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Biomedical subjects

F M Pulcinelli

Publications and source records attributed to F M Pulcinelli.

46 records · Page 3Linked to original sources
PubMed1992

Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation.

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.

Antigens, CD↗
PubMed1992

Use of Zn-pyrophosphatase in the high-performance liquid chromatographic analysis of cell extracts containing 32P-labelled inositol phosphates.

A simple method is described for eliminating the interference of pyrophosphate and pyrophosphorylated nucleosides in the high-performance liquid chromatographic determination of inositol 1,3,4-triphosphate and inositol 1,4,5-triphosphate of 32P-labelled extracts of cells. Treatment of the extract with pyrophosphatase, but substituting Zn2+ for Mg2+ as the cofactor, converts all nucleoside triphosphates and pyrophosphate to their di- and monoesters. Such change shifts their position in the elution profile, allowing a clear identification and quantification of the inositol phosphates. Typical overall recoveries near 80% or higher of added markers.

Chromatography, High Pressure Liquid↗
PubMed1992

Hydrogen peroxide triggers activation of human platelets selectively exposed to nonaggregating concentrations of arachidonic acid and collagen.

The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen, it induced platelet aggregation and serotonin release in a dose-dependent fashion. The increase in platelet aggregation was associated with thromboxane A2 production and was prevented by 100 mumol/L aspirin. The amplification of platelet response by H2O2 was also inhibited 2 hours after 300 mg aspirin was given to healthy subjects. H2O2 alone did not affect intraplatelet Ca++ influx or mobilization but, combined with subthreshold concentrations of arachidonic acid, it increased Ca++ mobilization. In platelets prelabeled with tritiated arachidonic acid, H2O2 induced tritium release in a dose-dependent fashion; this effect was prevented by mepacrine, an inhibitor of the phospholipase A2 enzyme. Platelet function was not affected by using H2O2 in combination with other agonists such as thrombin, calcium ionophore, or adenosine diphosphate. This study suggests that H2O2 triggers activation of platelets preexposed to agonists at subthreshold levels by stimulating arachidonic acid metabolism, likely by stimulating the phospholipase A2 enzyme. The stimulation of platelets by concentrations of H2O2 similar to those released by activated leukocytes may give new insights into the functional cooperation between leukocytes and platelets.

Arachidonic Acid↗
PubMed1991

Differential platelet proaggregating activity of three Burkitt's lymphoma-derived human cell lines.

We demonstrate a differential platelet in vitro proaggregating activity in three Burkitt's lymphoma--derived human B cell lines, i.e. Daudi, Raji and P3H-R1. Functional and ultrastructural findings indicated the ability of Daudi cells to induce a marked secondary irreversible platelet aggregation, while the Raji cells only induced a primary-type reversible platelet response; no evidence of proaggregating activity has been obtained for P3H-R1 cells. Luminometric assays indicated that contact of Daudi and Raji, but not P3H-R1, cells with platelet rich plasma (PRP) or platelet poor plasma (PPP) was followed by ADP release, in the range of 2,2-3,5 microM and 0.4-0.6 microM respectively for Daudi and Raji cells. After preincubation of PRP with apyrase Daudi cells induced a reversible platelet response similar to that obtained with the use of Raji cells: then, the irreversible complete platelet response induced by Daudi cells was to be related to ADP release from degranulating platelets. Experiments in gel-filtered platelet systems showed that the plasma co-factor inducing ADP release from Daudi and Raji cells was not fibrinogen. Specific inhibition of platelet thrombin receptors, as well as of cycloxygenase and lipoxygenase pathways, did not modify the proaggregating activity of Daudi and Raji cells. Work is in progress to characterize the plasma factor interacting with Daudi and Raji, but not P3H-R1 cells, and the differences between the three cell lines which support this differential interaction.

Adenine Nucleotides↗
PubMed1991

Blood leukotrienes in headache: correlation with platelet activity.

