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Giuseppe De Luca,
Monica Verdoia,
Ettore Cassetti,
Alon Schaffer,
Gabriella Di Giovine,
Alessandra Bertoni,
Clara Di Vito,
Sara Sampietro,
Gianluca Aimaretti,
Giorgio Bellomo,
Paolo Marino, Fabiola Sinigaglia
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ABSTRACT: Platelets play a central role in the pathogenesis of coronary artery disease (CAD). Mean platelet volume (MPV) is an indicator of platelet activation, and has been demonstrated to be correlated with platelet reactivity. Diabetic patients have been shown to have larger MPV, that may contribute to higher platelet reactivity and atherothrombotic complications observed in these patients. Therefore, the aim of the current study was to investigate whether MPV is associated with platelet reactivity and the extent of CAD among diabetic patients. We performed a cohort study including 1016 consecutive diabetic patients undergoing coronary angiography at the University Hospital 'Maggiore della Carita', Novara, Italy. CAD is defined as stenosis above 50% in at least one coronary vessel at coronary angiography. Platelet reactivity was evaluated in 50 diabetic patients without history of CAD and who were free (in the past month) from medications which may affect platelet aggregation. Platelet aggregation was evaluated by light transmission aggregometry after stimulation with 1 μg/ml collagen type I. We additionally evaluated platelet surface expression of P-selectin after stimulation with U46619 (a stable synthetic analogue of the prostaglandin PGH2) and plasma concentration of thromboxane B2 (TxB2). Patients were grouped according to tertile values of MPV (<10.6 fl, group 1; 10.6-11.3 fl, group 2; >11.4 fl, group 3). MPV was associated with age (P = 0.011), baseline fasting glucose (P = 0.044), glycosylated haemoglobin (P = 0.005), creatinine (P = 0.052) and haemoglobin (P = 0.003), but inversely related to platelet count (P < 0.001) and triglycerides (P = 0.031). Larger MPV was associated with therapy with statins (P = 0.012) and diuretics (P = 0.021). CAD was observed in 826 patients (81.3%). MPV was not associated with the prevalence of CAD [odds ratio (OR), 0.85 (0.7-1.03), P = 0.11]. The results were confirmed in terms of severe CAD [OR, 1.03 (0.88-1.21), P = 0.7]. The absence of any significant relationship between MPV and CAD was confirmed after correction for baseline confounding factors [OR, 0.9 (0.75-1.08), P = 0.19]. Finally, MPV was not related to platelet reactivity. This is the first study showing that in diabetic patients MPV is not related to platelet reactivity and the prevalence and extent of CAD. Therefore, MPV may not be considered a risk factor for CAD among diabetic patients.
Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 05/2013; · 1.25 Impact Factor
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Paolo E Porporato,
Nicoletta Filigheddu,
Simone Reano,
Michele Ferrara,
Elia Angelino,
Viola F Gnocchi,
Flavia Prodam,
Giulia Ronchi,
Sharmila Fagoonee,
Michele Fornaro,
Federica Chianale,
Gianluca Baldanzi,
Nicola Surico, Fabiola Sinigaglia,
Isabelle Perroteau,
Roy G Smith,
Yuxiang Sun,
Stefano Geuna,
Andrea Graziani
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ABSTRACT: Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.
The Journal of clinical investigation 01/2013; · 15.39 Impact Factor
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ABSTRACT: Phytoestrogens are plant-derived polyphenolic compounds that exert beneficial effects on human health, mostly related to their estrogen mimetic activity. In particular a strong correlation between phytoestrogens intake and a lower risk of cardiovascular diseases has been reported. The flavanone 8-prenylnaringenin, extracted from hop flowers, has been identified as a novel phytoestrogen, unique with respect to estrogen receptors specificity and potency. However, to date no investigations on the 8-prenylnaringenin role in modulating platelet function have been undertaken.
We evaluated the effect of 8-prenylnaringenin on platelet aggregation, intracellular calcium mobilization and protein phosphorylation triggered by thrombin and collagen, and platelet adhesion and dense granule secretion triggered by collagen.
8-Prenylnaringenin inhibited platelet aggregation induced by different agonists and platelet adhesion to collagen matrix. 8-Prenylnaringenin directly increased intracellular cAMP and cGMP levels and thus promoted VASP phosphorylation. However, these molecular events were not responsible for the inhibitory action of 8-prenylnaringenin on platelets. Moreover, 8-prenylnaringenin inhibited the phosphorylation of Pyk2, Akt, and ERK1/2. Finally, 8-prenylnaringenin suppressed the mobilization of calcium and the secretion of dense granules. All these effects were independent of estrogen receptors recruitment.
