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Biomedical subjects

C Balduini

Publications and source records attributed to C Balduini.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence for a glycoprotein IIb-IIIa- and aggregation-independent mechanism of phosphatidylinositol 3',4'-bisphosphate synthesis in human platelets.

The synthesis of phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2) in 32P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P2, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P2 induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P2 induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P2 was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P2 induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P2 production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P2. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P2 in thrombin-stimulated platelets.

Blood Platelets↗
PubMed1995

Oxidation state of glutathione and membrane proteins in human red cells of different age.

In this study the oxidation state of glutathione and membrane proteins was analyzed in red cells of different age in basal conditions. Red cells of different age were prepared by centrifugation and separated according to their density by two procedures: on self-forming gradients of autologous plasma (Murphy's procedure) and on discontinuous Stractan gradients. The efficiency of the two procedures in the isolation of senescent cells was compared. The results indicate that, despite the evidence that total cell GSH decreases with aging, its concentration, evaluated in the cell preparations of different ages, remains constant throughout the red cell life, when correlated with cell water content. Glutathione disulfide concentration increases with aging. The oxidation state of membrane proteins does not seem to change during the red cell life span.

Adult↗
PubMed1995

Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets.

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.

Blood Platelets↗
PubMed1994

Glycoprotein IIb-IIIa and the translocation of Rap2B to the platelet cytoskeleton.

The stimulation of human platelets with physiological agonists results in the incorporation of several proteins into the cytoskeleton, fibrinogen binding, and platelet aggregation. We recently demonstrated that the Ras-related low molecular weight GTP-binding protein Rap2B associates with the cytoskeleton in activated platelets and that this interaction requires platelet aggregation. In the present study we demonstrate that agonist-induced actin polymerization is necessary for the translocation of Rap2B to the cytoskeleton, suggesting that Rap2B interacts with the newly formed actin filaments. Moreover, the association of Rap2B with Triton X-100-insoluble material from platelets was totally blocked by treatment of intact platelets with monoclonal antibodies against the fibrinogen receptor glycoprotein IIb-IIIa. Platelets from patients affected by Glanzmann thrombastenia, a genetic disorder in which platelet plasma membranes lack glycoprotein IIb-IIIa but possess normal levels of Ras-related proteins, failed to incorporate Rap2B into the cytoskeleton upon activation by thrombin. Comparative immunoblotting revealed that the translocation of Rap2B to the cytoskeleton during platelet aggregation was accompanied by the simultaneous translocation of glycoprotein IIb-IIIa. Moreover, the cytoskeleton from aggregated platelets contained Rap2B and glycoprotein IIb-IIIa in comparable amounts. These results demonstrate the association of Rap2B and glycoprotein IIb-IIIa and their translocation to the cytoskeleton in aggregated human platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Effect of cAMP on the association of small GTP-binding proteins with the cytoskeleton of human platelets.

Following activation of human platelets changes in cytoskeletal organization occur: some proteins, which are present in the cytosol or membrane-associated in resting platelets, are recovered in the Triton-insoluble residue in activated cells. Assembly and disassembly of complex effector units on the membrane and inside cells is under the control of low molecular weight GTP-binding proteins, particularly those in the ras family. We investigated the interaction of small GTP-binding proteins with the platelet cytoskeleton and the effect of high cAMP levels on these interactions. At least two GTP-binding proteins of 24 and 28 kDa were detected in the Triton-insoluble residue of resting platelets. Stimulation of platelets with thrombin or concanavalin A (Con A), under non-aggregating conditions, resulted in increased 24 kDa protein-bound GTP, which also contained a significant amount of rap1B. High cAMP levels differently affected this interaction depending on the type of agonist used. cAMP increased association of G-proteins with the cytoskeleton following Con A-activation, while it decreased G-proteins interaction after thrombin stimulation. The activation did not influence the cAMP-dependent phosphorylation of rap1B. No phosphoprotein corresponding to rap1B could be detected in the Triton-insoluble residues, however. These findings could be related to the different mechanisms of cytoskeletal protein recruitment in platelets activated with either thrombin or Con A.

Blood Platelets↗
PubMed1993

Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation.

The intracellular distribution of the low-molecular-weight GTP-binding protein rap2B was investigated in resting and agonist-activated human platelets. In both cases, platelets were lysed by Triton X-100, and cell fractions were obtained by differential centrifugations. Using a specific polyclonal antiserum, we found that rap2B in resting platelets was completely detergent-soluble. When platelets were aggregated with thrombin, the thromboxane analogue U46619, or the Ca(2+)-ATPase inhibitor thapsigargin, a significant amount of rap2B became associated with the cytoskeleton. This association was paralleled by a decrease of rap2B in the Triton X-100-soluble fraction. Translocation of rap2B to the cytoskeleton strictly depended on platelet aggregation, and maximal incorporation was found when approximately 50% aggregation was measured. Inhibition of fibrinogen binding to the glycoprotein IIb-IIIa complex completely prevented the interaction of rap2B with the cytoskeleton. These results clearly demonstrate that changes in the intracellular localization of rap2B occur during platelet activation and represent evidence that this low molecular weight GTP-binding protein may be involved in platelet function.

Blood Platelets↗
PubMed1993

Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A-stimulated platelets.

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca2+. This effect was due to two different processes: Ca2+ mobilization from internal stores and Ca2+ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca2+ from internal storage sites occurred sooner than the opening of plasma membrane Ca2+ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca2+ concentration was antagonized and reversed by methyl alpha-D-mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl-concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl-concanavalin A, per se, was unable to induce Ca2+ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca2+ from the same internal stores from which Ca2+ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca2+ release after stimulation of platelets with ConA, ConA was able to cause a full discharge of Ca2+ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.

