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ABSTRACT: The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.
Journal of Biological Chemistry 02/2003; 278(1):131-8. · 4.77 Impact Factor
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Gianluca Baldanzi,
Nicoletta Filigheddu,
Santina Cutrupi,
Filomena Catapano,
Sara Bonissoni,
Alberto Fubini,
Daniela Malan,
Germano Baj,
Riccarda Granata,
Fabio Broglio,
Mauro Papotti,
Nicola Surico,
Federico Bussolino,
Jorgen Isgaard,
Romano Deghenghi, Fabiola Sinigaglia,
Maria Prat,
Giampiero Muccioli,
Ezio Ghigo,
Andrea Graziani
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ABSTRACT: Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein-coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.
The Journal of Cell Biology 01/2003; 159(6):1029-37. · 10.26 Impact Factor
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ABSTRACT: Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcgammaRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca(2+) concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcgammaRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the G(i)-coupled ADP receptor P2Y12, but was not affected by blockade of the G(q)-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca(2+) mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca(2+) signaling. In platelets stimulated by cross-linking of FcgammaRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G(i)-coupled alpha(2A)-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G(q)-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G(q)-coupled thromboxane A(2) receptor by was completely inhibited by ADP scavengers under conditions in which intracellular Ca(2+) mobilization was unaffected. Inhibition of -induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G(i)-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.
Journal of Biological Chemistry 05/2002; 277(14):12009-15. · 4.77 Impact Factor
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ABSTRACT: Stimulation of human platelets with von Willebrand factor (vWF) induces the rapid tyrosine phosphorylation of several proteins, but very little is known on the tyrosine kinases involved in this process. In the present work, we investigated and compared the activation of two related tyrosine kinases expressed in platelets: the proline-rich tyrosine kinase 2 (Pyk2) and the focal adhesion kinase (FAK). Both kinases were tyrosine phosphorylated upon vWF interaction with glycoprotein Ib-IX-V complex, but with different mechanisms. Tyrosine phosphorylation of FAK was totally dependent on thromboxane A2 production, and was inhibited by the integrin alphaIIbeta3 antagonist RGDS peptide. Moreover, chelation of intracellular calcium or inhibition of protein kinase C (PKC) totally blocked vWF-induced tyrosine phosphorylation of FAK, indicating that this event is downstream phospholipase A2 and phospholipase C activation. By contrast, tyrosine phosphorylation of Pyk2 was only partially reduced by aspirin and RGDS, and was not affected by either calcium chelation or PKC inhibition, suggesting that activation of this kinase does not require phospholipase-mediated signalling. Both FAK and Pyk2 translocated to the cytoskeleton upon vWF stimulation of human platelets by a mechanism depending on agonist-induced actin polymerisation. Prevention of cytoskeletal relocation of Pyk2 and FAK by cytochalasin D totally blocked vWF-induced tyrosine phosphorylation of both kinases. Finally, phosphorylation of Pyk2 induced by vWF, but not by thrombin, was inhibited by piceatannol, suggesting that this kinase lies downstream Syk. These results demonstrate that both Pyk2 and FAK are involved in platelet stimulation by vWF, but indicate that only Pyk2 may play a role in the early signal transduction events activated by ligand binding to glycoprotein Ib-IX-V.
Thrombosis and Haemostasis 04/2002; 87(3):509-17. · 5.04 Impact Factor
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ABSTRACT: Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined
mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, FcγRIIA, in GPIb-IX-V
signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of FcγRIIA by a Src kinase.
Treatment of platelets with the anti-FcγRIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced
pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets,
lacking FcγRIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets
stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid
and synthesis of TxA2induced by vWF are not affected by the anti-FcγRIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation
of FcγRIIA, as well as of Syk and PLCγ2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by
piceatannol does not affect vWF-induced tyrosine phosphorylation of FcγRIIA but prevents phosphorylation of PLCγ2. Pleckstrin
phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin
phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLCγ2
plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through
endogenous TxA2 and FcγRIIA.
Journal of Biological Chemistry 07/2001; 276(28):26022-26029. · 4.77 Impact Factor
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ABSTRACT: The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPS. We also found that polyhistidine-tagged rap2 immobilized on Ni2+-Sepharose and loaded with either GDP or GTPS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein. J. Cell. Biochem. 75:675–685, 1999. © 1999 Wiley-Liss, Inc.
Journal of Cellular Biochemistry 12/1999; 75(4):675 - 685. · 2.87 Impact Factor
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ABSTRACT: Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.
Biochemical and Biophysical Research Communications.
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ABSTRACT: We studied the influence of the occupancy of the fibrinogen receptor (GP IIb-IIIa complex)on two early aspects of agonist induced platelet activation: the increase of the intracellular Ca2+ concentration and the cytoskeleton reorganization. A monoclonal antibody, a peptide containing the RGD sequence and fibrinogen purified from human plasma were used as GP IIb-IIIa ligands. The obtained results demonstrated that fibrinogen receptor occupancy inhibits Ca2+ movement and cytoskeleton reorganization caused by low thrombin concentration and ADP.
Biochemical and Biophysical Research Communications 154(1):258-264. · 2.48 Impact Factor