8-Prenylnaringenin exerted anti-aggregatory and anti-adhesive effects on human platelets, independently of estrogen receptors, acting as an inhibitor of multiple proteins essential for the morphological and biochemical transformations that occur during platelet activation and aggregation.
8-Prenylnaringenin may represent a useful tool in the therapy and prevention of vascular diseases associated with platelet aggregation, such as atherosclerosis, myocardial infarction, coronary artery disease, and thrombosis.
Biochimica et Biophysica Acta 07/2012; 1820(11):1724-33. · 4.66 Impact Factor
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ABSTRACT: Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, are the most abundant steroids circulating in human blood. DHEA stimulates endothelial cells to release high amounts of nitric oxide in the circulation. Nitric oxide activates guanylyl cyclase in platelets thus decreasing the responsiveness of these cells to physiological agonists. However, the impact of DHEA-S and DHEA on platelet function and their possible role in modulating the response of human platelets to physiological agonists were not yet investigated. Here, DHEA-S, but not DHEA, inhibited in vitro thrombin-dependent platelet aggregation in a dose-dependent manner. DHEA-S exerted this effect by decreasing thrombin-dependent dense granule secretion, and so impairing the positive feed-back loop provided by ADP. Furthermore, DHEA-S inhibited thrombin-dependent activation of Akt, ERK1/2, and p38 MAP kinase. Although both DHEA-S and DHEA directly activated in platelets the inhibitory cGMP/PGK/VASP pathway, these events were not responsible for the inhibitory action of DHEA-S in platelets. In addition DHEA-S acted in synergism with nitric oxide in inhibiting platelet aggregation. In conclusion DHEA-S inhibited platelet activation caused by a mild stimulus without completely hampering platelet functionality and thus DHEA-S may participate in the physiological mechanisms that maintain circulating platelets in a resting state. The role played by DHEA-S could be relevant mainly when the functionality of the vascular endothelium is compromised.
Steroids 12/2011; 77(3):260-8. · 2.83 Impact Factor
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Gianluca Baldanzi,
Andrea Pighini,
Valentina Bettio,
Elena Rainero,
Sara Traini,
Federica Chianale,
Paolo E Porporato,
Nicoletta Filigheddu,
Riccardo Mesturini,
Shuping Song,
Tamas Schweighoffer,
Laura Patrussi,
Cosima T Baldari,
Xiao-Ping Zhong,
Wim J van Blitterswijk, Fabiola Sinigaglia,
Kim E Nichols,
Ignacio Rubio,
Ornella Parolini,
Andrea Graziani
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ABSTRACT: Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.
The Journal of Immunology 11/2011; 187(11):5941-51. · 5.79 Impact Factor
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ABSTRACT: We demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) activated dose-dependently washed human platelets and increased intracellular calcium levels. Moreover 2-AG activated protein kinase C measured as p47pleckstrin phosphorylation. These parameters were prevented by the tromboxane A2 receptor antagonist SQ29548, by phospholipase C pathway (U73122) and protein kinase C (GF109203X) inhibitors. No effect on 2-AG-induced platelet activation and calcium elevation in the presence of inhibitors of fatty acid amide hydrolase or monoacylglycerol lipase was observed. In addition we have shown that 2-AG dose-dependently increased NO and cGMP levels. These effects were abolished by U73122, GF109203X, EGTA and the intracellular calcium chelator BAPTA/AM. Moreover, 2-AG enhanced eNOS activity through the phosphorylation of its positive regulatory residue ser1177 and by dephosphorylation of the negative one thr495. The eNOS ser1177 phosphorylation was inhibited by U73122 and GF109203X but it was unaffected by the PI3K/AKT pathway inhibitors LY294002 and MK2206. The dephosphorylation of thr495 was reversed by low concentrations of calyculin A. Taken together these data suggest that 2-AG behaves as a true platelet agonist stimulating PKC activation and calcium elevation. Likely 2-AG can modulate platelet activation by increasing NO levels through eNOS activation.
Current neurovascular research 06/2011; 8(3):200-9. · 3.23 Impact Factor
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Federica Chianale,
Elena Rainero,
Cristina Cianflone,
Valentina Bettio,
Andrea Pighini,
Paolo E. Porporato,
Nicoletta Filigheddu,
Guido Serini, Fabiola Sinigaglia,
Gianluca Baldanzi,
Andrea Graziani
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ABSTRACT: Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into phosphatidic acid (PA), acting as molecular switches between
DAG- and PA-mediated signaling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKα
are required for growth-factor-induced cell migration and ruffling, through the control of Rac small-GTPase activation and
plasma membrane localization. Herein we unveil a signaling pathway through which DGKα coordinates the localization of Rac.
