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F M Pulcinelli

Publications and source records attributed to F M Pulcinelli.

At least 37 records · Page 2Linked to original sources
PubMed1998

Shape change is independent of tyrosine phosphorylation of p130 in human platelets.

It has been previously suggested that tyrosine phosphorylation of p62, p68, and p130 might be necessary for the platelet shape change to occur. In preliminary studies we observed that high concentrations (30 microM) of a protein kinase C inhibitor, Ro 31-8220, selectively suppressed p130 tyrosine phosphorylation induced by thrombin, the thromboxane synthetic analogue (U46619) and ADP. Therefore, we have investigated the correlation, if any, between p130 tyrosine phosphorylation and platelet shape change induced by the same agonists in the presence of Ro 31-8220. Our results demonstrated that high concentrations of this compound almost completely abolished p130 tyrosine phosphorylation, whereas they had no effect on platelet shape change, thus proving a dissociation between these two phenomena. Our data support the hypothesis that a role in platelet shape change might be played by tyrosine phosphorylation of proteins other than p130.

Blood Platelets↗
PubMed1998

Concomitant activation of Gi and Gq protein-coupled receptors does not require an increase in cytosolic calcium for platelet aggregation.

U46619 is a potent platelet agonist, its binding to the thromboxane A2 receptor resulting in Gq-binding protein-mediated responses; nevertheless, it is unable to cause platelet aggregation, unless released ADP is present. In this study we demonstrate that Gi activation is the step U46619 lacks to cause platelet aggregation; in fact, when platelets were treated with an ADP scavenger system, the response to U46619 was restored by the addition of epinephrine, which activates platelets via a Gi protein. The concomitant activation of Gi and Gq proteins does not require increased cytosolic calcium to cause aggregation, as assessed by the fact that platelets treated with the intracellular calcium chelator BAPTA were able to respond to U46619 provided ADP or epinephrine was present. Moreover, as the calcium ionophore ionomycin, at low concentrations, potentiated the response to U46619 but not to epinephrine, we may conclude that calcium influx preferentially activates a Gi downstream signalling pathway.

Adenosine Diphosphate↗
PubMed1998

Hydrogen peroxide is involved in collagen-induced platelet activation.

In this study, we investigated whether (1) collagen-induced platelet aggregation is associated with a burst of H2O2, (2) this oxidant species is involved in the activation of platelets, and (3) the pathways of platelet activation are stimulated by H2O2. Collagen-induced platelet aggregation was associated with production of H2O2, which was abolished by catalase, an enzyme that destroys H2O2. H2O2 production was not observed when ADP or thrombin were used as agonists. Catalase inhibited dose-dependently thromboxane A2 production, release of arachidonic acid from platelet membrane, and Inositol 1,4,5P3 (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This study suggests that collagen-induced platelet aggregation is associated with a burst of H2O2 that acts as a second messenger by stimulating the arachidonic acid metabolism and phospholipase C pathway.

Blood Platelets↗
PubMed1998

Effects on platelet function of combination etoposide and carboplatin chemotherapy in pediatric oncology patients.

The effects of a therapeutic course of the combination of carboplatin and etoposide on platelet function have been evaluated in 10 pediatric patients with brain tumors. Platelet count, in vitro aggregation tests, P-selectin expression and agonist-induced ATP release were evaluated before, and 7 and 15 days after one cycle of chemotherapy. The analysis of the results demonstrated the presence of an in vitro platelet aggregation defect in response to collagen and arachidonic acid in all patients 7 days after therapy. A concomitant decrease of collagen- and arachidonic acid-induced ATP release was also observed. Both platelet aggregation and ATP release returned to baseline values 15 days after chemotherapy administration. Conversely, in vitro platelet aggregation and secretion induced by ADP and epinephrine were unaltered by carboplatin and etoposide administration. Furthermore, P-selectin expression was negative at baseline and did not change after chemotherapy. These results support the hypothesis that combination etoposide and carboplatin chemotherapy in pediatric patients is responsible for possible disturbances in biochemical pathways required for platelet secretion and aggregation.

Journal Article↗
PubMed1998

Nickel enhances collagen-induced platelet activation acting by increasing the organization of the cytoskeleton.