Blood Platelets↗
PubMed1992

Effect of oxygen tension and lactate concentration on keratan sulphate and chondroitin sulphate biosynthesis in bovine cornea.

Calf cornea slices were incubated with [U-14C]glucose, in varying pO2 or lactate concentrations. Acid glycosaminoglycans were separated by ion-exchange chromatography after papain digestion. The percentage radioactivity incorporated into keratan sulphate increased markedly with decreased oxygen tension, whereas a concomitant relative decrease of the biosynthesis of glycosaminoglycuronans occurred. Similar results were obtained with increased lactate concentration. Our findings support the idea that keratan sulphate is a functional substitute for chondroitin sulphate in conditions of oxygen lack (Scott, J.E. and Haigh, M. (1988) J. Anat. 158, 95-108).

Animals↗
PubMed1992

Stimulation of human platelets with concanavalin A involves phospholipase C activation.

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.

Biological Transport↗
PubMed1991

Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein.

Platelet membrane glycoprotein IIb-IIIa has been widely studied in the last years because of its role as an activation-dependent, adhesive protein receptor. Recently we demonstrated that occupancy of glycoprotein IIb-IIIa-receptor sites by specific ligands exerts an inhibitory effect on platelet responses induced by mild stimulation, leading us to suppose that this event may interact with activation pathways. Although the mechanisms of signal transduction in human platelets are not completely elucidated, the hypothesis that GTP-binding proteins are involved is generally accepted. Our results demonstrate that platelet ConA receptors, known to be located mainly on GP IIb-IIIa, are able to bind [35S]GTP gamma S; the GTP-binding activity is specific and is due to the association with the receptors of two G-proteins, with apparent molecular masses of 25 and 21 kDa, respectively. After the purification of GP IIb-IIIa, a glycoprotein complex electrophoretically pure was obtained that was still associated with a GTP-binding activity, migrating in SDS-polyacrylamide gel electrophoresis as a narrow band of about 21 kDa.

Autoradiography↗
PubMed1991

An atypical myeloproliferative disorder with high thrombotic risk and slow disease progression.

Among 761 consecutive patients with chronic myeloproliferative disorders (CMD), it was found that 18 (nine men and nine women) did not fulfill at presentation the established diagnostic criteria for the typical forms. In seven patients, the diagnosis of CMD was made on the basis of an intense and persistent thrombocytosis that complicated splenectomy. The other 11 patients had various combinations of the following signs suggesting CMD: splenomegaly, bone marrow myeloid hyperplasia and/or slight myelofibrosis, mild thrombocytosis and/or leukocytosis, and rare immature myeloid cells in the peripheral blood. All patients were younger than 46 years of age (median age, 31.5 years; range, 20 to 45 years). A major thrombotic event was the most frequent presenting feature (eight of 18 cases), and thrombotic complications supervened in seven of the eight splenectomized patients (six in the portal system), raising the overall rate of patients with thrombotic events in their history to 11 of 18. At a median follow-up of 50 months (range, 24 to 241 months), three patients had died of thrombotic complications (two after splenectomy). The 15 surviving patients had stable disease, and 12 of them were not receiving cytoreductive therapy. Spontaneous growth of circulating burst-forming units erythroid was demonstrated in one patient, and erythroid responsiveness to erythropoietin appeared higher than in the normal controls in four. Spontaneous in vitro platelet aggregation in whole blood and/or platelet-rich plasma was seen in five of seven patients. It was concluded that a difficult to identify, slowly progressive form of CMD occurs in young people, that it carries a high risk of thrombosis, and that splenectomy is a high risk procedure in these cases.

Adult↗
PubMed1991

Evidence for membrane protein oxidation during in vivo aging of human erythrocytes.

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evident in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Oxidative lesion to membrane proteins in senescent erythrocytes.

The oxidative lesion undergone by membrane proteins in senescent human erythrocytes was evaluated by assaying their MetSO and thiol group content in ghosts and the amount of a coumarinyl derivative of maleimide, the DACM, bound by individual membrane proteins after treatment of erythrocytes of different age with this reagent. Quantitation of MetSO content of ghost membranes indicates an increase of the oxidative state of membrane proteins from young to mature and senescent erythrocytes, while thiol group assay does not show significant differences among erythrocytes of different age. Quantitation of DACM bound in intact cells by individual membrane proteins shows a decreased accessibility of thiol groups of band 3 protein and of the main proteins of the membrane skeleton in senescent erythrocytes, and this could be partly due to oxidation. The decreased reactivity to DACM of senescent erythrocyte band 3 seems to concern thiols located on the cytoplasmic domain of this protein, since the anion channel binds the same amount of the anion transport inhibitor EM, in mature and senescent erythrocytes.

Cytoplasm↗
PubMed1989

Fibronectin modulates the activation of human platelets.

In this paper we studied the effect of plasma fibronectin on platelet responsiveness to low doses of thrombin or ADP. Fibronectin causes: (i) a significant lowered cytoplasmic calcium movement in platelets activated both with low doses of thrombin and with ADP, (ii) a lowered decrease of the cAMP level induced by low thrombin, but not by ADP, (iii) a dramatic decrease of protein phosphorylation in low thrombin-treated platelets. The results reported here demonstrate that plasma fibronectin behaves like an inhibitor of mild activations of human platelets; its effect is probably mediated by the glycoprotein llb-llla complex in thrombin activated platelets, whereas a different and unknown mechanism must be supposed to explain the results obtained with ADP-activated platelets.

Adenosine Diphosphate↗