We show that upon hepatocyte growth-factor stimulation, DGKα, by producing PA, provides a key signal to recruit atypical PKCζ/ι
(aPKCζ/ι) in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. Then, DGKα-dependent activation
of aPKCζ/ι mediates the release of Rac from the inhibitory complex with RhoGDI, allowing its activation and leading to formation
of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKα as the central
element of a lipid signaling pathway linking tyrosine kinase growth-factor receptors to regulation of aPKCs and RhoGDI, and
providing a positional signal regulating Rac association to the plasma membrane.
Proceedings of the National Academy of Sciences 03/2010; 107(9):4182-4187. · 9.68 Impact Factor
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Federica Chianale,
Elena Rainero,
Cristina Cianflone,
Valentina Bettio,
Andrea Pighini,
Paolo E Porporato,
Nicoletta Filigheddu,
Guido Serini, Fabiola Sinigaglia,
Gianluca Baldanzi,
Andrea Graziani
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ABSTRACT: Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into phosphatidic acid (PA), acting as molecular switches between DAG- and PA-mediated signaling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKalpha are required for growth-factor-induced cell migration and ruffling, through the control of Rac small-GTPase activation and plasma membrane localization. Herein we unveil a signaling pathway through which DGKalpha coordinates the localization of Rac. We show that upon hepatocyte growth-factor stimulation, DGKalpha, by producing PA, provides a key signal to recruit atypical PKCzeta/iota (aPKCzeta/iota) in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. Then, DGKalpha-dependent activation of aPKCzeta/iota mediates the release of Rac from the inhibitory complex with RhoGDI, allowing its activation and leading to formation of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKalpha as the central element of a lipid signaling pathway linking tyrosine kinase growth-factor receptors to regulation of aPKCs and RhoGDI, and providing a positional signal regulating Rac association to the plasma membrane.
Proceedings of the National Academy of Sciences 02/2010; 107(9):4182-7. · 9.68 Impact Factor
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British Journal of Haematology 12/2009; 149(1):150-2. · 4.94 Impact Factor
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ABSTRACT: The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(2) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB(2) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.
Journal of Molecular Medicine 10/2008; 87(1):65-74. · 4.67 Impact Factor
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Federica Chianale,
Santina Cutrupi,
Elena Rainero,
Gianluca Baldanzi,
Paolo E Porporato,
Sara Traini,
Nicoletta Filigheddu,
Viola F Gnocchi,
Massimo M Santoro,
Ornella Parolini,
Wim J van Blitterswijk, Fabiola Sinigaglia,
Andrea Graziani
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ABSTRACT: Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.
Molecular Biology of the Cell 01/2008; 18(12):4859-71. · 4.94 Impact Factor
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Nicoletta Filigheddu,
Viola F Gnocchi,
Marco Coscia,
Miriam Cappelli,
Paolo E Porporato,
Riccardo Taulli,
Sara Traini,
Gianluca Baldanzi,
Federica Chianale,
Santina Cutrupi,
Elisa Arnoletti,
Corrado Ghè,
Alberto Fubini,
Nicola Surico, Fabiola Sinigaglia,
Carola Ponzetto,
Giampiero Muccioli,
Tiziana Crepaldi,
Andrea Graziani
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ABSTRACT: Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.
Molecular Biology of the Cell 04/2007; 18(3):986-94. · 4.94 Impact Factor
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ABSTRACT: The impact of estrogens on the viability of cardiovascular system and their ability to regulate platelet function is still an open and debated question. We have previously shown that estrogen is able to significantly potentiate the aggregation induced by low doses of thrombin and to initiate a rapid and reversible signaling pathway mediated by ERbeta-directed activation of the tyrosine kinases Src and Pyk2 at the level of the plasma membrane. Lipid rafts are critical, cholesterol-enriched membrane domains, which play a major role in blood platelet activation processes. In this work, we investigated the role of lipid rafts in 17beta-estradiol signaling in human platelets. We observed that membrane rafts were essential for both 17beta-estradiol-dependent potentiation of platelet aggregation induced by subthreshold concentrations of thrombin and 17beta-estradiol-induced phosphorylation of Src. 17beta-estradiol caused the reversible translocation of ERbeta to the raft fractions and promoted the rapid and transient recruitment to, and activation within the membrane raft domains of the tyrosine kinases Src and Pyk2. The raft integrity was essential with this respect, as these effects of 17beta-estradiol were completely inhibited by cholesterol depletion. This paper provides evidence for the first time that membrane lipid rafts coordinate estrogen signaling in human platelets.