In keratinocytes, osteoclasts and enterocytes, Ni2+ acts as an agonist working through selective activation of the polyvalent cation-sensing receptor. We report here that while Ni2+ alone had no direct ability to induce platelet aggregation or secretion, Ni2+ pretreatment produced these responses when platelets were stimulated with subthreshold concentrations of collagen. In addition, pretreatment with Ni2+ significantly enhanced collagen-induced phospholipase C activation and calcium mobilization. Platelet adhesion to collagen was increased and the inhibition of collagen-induced adhesion normally seen after cytochalasin D treatment was significantly diminished. When Ni2+ was added to platelets alone, tyrosine phosphorylation of p60src was increased. Moreover, Ni2+ enhanced the amount of protein, especially actin, found in the low-speed Triton X-100 insoluble cytoskeleton. Our results indicate that nickel, possibly acting via a platelet cation sensing receptor analogous to that which has been described in other cell types, may cause a rapid tyrosine kinase-dependent cytoskeleton reorganization leading to enhanced adhesion of platelets to collagen and increasing collagen-dependent responses.

Blood Platelets↗
PubMed1998

Platelet activation and cytokine production during hypothermic cardiopulmonary bypass--a possible correlation?

Cardiopulmonary bypass (CPB) is associated with impaired platelet function and a systemic inflammatory response. The present study was designed to evaluate whether any correlation between platelet activation and inflammatory response during CPB exists. The results obtained from 8 patients undergoing hypothermic CPB for cardiac surgery showed the occurrence of a moderate degree of platelet activation during CPB, demonstrated by an increase of platelet CD62P expression in correlation with an increase of beta-thromboglobulin levels, with a concomitant decrease of in vitro platelet response. Plasma IL-1beta levels significantly increased during CPB, with a peak between 1 and 4 h after CPB. Similarly, IL-6 levels were elevated 30 min from CPB starting, peaked at 4 h, and remained elevated after 24 h. A direct correlation was found between plasma IL-1beta and IL-6 levels. A significant correlation between plasma IL-1beta and beta-thromboglobulin levels was also found. In turn, plasma beta-thromboglobulin levels correlated with CD62P expression on activated platelets. An inverse correlation was found between in vitro platelet aggregation and plasma IL-1beta or IL-6 levels. From the present results it may be speculated that platelet activation during CPB may contribute, through the release of IL-1beta, to activation of endothelial cells and subsequent release of other cytokines with chemotactic and pro-inflammatory properties, thus playing an important role in the inflammatory response associated with CPB.

Aged↗
PubMed1997

Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated.

BACKGROUND: Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation. METHODS AND RESULTS: Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (-74%, P < .005), catalase (-67%. P < .005). SOD plus catalase (-82%, P < .005), and the hydroxyl radical (OH0) scavengers mannitol (-66%, P < .005) and deoxyribose (-55%, P < .005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (0-2), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2.3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH0. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin: by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium an inhibitor of flavoprotein-dependent enzymes: and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase. CONCLUSIONS: This study shows that platelets exposed to A-R intrinsically generated 0-2 and OH0, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.

Acridines↗
PubMed1997

Mechanism of the persisting TxA2 receptor antagonism by picotamide.

Picotamide is a dual TxA2 receptor antagonist/TxA2 synthetase inhibitor. As the picotamide effect is maximum only after 5-10 min of incubation, aim of the present work was to study whether the effect of picotamide could be observed even after wash out of the drug from the external buffer. Platelet aggregation, serotonin release and changes in intracellular calcium were analyzed in platelets treated with picotamide and then gel-filtered, in comparison with gel-filtered platelets treated with picotamide. To exclude the possibility that the inhibitory effect of picotamide is due to its intracytosolic storage, serotonin release in digitonin-permeabilized platelets was measured. Platelet aggregation and serotonin release in response either to U46619, a synthetic agonist of the thromboxane A2 receptor, or arachidonic acid or collagen, as well as the changes in intracellular calcium concentration after U46619 or arachidonic acid stimulation, were inhibited by picotamide even when it had been washed out by gel-filtration. Both inhibitions were very similar to that observed in experiments in which picotamide was present in the medium. As the serotonin release in digitonin permeabilized platelets presented the same inhibition it may be excluded that picotamide effect is consequent upon the cytosolic storage of the drug. Since our results clearly indicate that the action of picotamide persists even after washing out of the drug from the medium, the idea that this antagonistic effect may be dependent on its binding to platelet plasma membrane rather than on its cytosolic concentration, is strongly substantiated.

Blood Platelets↗
PubMed1997

Proinflammatory cytokines and hemostatic system in patients with chronic obstructive pulmonary disease.

