtex Suprarrenal/metabolismo , Encéfalo/metabolismo , Canais de Cálcio , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares , Animais , Bovinos , Heparina/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Magnésio/farmacologia , Masculino , Polietilenoglicóis , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/isolamento & purificaçãoRESUMO
Cell surface receptors are targets for the pharmacological manipulation of physiological processes and thus represent a key direction for the development of selective therapeutic agents. Traditional pharmacological techniques, together with the development of synthetic ligands, have led to the identification of differences in receptor recognition properties and the proposal of multiple receptor subtypes. Molecular biological studies have confirmed the existence of receptor subtypes within a single species by demonstrating differences in receptor primary sequences. However, equivalent receptors between species also show differences in primary structure, albeit to a much lower degree. This review by Judith Hall and colleagues addresses the question of how differences in receptor primary structure between species relate to changes in pharmacology. The relevance of this to the choice of screens in the testing of potential therapeutic drugs is discussed.
Assuntos
Receptores de Superfície Celular/efeitos dos fármacos , Animais , Desenho de Fármacos , Humanos , Especificidade da EspécieRESUMO
Preterm labour is a major cause of perinatal mortality and morbidity, but its prevention is difficult because most of the available drugs lack uterine selectivity and have potentially serious side-effects for the mother or the foetus. In this article, Andrés López Bernal and colleagues discuss new evidence that shows pregnancy is associated with changes in G protein signalling and second messenger formation in human myometrium. During gestation uterine relaxation is favoured by a pronounced increase in G alpha s levels, thereby facilitating the effect of agonists that increase cAMP formation. The change in G alpha s is reversed in spontaneous labour enabling the uterus to become responsive to contractile agents. Although it is not established that these changes in G protein function are causally related to the spontaneous onset of labour, nevertheless they provide a novel viewpoint towards increased understanding of the cellular mechanisms of uterine contractility, which may result in better drugs for the management of preterm labour.
Assuntos
Trabalho de Parto Prematuro/prevenção & controle , Feminino , Proteínas de Ligação ao GTP/fisiologia , Humanos , Miométrio/metabolismo , Trabalho de Parto Prematuro/metabolismo , Gravidez , Estimulação QuímicaRESUMO
Proteomic technology has the potential to transform the way we analyze platelet biology, through the determination of platelet protein composition and its modification upon stimulation and with disease. We are a considerable way from achieving these goals, however, because of significant limitations in current methodology. It is therefore important to consider the extent to which these aims can be met and the way that proteomic data should be presented and used. These issues are discussed in the present paper by the Platelet Physiology Subcommittee of the ISTH Scientific Standardisation Committee (SSC). It is recommended that proteomic information be combined with data from other experimental approaches to establish a database on protein expression and function in platelets.
Assuntos
Plaquetas/química , Proteoma , Bases de Dados de Proteínas , Guias como Assunto , Humanos , Proteômica/métodosRESUMO
This review summarizes recent developments in our understanding of the molecular basis of platelet activation by two distinct types of surface receptor, the immunoglobulin GPVI, and the integrin alphaIIb beta3 (also known as GPIIbIIIa). These two classes of receptor signal through similar yet distinct tyrosine kinase-based signaling cascades leading to activation of phospholipase C gamma2. The significance of these signaling cascades in platelet adhesion and platelet aggregation at arterial rates of shear is discussed.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Animais , Colágeno/metabolismo , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Biológicos , Dados de Sequência Molecular , Ativação Plaquetária , Adesividade Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Quinases da Família src/metabolismoRESUMO
Phospholipase C gamma (PLCgamma) isoforms are critical for the generation of calcium signals in haematopoietic systems in response to the stimulation of immune receptors. PLCgamma is unique amongst phospholipases in that it is tightly regulated by the action of a number of tyrosine kinases. It is itself directly phosphorylated on a number of tyrosines and contains several domains through which it can interact with other signalling proteins and lipid products such as phosphatidylinositol 3,4,5-trisphosphate. Through this network of interactions, PLCgamma is activated and recruited to its substrate, phosphatidylinositol 4,5-bisphosphate, at the membrane. Both isoforms of PLCgamma, PLCgamma1 and PLCgamma2, are present in haematopoietic cells. The signalling cascade involved in the regulation of these two isoforms varies between cells, though the systems are similar for both PLCgamma1 and PLCgamma2. We will compare these cascades for both PLCgamma1 and PLCgamma2 and discuss possible reasons as to why one form of PLCgamma and not the other is required for signalling in specific haematopoietic cells, including T lymphocytes, B lymphocytes, platelets, and mast cells.
Assuntos
Células Sanguíneas/fisiologia , Isoenzimas/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Linfócitos B/imunologia , Plaquetas/fisiologia , Isoenzimas/química , Substâncias Macromoleculares , Camundongos , Modelos Biológicos , Fosfolipase C gama , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Fosfolipases Tipo C/químicaRESUMO
BACKGROUND: The C-type lectin-like receptor 2 (CLEC-2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen-activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC-2 signaling is not known. OBJECTIVES: We sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC-2. RESULTS: The CLEC-2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal-related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC-2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31-8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/ß (GSK3α/ß). Phosphorylation of GSK3α/ß was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/ß inhibitors. CONCLUSIONS: These results demonstrate that CLEC-2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/ß, and enhanced platelet aggregation and secretion.