Biochimica et Biophysica Acta 03/2007; 1773(2):273-8. · 4.66 Impact Factor
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ABSTRACT: Circulating HGF is significantly increased in a number of thrombus-associated disorders. Since platelets play a pivotal role in thrombogenesis, the ability of HGF to interact with human platelets was investigated. This paper shows for the first time that human platelets express HGF receptor, the tyrosine kinase encoded by c-MET gene. At physiological concentrations HGF was found to inhibit both glycoprotein (alpha)IIb(beta)3 activation and thrombin-dependent platelet aggregation in a dose- and time-dependent manner. These results suggest that circulating HGF may counteract thrombogenesis by negatively modulating platelet functions.
FEBS Letters 09/2005; 579(20):4550-4. · 3.54 Impact Factor
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ABSTRACT: Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.
Journal of Biological Chemistry 08/2005; 280(26):24386-95. · 4.77 Impact Factor
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ABSTRACT: Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists,
including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the Gq-coupled P2Y1 purinergic receptor but not upon inhibition of the Gi-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of Gz by epinephrine but not upon co-stimulation of Gq by the thromboxane analogue U46619. Platelet aggregation induced by TPO and Gi stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP,
TPO was unable to induce integrin αIIbβ3 activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of
integrin αIIbβ3 was blocked by antagonists of the Gq-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out
activation of integrin αIIbβ3 induced by TPO and Gi stimulation occurred independently of thromboxane A2 production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and Gi activation of integrin αIIbβ3 was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that
TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon
blockade of the Gq-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent
on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon
blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and
P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results
indicate that TPO can integrate Gi, but not Gq, stimulation and can efficiently support integrin αIIbβ3 activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol
3-kinase and the small GTPase Rap1B.
Journal of Biological Chemistry 06/2005; 280(26):24386-24395. · 4.77 Impact Factor
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Laura Moro,
Stefania Reineri,
Daniela Piranda,
Daniela Pietrapiana,
Paolo Lova,
Alessandra Bertoni,
Andrea Graziani,
Paola Defilippi,
Ilaria Canobbio,
Mauro Torti, Fabiola Sinigaglia
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ABSTRACT: The impact of estrogens on the cardiovascular system and their ability to regulate platelet function are matters of controversy. The recent finding that estrogen receptors are expressed in human platelets renders these cells an excellent model for studying the nongenomic effects of these hormones. In this work, we investigated 17beta-estradiol-dependent signaling in platelets from adult healthy men. 17beta-estradiol caused the rapid phosphorylation of the tyrosine kinases Src and Pyk2 and the formation of a signaling complex, which included Src, Pyk2, and the phosphatidylinositol 3-kinase. Both these events were dependent on estrogen receptor beta engagement. We found that estrogen receptor beta was membrane-associated in platelets. On treatment with 17beta-estradiol, Src and Pyk2 activation occurred in the membrane fraction but not in the cytosol. In contrast, no significant activation of phosphatidylinositol 3-kinase was detected in estrogen-treated platelets. 17beta-estradiol did not induce any platelet response directly, but it strongly potentiated the activation of integrin alpha(IIb)beta3 and the platelet aggregation induced by subthreshold concentrations of thrombin. These effects were dependent on estrogen receptor beta recruitment and were associated with a strong synergistic effect with thrombin on Src activation. Taken together, these results indicate that 17beta-estradiol can modulate platelet function by exercising a proaggregating role.
Blood 02/2005; 105(1):115-21. · 9.90 Impact Factor
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ABSTRACT: Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.
Oncogene 07/2004; 23(28):4828-38. · 6.37 Impact Factor
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[hide abstract]
ABSTRACT: Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.
Journal of Biological Chemistry 07/2004; 279(24):25299-306. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and
PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced
activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4
by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1-
and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced
the increase of intracellular Ca2+ concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses
of agonists able to elicit comparable intracellular Ca2+ increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or
phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited.
By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant
stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B.
A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human
platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation
of platelets with the anti-GPIbα monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally
abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular
portion of GPIbα by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase
and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be
activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers.
These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this
GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.
Journal of Biological Chemistry 06/2004; 279(24):25299-25306. · 4.77 Impact Factor