The prothrombotic state frequently observed in patients with chronic obstructive pulmonary disease (COPD) may be related to a systemic inflammatory response. In the present study plasma interleukin-6 (IL-6), IL-2 and IL-1beta levels have been investigated in 33 patients with COPD. In vitro platelet activity, plasma C-reactive protein (CRP) and fibrinogen levels were also determined in all patients. The results obtained demonstrated that plasma IL-6 levels were significantly higher in the patient group compared with a control population age and sex-matched ( P < 0.02), while plasma IL-2 and IL-1beta levels were not significantly modified. An overall condition of platelet hyperactivity in COPD patients was also observed. A comparative analysis of platelet activity and blood gas levels demonstrated a correlation between platelet hyperactivity and a severe impairment of oxygen ( P < 0.001) and carbon dioxide tensions ( P < 0.01). Finally, a statistical analysis of the population under study showed the presence of a significant correlation between elevated plasma IL-6 ( P < 0.001) and IL-1beta levels ( P < 0.007), and an increased sensitivity of platelets to arachidonic acid, suggesting a possible correlation between the inflammatory response and the prothrombotic state observed in patients with COPD.

Journal Article↗
PubMed1996

Effects of storage on in vitro platelet responses: comparison of ACD and Na citrate anticoagulated samples.

The present study was designed to evaluate whether the use of acid citrate dextrose (ACD) Formula A may enhance the survival of platelets during storage, thus allowing the continuance of platelet studies over the period of 2-3 hours usually recommended. For this purpose the effects of time on in vitro platelet response to several agonists have been investigated in platelet-rich plasma (PRP) obtained from blood samples anticoagulated with either Na citrate or ACD Formula A. The analysis of the data obtained in in vitro platelet aggregation studies using various parameters and at different time points demonstrated that storage of PRP obtained from citrated samples caused a marked reduction of platelet responses. This reduction was already evident after 6 hours, and a strong decrease was observed after 8 hours with all the agonists used. On the other hand, storage of ACD anticoagulated blood did not cause any significant decrease of platelet responsiveness up to 6 hours. A reduction of platelet aggregation became evident only after 8 hours, but not to the same extent as the one observed in citrated samples. Therefore, it may be concluded that the use of ACD Formula A as anticoagulant is capable of maintaining a normal platelet responsiveness up to 6-8 hours, thus permitting the investigation of platelet function for periods of time over those commonly recommended.

Adenosine Diphosphate↗
PubMed1995

Protein kinase C activation is not a key step in ADP-mediated exposure of fibrinogen receptors on human platelets.

UNLABELLED: A selective inhibitor of protein kinase C (PKC), Ro 31-8220, blocks pleckstrin (P47) phosphorylation in platelets activated with either ADP, ADP plus synthetic thromboxane agonist U46619 and ADP plus U46619 plus epinephrine, while inducing a weak inhibition of platelet aggregation, and no significant effect on the fibrinogen binding. In platelets activated by U46619 alone, P47 phosphorylation, platelet aggregation, fibrinogen binding and serotonin release are all inhibited by Ro 31-8220. In the presence of an ADP scavenger system, U46619 induces pleckstrin phosphorylation, serotonin release and calcium mobilization but not platelet aggregation and fibrinogen binding, unless epinephrine is added. IN CONCLUSION: (1) PKC activation is required for ADP secretion; (2) ADP or epinephrine are essential for fibrinogen receptor exposure induced by U46619; (3) fibrinogen receptor exposure induced by ADP is independent of activation of PKC.

Adenosine Diphosphate↗
PubMed1995

Acid citrate dextrose (ACD) formula A as a new anticoagulant in the measurement of in vitro platelet aggregation.

To evaluate whether the use of ACD Formula A may affect in vitro platelet function, blood samples were obtained from 21 healthy blood donors and anticoagulated in ACD (acid-citrate dextrose, NIH Formula A), Na citrate 3.8%, and K3EDTA. Platelet count, mean platelet volume, and in vitro platelet aggregation were evaluated on each sample. No significant difference was observed in platelet count and mean platelet volume among the different samples. Conversely, the ACD treated platelets showed a higher reactivity to the agonists as demonstrated by a significant increase of the maximum percentages of aggregation induced by ADP, epinephrine, and collagen, as well as a significant decrease of secondary aggregation thresholds to ADP and epinephrine. In conclusion, it may be speculated that ACD Formula A is capable of better maintaining the intraplatelet signal transduction mechanisms during PRP preparation, thus improving the overall responsiveness of platelets.

