Impact of diabetes on fibrinogen levels and its relationship with platelet reactivity and coronary artery disease: A single-centre study.

BACKGROUND: Previous reports have suggested an association between elevated fibrinogen and CAD. Few studies have so far investigated the impact of diabetes on fibrinogen levels and its association with coronary artery disease (CAD) and platelet reactivity in diabetic patients that are therefore the aims of the current study. METHODS: We measured fibrinogen in 3280 consecutive patients undergoing coronary angiography. Samples were collected at admission for fibrinogen levels assessment. Coronary disease was defined for at least 1 vessel stenosis >50% as evaluated by QCA. RESULTS: Diabetes was observed in 1201 out of 3280 patients. Diabetic patients were older with more hypercholesterolemia, hypertension, higher BMI, more renal failure, previous MI or coronary revascularization (p<0.001, respectively) and smoking (p=0.001). Diabetic patients were more often on ACE-inhibitors, ARBs, b-blockers, calcium-antagonists, diuretics, statins (p<0.001, respectively), and ASA (p=0.004). Diabetic patients displayed higher glycaemia and HbA1c (p<0.001), higher creatinine and triglycerides (p<0.001) but lower total and HDL cholesterol (p<0.001) and haemoglobin (p<0.001). Diabetic patients had higher fibrinogen levels (p=0.003), however neither diabetes nor glucose homeostasis parameters resulted as independent predictors of hyperfibrinogenemia. Furthermore, among diabetic patients, higher fibrinogen levels did not affect platelet reactivity and were not associated with the prevalence of CAD (adjusted OR[95%CI]=0.99 [0.82-1.19], p=0.9). Similar results were found for severe CAD (adjusted OR[95%CI]=0.94 [0.82-1.08], p=0.40). CONCLUSIONS: Our study showed that diabetes and glycaemic control are not independent predictors of hyperfibrinogenemia. Among diabetic patients, elevated fibrinogen is not associated with platelet reactivity and the prevalence and extent of CAD.

Impact of age on mean platelet volume and its relationship with coronary artery disease: a single-centre cohort study.

UNLABELLED: Elderly patients represent a high risk category among subjects with atherosclerosis, due to the presence of comorbidities and suboptimal response to antiplatelet drugs. Mean platelet volume (MPV) has been indicated as a marker of platelet reactivity, with contrasting data on its role on coronary artery disease. Aim of the present study was to evaluate the impact of age on the MPV and its role on the extent of coronary artery disease (CAD). METHODS: Our population is represented by a cohort of 3750 patients undergoing coronary angiography. Elderly were defined according to age ≥ 75 years. MPV was measured at admission. Significant coronary artery disease was defined as a stenosis >50% in at least 1 coronary vessel, while severe CAD was defined as left main and/or three-vessel disease. RESULTS: A total of 1170 out of 3750 (31.2%) patients were ≥ 75 years old. Advanced age was associated with female gender (p<0.001), hypertension (p<0.001), renal failure (p<0.001), previous myocardial infarction (p=0.03) coronary artery bypass grafting (p<0.001) indication to angiography (p<0.001), therapy with angiotension-receptor blockers, (p=0.003), nitrates, diuretics and calcium-antagonists (p<0.001), serum creatinine (p<0.001), fibrinogen (p<0.001) and C reactive protein (p=0.02), but inversely to percutaneous coronary interventions (p=0.02), dyslipidemia, family history of CAD and smoking (p<0.001, respectively), use of statins (p=0.02) and beta blockers (p=0.003), haemoglobin, total cholesterol and triglycerides (p<0.001, respectively), white blood cells (p=0.009) and platelet count (p=0.006). Elderly patients displayed a significantly larger platelet volume (p<0.001), with a direct linear relationship between age and the MPV (r=0.08, p<0.001), with age being confirmed as an independent predictor of larger MPV (≥10.85fl) at multivariate analysis (adjusted OR [95% CI]=1.18 [1.01-1.40], p=0.04). Among the elderly, MPV value above the median (≥10.85fl) was not associated with a higher prevalence of coronary artery disease (77.3 vs. 79.4%, p=0.39, adjusted OR [95% CI]=0.94 [0.66-1.33], p=0.71), or higher prevalence of severe CAD (35.2 vs. 32.4%, p=0.28, adjusted OR [95% CI]=1.34 [0.99-1.82], p=0.06). CONCLUSION: Advanced age was directly associated with larger mean platelet volume that, however, did not contribute to explain the higher prevalence and extent of coronary artery disease observed in elderly patients.

Eosinophils count and periprocedural myocardial infarction in patients undergoing percutaneous coronary interventions.