Platelet hyperactivity, one of the commonest findings associated with migraine, has been related to increased release of biologically active substances such as catecholamines and arachidonic acid metabolites, which seem to play a role in the pathogenesis of migraine. In this study, in vitro platelet aggregation tests were performed on samples from patients with different types of headache. The presence of platelet hyperactivity was clearly demonstrated in 11 patients with classical migraine between attacks, but not in 4 patients between attacks of common migraine. Nevertheless, the presence of a marked platelet hyporesponsivity was found during the attack phase of both classical and common migraine. No difference in platelet aggregability was found between attack and post-attack phases in 5 patients with cluster headache. Blood leukotrienes were analyzed in 8 patients with classical migraine and in the 5 patients with cluster headache. During the attack phase of classical migraine both LTC4 and LTB4 were present in the peripheral blood, while the post-attack phase was characterized by the disappearance of LTC4 and the presence of LTB4 and its transisomer delta 6-trans-LTB4. Blood leukotrienes were constantly absent during both phases of cluster headache. Incubation of normal platelets with LTC4 or delta 6-trans-LTB4 was followed by inhibition of platelet response to epinephrine. delta 6-trans-LTB4, at higher concentrations, induced the opposite effect. A possible role of blood leukotrienes in the changes occurring in platelet aggregability during the different phases of classical migraine, is discussed.

Adult↗
PubMed1990

Blood cellular and biochemical changes after extracorporeal shock wave lithotripsy.

The present study deals with blood cell behaviour and related hematochemical changes occurring in man after exposure to extracorporeal shock wave lithotripsy (ESWL). The following parameters have been investigated, before and after treatment, in a longitudinal study carried out on 58 patients of our Center: RBC count, Ht, Blood Hb, Plasma Hb, autohemolysis after 72 hours, bilirubinemia, WBC count, platelet count, in vitro platelet aggregation, beta-TG, sideremia and urinary post-exposure transferrin concentration. After exposure to ESWL, with pulse numbers ranging from 350 to 3,000, 18-20 KV, direct and indirect evidences of hemolysis have been found when number of SW exceeded 1,000. The hemolysis rate in different models of in vitro exposure to ESWL of blood samples was also investigated. Increase of WBC count, and changes of the platelet in vitro aggregation patterns were also observed. Changes in sideremia values have been related to urinary loss of transferrin.

Blood Cell Count↗
PubMed1988

In vitro enhancement of human platelet aggregation by somatostatin.

Very low concentrations of somatostatin (S-14) strongly potentiate the in vitro aggregation induced by collagen, ristocetin and arachidonic acid, but not that induced by ADP or epinephrine, in both human platelet rich plasmas and gel-filtered platelet preparations. Desensitization phenomena may be induced either by repeated addition of S-14 or long lasting contact between S-14 and platelets.

Adenosine Diphosphate↗
PubMed1987

A multiparametric index of platelet in vitro aggregation in cerebrovascular disease.

148 patients with various forms of cerebrovascular disease (CVD) were studied by means of a multiparametric analysis of in vitro platelet aggregation, based on the following six parameters: ADP and epinephrine primary and secondary aggregation thresholds and percent maximum aggregation induced by optimal concentrations of ADP and epinephrine. These patients were assigned to four study groups, according to clinical diagnosis supported by CT scan, of transient ischemic attack and reversible neurological deficit (TIA-RIND), or completed stroke, in the presence or absence respectively of antiplatelet medical treatment at the time of the study. A statistically significant increase of the in vitro platelet aggregation was found in 44.4% of the untreated TIA-RIND patients and in 33.9% of the untreated stroke patients. However this last group showed a higher percentage of very marked hyperaggregation. Differences between the two treated study groups and controls were not significant. No difference was found in collagen- and ristocetin-induced aggregation between the patient groups and the controls.

Adult↗
PubMedDate not supplied

Human sperm cryopreservation and reactive oxygen species (ROS) production.

The aim of this work was to establish whether cryopreservation procedure can trigger the production of Reactive Oxygen Species (ROS) in selected sperm populations. Semen samples were obtained from 45 subjects attending our Department of Medical Pathophysiology. Motile sperm suspensions were obtained by swim-up in Tyrode's salt solution. After dilution with TEST yolk buffer freezing medium, they were cryopreserved in liquid nitrogen. In addition to motility assessment, in basal and freeze/thaw conditions ROS detection and the Hypoosmotic Viability Test were also carried out. In 19 subjects (42.2%) there was already evidence of ROS production prior to cryopreservation, which increased after thawing. In 9 subjects (20.0%) there was no ROS production prior to cryopreservation, however, after freezing/thawing we detected evidence of the presence of ROS. It seems, therefore, that cryoprocedure can indeed provoke or increase ROS production in some semen samples. In ROS producing subjects, the post-show recovery of sperm motility and vitality was significantly lower compared to ROS-free subjects. This was probably due to damage by oxidative stress leading to lipid peroxidation of the sperm membrane. Moreover, in some ejaculates, ROS overproduction or scavenger system failure can be regarded as a cryopathogenetic factor affecting "sperm quality" recovery.

Adult↗