Adult↗
PubMed1995

Fibrinogen binding is independent of an increase in intracellular calcium concentration in thrombin degranulated platelets.

In a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C (PLC) activation and calcium mobilization in TDP activates by ADP, epinephrine and U46619. ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

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

Effect of picotamide on the calcium mobilization and phospholipase C activation in human platelets.

The effect of picotamide (G137 or N,N'-bis-3-picolyl-4-methoxyisophthalamide), a dual thromboxane A2 (TxA2) synthetase inhibitor/TxA2 endoperoxide receptor antagonist, on the phospholipase C (PLC) activation and calcium mobilization in human platelets stimulated by arachidonic acid (AA) and TxA2 receptor synthetic agonist U46619, has been studied. Preincubation with picotamide (10(-4) M) for 1 and 3 min significantly reduced (p < 0.03 and p < 0.005 respectively) the calcium concentration changes induced in gel-filtered platelets (GFPs) by U46619 125 nM and 250 nM. Picotamide also reduced the calcium concentration changes induced in GFPs by AA 75 and 150 microM and by ADP 5 and 10 microM. In thrombin degranulated platelets picotamide inhibited the effect of U46619 up to 500 nM. The PLC activation, as indicated by inositol-1,3,4 P3 (Ins 1,3,4 P3) formation in response to U46619 250 nM and AA 150 microM was also inhibited by picotamide. These results may suggest a dual effect of picotamide on the receptor/effector systems through which TxA2 mediates platelet activation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Intracellular signalling of epinephrine in rat hepatocytes during fetal development and hepatic regeneration.

The changes in intracellular calcium concentration and IP3 production after the addition of epinephrine were analysed in adult, fetal (20th-22nd day of intrauterine life), and regenerating rat hepatocytes (4 h-24 h after partial hepatectomy) to determine whether the signal transduction is the same in quiescent proliferating and differentiating cells. The epinephrine treatment causes a significative cytosolic calcium transient in hepatocytes isolated in the last day of fetal life (22-day old) and in the early stage of regeneration (4 h). This effect is not significant in the previous stage of fetal life (20-day old) and at the onset of M phase of cell cycle after partial hepatectomy (24 h). [3H]myo inositol incorporation into IP3 and IP4 is higher in 20 day fetal and regenerating hepatocytes with respect to the control. In these cells the epinephrine does not affect basal level of IP3 and IP4, while it causes a substantial increase of these inositol phosphates in adult hepatocytes. [3H]myo inositol incorporation into PIP2 is very low at the 20th day of fetal life. Epinephrine has no effect on this parameter in fetal and regenerating hepatocytes. Our results show that the epinephrine signal is mediated differently in proliferating and in quiescent hepatocytes.

Animals↗
PubMed1993

Chronic obstructive pulmonary disease. A possible correlation between platelet aggregation tests and respiratory indexes. ECOB Group.

It has been suggested that thrombotic events occurring in patients with chronic obstructive pulmonary disease could be partially due to abnormalities in blood coagulation and fibrinolysis, as well as to changes in platelet function. In the present study in vitro platelet aggregation, in addition to clotting activity, has been studied in 20 patients with chronic obstructive pulmonary disease and different degrees of alteration of blood gas analysis. The results obtained suggest that the presence of a condition of platelet hypoaggregability characterizes patients with marked hypercapnia and/or hypoxemia, whereas the improvement of respiratory indexes is followed by a trend to normalization, or, more often, to an increase of platelet activity.

Aged↗
PubMed1993

Circadian variations in platelet aggregability in non insulin dependent diabetes patients (NIDDM).

Circadian rhythms of platelet activity have been extensively investigated in recent years in view of a possible correlation with the increased morning frequency of thrombotic events. Since a role of enhanced platelet activity has been suggested in diabetic macro- and microvascular disease, the aim of the present study was to analyze the circadian variations of in vitro platelet aggregability in patients with non insulin-dependent diabetes mellitus. Eighteen in-patients, ranging in age from 49 to 73 years, all in good metabolic control, were included in the study. Aggregometric patterns were analyzed in blood samples collected at 0700, 0900, 1100 and 1700. A significant increase of the threshold concentrations for ADP-collagen- and arachidonic acid-induced platelet aggregation has been observed in the interval 0700-1100 or 0700-1700, suggesting, also in diabetic patients as in normal subjects, a maximum of platelet activity in the morning, regardless of postural changes and timing of meals.

Aged↗