BACKGROUND: Eosinophils have been involved in a wide spectrum of pro-inflammatory and pro-thrombotic conditions, with the development of cardiovascular complications in a significant proportion of hypereosinophilic patients. However, no study has so far evaluated the impact of eosinophils levels on periprocedural myocardial infarction (PMI) in patients undergoing non-urgent percutaneous coronary interventions (PCI), that was, then, aim of current study. METHODS: In a consecutive cohort of patients, myonecrosis biomarkers were dosed at intervals from 6 to 48 h after PCI. Periprocedural myonecrosis was defined as troponin I increase by 3 times the ULN or by 50% of an elevated baseline value, whereas PMI as CKMB increase by 3 times the ULN or 50% of baseline. RESULTS: Our population is represented by 1543 patients who were divided according to tertiles of absolute eosinophils count (AEC ≤ 0.1; 0.1-0.2; >0.2 × 10ˆ3/ml). Higher AEC was related to male gender (p = 0.002), arterial hypertension (p = 0.02), diabetes (p = 0.001), previous coronary revascularization (p = 0.003 for PCI, p = 0.03 for CABG), treatment with ARBs, beta-blockers, diuretics and ASA (p < 0.001), statins (p = 0.02), calcium antagonists (p = 0.05), glycosylated hemoglobin (p < 0001), creatinine levels (p = 0.001) and platelet count (p = 0.01), while inversely with acute presentation (p < 0.001), glycemia (p = 0.03), HDL-cholesterol and C-reactive protein (p = 0.02). AEC related with multivessel coronary artery disease (p = 0.05), lesion length (p = 0.01), drug eluting stents implantation (p = 0.001) and use of kissing balloon technique (p = 0.05), while inversely to intracoronary thrombus (p < 0.001) and thrombectomy (p = 0.04). AEC did not influence the occurrence of PMI (p = 0.06, adjusted OR [95% CI] = 1.06 [0.86-1.31], p = 0.57) or myonecrosis (p = 0.15, adjusted OR [95% CI] = 1.06 [0.88-1.27], p = 0.53). Results were confirmed at subgroup analysis in higher-risk subsets of patients. CONCLUSION: In patients undergoing non-urgent PCI, eosinophils levels are not associated with the occurrence of periprocedural myocardial infarction or myonecrosis.

The diacylglycerol kinase α/atypical PKC/ß1 integrin pathway in SDF-1α mammary carcinoma invasiveness.

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5ß1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of ß1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and ß1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - ß1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.

Impact of diabetes on uric acid and its relationship with the extent of coronary artery disease and platelet aggregation: a single-centre cohort study.

BACKGROUND: Serum uric acid (SUA) elevation has been associated with the main determinants of atherosclerosis and metabolic syndrome, although an independent relationship between SUA and coronary artery disease (CAD) has never been confirmed. Recent reports suggested a central role of SUA in diabetic patients, possibly being an early marker of impaired glucose metabolism and best predicting the risk of cardiovascular events in these patients. Aim of current study was to evaluate the relationship between diabetes and uric acid and its association with the extent of CAD and platelet aggregation among diabetics. METHODS: In diabetic patients undergoing coronary angiography, fasting samples were collected for uric acid levels assessment. Coronary disease was defined for at least 1 vessel stenosis>50% as evaluated by QCA. RESULTS: Diabetes was observed in 1173 out of 3280 (35.7%) diabetes was related to age, hypercholesterolemia, hypertension, BMI, renal failure, previous MI or coronary revascularization (p<0.001, respectively) and smoking (p=0.001). Diabetics were more frequently treated with ACE-inhibitors, ARBs, b-blockers, calcium-antagonists, diuretics, statins (p<0.001, respectively), and ASA (p=0.004). Diabetics displayed higher glycemia and HbA1c (p<0.001), higher creatinine and triglycerides (p<0.001) but lower total and HDL cholesterol (p<0.001) and haemoglobin (p<0.001). No significant difference was found in SUA levels between diabetic and non diabetic patients (p=0.09). In fact, we identified age, renal failure, hypertension, smoking, BMI, use of diuretics, statins, haemoglobin, triglycerides and HDL cholesterol levels as independent predictors of higher levels of uric acid (3rd tertile,≥6.7mg/dl or 0.39mmol/l). Among diabetic patients, no relationship was found between uric acid and the extent of coronary artery disease (p=0.27; adjusted OR [95%CI]=0.93 [0.76-1.1], p=0.48), or severe (LM-trivessel) CAD (P=0.05; adjusted OR [95%CI]=1.01 [0.86-1.18], p=0.94). Furthermore, SUA levels did not influence platelet aggregation. CONCLUSION: Ageing, BMI, renal failure, hypertension, smoking, use of statins and diuretics, haemoglobin, HDL cholesterol and tryglicerides levels but not diabetes or glycemic control are independent predictors of hyperuricemia. Among diabetic patients, higher SUA is not independently associated with the extent of CAD or with platelet aggregation.

