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Biomedical subjects

M Torti

Publications and source records attributed to M Torti.

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

Activation of poly(ADP-ribose)polymerase in apoptotic human cells.

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible in HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation process. To this purpose, we have analysed, by means of the activity gel technique; the structural and catalytical properties of poly(ADP-ribose)polymerase. In extracts from cells where etoposide-induced DNA fragmentation occurred, we have shown that the label of the autoribosylated form of the enzyme is greatly increased even if the amount of the protein remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inhibitory effect on the endonuclease(s) responsible for the fragmentation of DNA during apoptosis. After incubation of cells with zinc/VP-16 we have found the occurrence of apoptotic parameters even in the absence of internucleosomal DNA cleavage. The inhibition of DNA fragmentation prevents the activation of poly(ADP-ribose)polymerase activity. These results indicate that the activation of the enzyme towards the automodification reaction is strictly dependent on the appearance of DNA internucleosomal fragments and could represent a way to control enzyme activity.

Adenosine Monophosphate↗
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

Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets.

Thrombin activates phospholipase C in human platelets, but the specific isoenzymes activated and the signal pathway used are unknown. Using specific antibodies, we found that phospholipase C-gamma 1 and the p21ras GTPase-activating protein, rasGAP, are present in human platelets. Furthermore, phospholipase C-gamma 1 was detectable, based on enzyme activity and Western blot analysis, in immunoprecipitates of rasGAP, suggesting that these two proteins form tight complexes. The pool of phospholipase C-gamma 1 associated with rasGAP was phosphorylated but not through tyrosine phosphorylation. Although thrombin stimulation had no effect on the level of phosphorylation of phospholipase C-gamma 1 and only slightly increased the tyrosine phosphorylation of rasGAP, the agonist induced the association of rasGAP with rap1B, as indicated by the appearance of rap1B on a Western blot of rasGAP immunoprecipitates. Our results suggest the formation of a signaling complex involving rasGAP, phospholipase C-gamma 1, and rap1B that might be important in the cascade leading to platelet activation.

Blood Platelets↗
PubMed1992

Defect of platelet aggregation and adhesion induced by autoantibodies against platelet glycoprotein IIIa.

A young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient's plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient's plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.

Adult↗
PubMed1992

Epinephrine induces association of pp60src with Gi alpha in human platelets.

Using specific antibodies against the alpha subunit of the inhibitory GTP-binding protein Gi, we analyzed the association of Gi alpha with other cellular components in human platelets. Three tyrosine phosphorylated proteins with molecular mass of 63, 58, and 55 kDa were specifically associated with Gi alpha in resting platelets. Stimulation of platelets with epinephrine, but not with thrombin, induced an increase of the reactivity of the 63- and 55-kDa proteins to anti-phosphotyrosine antibodies on western blotting. By in vitro kinase assay we found that epinephrine induced the association of kinase activity with Gi alpha and that the 63-kDa protein was phosphorylated by this activity. The association of kinase activity with Gi alpha in epinephrine-stimulated platelets paralleled the association of pp60src with Gi alpha, as detected by western blotting analysis using specific anti-pp60src monoclonal antibodies. The interaction of pp60src with Gi alpha may play a role in the mechanism of platelet activation by epinephrine or in the epinephrine-induced potentiation of the action of other platelet agonists.

Blood Platelets↗
PubMed1992

Erythropoietin rapidly induces tyrosine phosphorylation in the human erythropoietin-dependent cell line, UT-7.

UT-7 is a human megakaryoblastic cell line capable of growing in interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (Epo) (Cancer Res 51:341, 1991). We used this cell line and a selected Epo-dependent subcell line (UT-7/Epo) to study the early signal transduction events induced by Epo. When UT-7 cells were exposed to Epo, tyrosine phosphorylation of several proteins (with molecular weight equivalent to that of p85, p110, and p145) was observed. Protein phosphorylation occurred in both a dose- and time-dependent manner. p85 showed a marked increase in phosphotyrosine content within 30 seconds; maximal phosphorylation was observed at 1 minute. Subsequently, tyrosine phosphorylation of p110 and p145 was observed, beginning at 1 minute and reaching plateau at 5 minutes. The degree of phosphorylation of these three proteins gradually decreased thereafter. In addition, in UT-7/Epo cells, Epo induced tyrosine phosphorylation of other proteins that were not observed in Epo-induced UT-7 cells. The concentration of Epo required to induce tyrosine phosphorylation was in the same range of concentration required to stimulate cell growth. Epo was also able to activate p21ras as measured by exchange of guanosine diphosphate for guanosine triphosphate. These data show that tyrosine phosphorylation and P21ras activation are early signals in the Epo-induced mitogenic pathway.

Erythropoietin↗
PubMed1992

Erythropoietin induces p21ras activation and p120GAP tyrosine phosphorylation in human erythroleukemia cells.

Erythropoietin is the major regulator of the proliferation and differentiation of erythroid precursors, but little is known about its molecular mechanism of action. Using a human erythroleukemic cell line (HEL), we investigated whether p21ras is involved in erythropoietin signal transduction. We found that stimulation of HEL cells with erythropoietin induces a 5-fold increase in the amount of GTP bound to the endogenous p21ras. This effect is dose-dependent and occurs very rapidly. We also observed that erythropoietin causes tyrosine phosphorylation of several proteins in a time-dependent manner that correlates with the p21ras activation. Moreover, inhibition of tyrosine kinases by genistein totally prevents the erythropoietin-induced accumulation of a p21ras.GTP complex. By using an antiserum against the GTPase-activating protein, we found that p120GAP is rapidly phosphorylated in tyrosine in response to erythropoietin. Furthermore, the ability of a lysate from erythropoietin-stimulated HEL cells to induce in vitro hydrolysis of GTP bound to p21ras was strongly reduced. These results demonstrate that activation of p21ras is an early event in the erythropoietin signal transduction pathway, and they suggest that accumulation of the p21ras.GTP complex may be triggered by inhibition of GTPase-activating protein activity.

Electrophoresis, Polyacrylamide Gel↗
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↗
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↗
PubMed1989

The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets.

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.

Blood Platelets↗
PubMed1988

Interrelation of platelet aggregation, release reaction and thromboxane A2 production.

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.

Adenosine Diphosphate↗
PubMed1988

Effect of GPIIb-IIIa complex ligands on calcium ion movement and cytoskeleton organization in activated platelets.

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.

Adenosine Diphosphate↗