Diabetes, glucose control and mean platelet volume: a single-centre cohort study.

UNLABELLED: Diabetes is a major determinant of cardiovascular risk, mainly due to higher prothrombotic status and enhanced platelet reactivity. Mean platelet volume (MPV) has been suggested as indicator of platelet reactivity and moreover, diabetics have been shown to have larger MPV. The aim of our study was to evaluate the impact of diabetes and glycemic control on MPV in a large cohort of patients. METHODS: Our population is represented by 3414 patients undergoing coronary angiography at Azienda Ospedaliera-Universitaria, "Maggiore della Carità", Novara, Italy. We obtained a fasting blood sample for glycemic assessment and for MPV evaluation. History of diabetes and pharmacological treatment, together with main cardiovascular risk factors were recorded. New diagnosis of diabetes was defined as nonfasting glucose >200mg/dL, fasting glucose ≥126mg/dL, or HbA1c >48mmol/L. RESULTS: Diabetes was observed in 1272 patients (37.2%). Diabetes was related to older age, waist circumference, arterial hypertension, smoking, hypercholesterolemia, renal failure, previous MI and PCI, therapy with ACE-inhibitors, ARBs, beta-blockers, diuretics, statins (respectively p<0.001) and ASA (p=0.004). Diabetics had lower haemoglobin (p<0.001), higher fibrinogen (p=0.001) and worst lipid profile (p<0.001). MPV was related with diabetes mellitus (p<0.001) and glycemic control (p=0.05; at linear regression r=0.07; p<0.001 for fasting glycaemia; r=0.09; p<0.001 for HbA1c, respectively). However, this relationship was not confirmed at multivariate analysis (OR[95%CI]=1.2[0.97-1.5], p=0.09 for diabetes, OR[95%CI]=1.05[0.96-1.15], p=0.25 for HbA1c). Independent predictors of MPV above median value (10.8fL) resulted to be age (OR[95%CI]=1.02[1.01-1.03], p=0.002), treatment with ARBs (OR[95%CI]=1.4[1.1-1.8], p=0.007) and haemoglobin levels (OR[95%CI]=1.2[1.15-1.23], p<0.001), while inverse relationship was found with total cholesterol (OR[95%CI]=0.99[0.99-1], p=0.002). CONCLUSION: Larger MPV is associated with ageing, treatment with ARBs, cholesterol and haemoglobin levels. Diabetes mellitus and glycemic control are not independently associated with larger platelet size.

Benefits from new ADP antagonists as compared with clopidogrel in patients with stable angina or acute coronary syndrome undergoing invasive management: a meta-analysis of randomized trials.

AIMS: New P2Y12 receptor inhibitors have provided new and more potent antiplatelet strategies, although raising several concerns on possible increase of bleedings. The aim of current meta-analysis was to evaluate the efficacy and safety of new adenosine diphosphate (ADP) receptor antagonists as compared with clopidogrel in elective or ACS patients managed invasively. METHODS AND RESULTS: Literature archives (Pubmed, EMBASE, Cochrane) and main scientific sessions abstracts were scanned for randomized trials comparing new ADP antagonists with clopidogrel in patients with acute coronary syndromes or stable angina. Primary endpoint was mortality. Secondary endpoints were: (1) nonfatal myocardial infarction (MI), (2) recurrent ischemia symptoms or ischemia-driven revascularization (RI/IDR), (3) stent thrombosis (ST), and (4) safety endpoints, defined as for TIMI major bleeding criteria. A total of 8 randomized clinical trials were finally included, for a total population of 67,851 patients. Mean follow-up was 7.6 months, ranging from 48 hours to 30 months. New ADP antagonists significantly reduced mortality {3.1% vs. 3.6%, odds ratio [OR] [95% confidence interval (CI)], 0.86 [0.79-0.94], P = 0.0008, P(het) = 0.18}, with greater impact of oral drugs. Similar benefits were found for MI [6.1% vs. 7%; OR (95% CI) (random-effect model) = 0.88 (0.79-0.98), P = 0.01, P(het) = 0.02], RI [2.7% vs. 3.1%; OR (95% CI) = 0.85 (0.77-0.93), P = 0.0005, P(het) = 0.09], or ST [1.1% vs. 1.7%; OR (95% CI) = 0.60 (0.51-0.71), P < 0.00001, P(het) = 0.13]. By meta-regression analysis, no relationship was observed between benefits in mortality, new MI, RI, and ST with new ADP antagonists and patients' risk profile [beta (95% CI) = -0.01 [-0.30 to 0.27], P = 0.94; beta (95% CI) = -0.05 [-1.49 to 1.43], P = 0.96); beta (95% CI) = 0.19 (-0.18 to 0.57), P = 0.31, and beta (95% CI) = -0.08 (-0.86 to 0.70), P = 0.84, respectively]. CONCLUSIONS: Present meta-analysis shows that the new ADP antagonists prasugrel, ticagrelor, and cangrelor are associated to significant reduction of mortality, reinfarction, RI, and ST respect to clopidogrel alone, without significant increase in bleeding complications.

A diet supplemented with ALA-rich flaxseed prevents cardiomyocyte apoptosis by regulating caveolin-3 expression.

AIMS: n-3 polyunsaturated fatty acids (PUFAs) induce beneficial effects on the heart, but the mechanisms through which these effects are operated are not completely clarified yet. Among others, cardiac diseases are often associated with increased levels of cytokines, such as tumour necrosis factor-α (TNF), that cause degeneration and death of cardiomyocytes. The present study has been carried out to investigate (i) the potential anti-apoptotic effects induced by the n-3 polyunsaturated α-linolenic acid (ALA) in experimental models of cardiac diseases characterized by high levels of TNF, and (ii) the potential role of caveolin-3 (Cav-3) in the mechanisms involved in this process. METHODS AND RESULTS: An ALA-rich flaxseed diet, administered from weaning to hereditary cardiomyopathic hamsters, prevented the onset of myocardial apoptosis associated with high plasma and tissue levels of TNF preserving caveolin-3 expression. To confirm these findings, isolated neonatal mouse cardiomyocytes were exposed to TNF to induce apoptosis. ALA pre-treatment greatly enhanced Cav-3 expression hampering the internalization of the caveolar TNF receptor and, thus, determining the abortion of the apoptotic vs. survival cascade. CONCLUSION: This study unveiled the Cav-3 pivotal role in defending cardiomyocytes against the TNF pro-apoptotic action and the ALA capacity to regulate this mechanism preventing cardiac degenerative diseases.

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice.

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 PI3Kß-, 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.

Alterations of negative regulators of cytokine signalling in immunodeficiency-related non-Hodgkin lymphoma.

We investigated immunodeficiency-related non-Hodgkin lymphoma for the presence of molecular alterations affecting negative regulators of the Janus family protein tyrosine kinase/signal transducer and activator of transcription pathway. Protein tyrosine phosphatase, non-receptor type 6/Src homology 2-containing tyrosine phosphatase-1 epigenetic silencing was recurrent in primary effusion lymphoma (100%), and diffuse large B-cell lymphoma (63%), with a higher prevalence in the non-germinal centre subtype, and was associated with the activation of the Janus family protein tyrosine kinase/signal transducer and activator of transcription 3 pathway. Suppressor of cytokine signalling (SOCS)1 and SOCS3 epigenetic silencing were occasionally detected, whereas SOCS1 was frequently mutated in diffuse large B-cell lymphoma and polymorphic post-transplant lymphoproliferative disorders, possibly as a cause of aberrant somatic hypermutation. However, the mutation profile of the coding region of the gene was different from that expected from the aberrant somatic hypermutation process, suggesting that, at least in some cases, SOCS1 mutations may have been selected for their functional activity.

The phytoestrogen 8-prenylnaringenin inhibits agonist-dependent activation of human platelets.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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.

Dehydroepiandrosterone-sulfate inhibits thrombin-induced platelet aggregation.

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.

SAP-mediated inhibition of diacylglycerol kinase α regulates TCR-induced diacylglycerol signaling.

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.

Expression of the endocannabinoid system in the bi-potential HEL cell line: commitment to the megakaryoblastic lineage by 2-arachidonoylglycerol.

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.

Diacylglycerol kinase-alpha mediates hepatocyte growth factor-induced epithelial cell scatter by regulating Rac activation and membrane ruffling.

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.

Ghrelin and des-acyl ghrelin promote differentiation and fusion of C2C12 skeletal muscle cells.

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.

Membrane lipid rafts coordinate estrogen-dependent signaling in human platelets.

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.

Met identification on human platelets: role of hepatocyte growth factor in the modulation of platelet activation.

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.