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Biomedical subjects

V Evangelista

Publications and source records attributed to V Evangelista.

13 recordsLinked to original sources
PubMed1992

Transcellular metabolism of arachidonic acid: increased platelet thromboxane generation in the presence of activated polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), in the presence of cytochalasin B, are able to induce activation of coincubated autologous platelets "via" cathepsin G released from the azurophilic granules. However, thromboxane (Tx) B2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed, the amount of TxB2 found in supernatants of platelet/PMN suspensions challenged with 1 mumol/L fMLP was twofold to fourfold higher than that measured when platelets were stimulated by supernatants from fMLP-activated PMN. In the present report, we analyzed the possibility that PMN-induced TxB2 production in this system is the result of transcellular metabolism of arachidonic acid (AA) between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labeled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. Our results showed that: (1) 3H-TxB2 and 3H-12-HHT are synthesized when 3H-AA-labeled PMN are activated mixed to unlabeled platelets; (2) total radioactivity released by fMLP-stimulated PMN is increased in the presence of platelets, whereas the membrane content of unesterified 3H-AA is reduced; (3) platelet cyclooxygenase inhibition completely prevents 3H-TxB2 synthesis; and (4) inhibition of cathepsin G-induced platelet activation with the antiprotease eglin C blocks the formation of 3H-TxB2. These data show that in the experimental system used, platelets use PMN-derived unmetabolized AA to synthesize TxB2.

Arachidonic Acids↗
PubMed1992

Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G.

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy.

Amino Acid Sequence↗
PubMed1992

Defibrotide inhibits platelet activation by cathepsin G released from stimulated polymorphonuclear leukocytes.

Defibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

Catalysis↗
PubMed1992

Confocal-line microscopy.

The confocal-line (CL) technique combines some of the characteristics of confocal-scanning microscopy with those of conventional-imaging methods. It is based on the introduction of line-shaped illumination and linear image detection, as an alternative to the current confocal-point (CP) approach. Although confocal only in one dimension, the proposed solution offers performance and features adequate to a variety of biological and non-biological applications and is also adaptable to an increased number of microscopical observations and measurements. The absence of moving components in the optical path and the use of electronic linear imagers permits flexible and fast operation that appears particularly relevant in many fields of basic and applied research. For instance, transmission, reflection and emission images can simultaneously be collected from the same area of the specimen, with slight adjustments to the optical setup. Useful extensions of CL microscopy to the field of spectral imaging are obtained with the introduction of a slit, a polychromator and an area detector, substituting for the linear imager. Prototype instrumentation has been constructed working from the cited principles and some tests have been performed on selected applications.

Autoradiography↗
PubMed1991

Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases.

Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN-platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.

Blood Platelets↗
PubMed1990

Platelet activation by polymorphonuclear leukocytes exposed to chemotactic agents.

Human platelets were loaded with aequorin, a Ca2(+)-sensitive photoprotein, and tested in the platelet-ionized calcium aggregometer for simultaneous recording of platelet aggregation and intraplatelet Ca2+ levels both in the presence and in the absence of autologous polymorphonuclear leukocytes. Cells were exposed to one of three chemotactic stimuli: platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (FMLP), or leukotriene B4 (LTB4). Platelets alone aggregated and showed intracellular Ca2+ movement only when exposed to PAF. Amplification of both platelet aggregation and intraplatelet Ca2+ movement was induced by PAF in the presence of leukocytes. Aggregation and intraplatelet Ca2+ mobilization were also observed in the presence of leukocytes activated by either FMLP or LTB4. Both parameters increased with the concentration of the stimuli and/or the number of leukocytes. Platelet thromboxane B2 production was also significantly increased in the presence of leukocytes. Addition of platelets at different times after leukocyte activation resulted in progressively reduced cytoplasmic Ca2+ increase. Cell-free supernatants prepared from FMLP-stimulated leukocytes were able to induce platelet aggregation, thromboxane B2 generation, and Ca2+ mobilization, although at a reduced degree as compared with intact leukocyte addition. The activity of leukocyte supernatants was stable at 37 degrees C for up to 30 min and was suppressed by trypsin inhibitor. Our study indicates that stimulated leukocytes release a soluble enzymatic activity able to activate platelets; cell-to-cell interaction may also play a role in this phenomenon. Platelet-leukocyte interaction could have physiopathological relevance and constitutes a new model for studying old and new platelet inhibitory drugs.

Blood Platelets↗
PubMed1989

Microspectroscopy of red blood cells.

Spectroscopic techniques have been widely employed to analyze properties of macromolecules and dynamics of intracellular events on bulk preparations of cells. The development of computer controlled microspectrophotometers has made possible the study of the same events in single cells, often providing significant and unexpected results. This paper briefly reviews experimental works carried out in our laboratories on single red blood cells. Microspectrophotometric techniques were applied which make use of the fact that ligand binding to intracellular haemoglobin is associated with optical changes. Information on the relative abundance of different haemoglobin components inside single erythrocytes of trout blood was obtained from spectra of air equilibrated samples, taking advantage of the extreme pH sensitivity of one of the four haemoglobin components. The kinetics of oxygen and carbon monoxide binding to haemoglobin has been followed and demonstrated to correspond to a zero order process, with a rate much slower than that characteristic for haemoglobin in solution. These results demonstrate that the process is diffusion limited; computer simulations suggest that ligand uptake is limited by the time required for the diffusion from the extracellular space of enough ligand molecules for total saturation of intraerythrocytic haemoglobin. Finally, oxygen dissociation curves in single red blood cells can be obtained by means of particular flow cell, with promising results for the study of physiological and pathological processes (namely red cell sickling in drepanocytosis).

Animals↗
PubMed1988

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a 'memory' of previous sickling cycles.

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer-controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a 'memory' of its previous cycle(s), possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

Anemia, Sickle Cell↗
PubMedDate not supplied

Measurement of ionized cytoplasmic calcium mobilization with the photoprotein aequorin in human polymorphonuclear leukocytes activated by platelet activating factor (PAF).

The use of the sensitive photoprotein aequorin as a Ca2+ indicator in human polymorphonuclear leukocytes (PMN) not pretreated with cytochalasin B and stimulated with platelet activating factor (PAF) may help cast more light on the relative importance of intracellular and extracellular Ca2+ in PMN function. PAF elicits Ca2+ mobilization in PMN (resuspended in the presence of 1 mM extracellular Ca2+), in a concentration-dependent manner. The Ca2+ chelator ethyleneglycoltetraacetic acid (EGTA) abolishes Ca2+ mobilization, suggesting that almost all Ca2+ mobilized by PAF derives from the external medium. Aggregation and enzymatic release parallel the Ca2+ mobilization triggered by PAF. In contrast PAF appears to be only a weak stimulus of superoxide anion production (compared to the phorbol ester phorbol 12-myristate 13-acetate (PMA] and leukotriene B4 (LTB4) synthesis (compared to the Ca2+ ionophore A23187). The fact that PAF elicits Ca2+ mobilization, aggregation, secretion and LTB4 generation in human PMN supports the role of this phospholipid as a powerful mediator of physiopathological events involving PMN activation.

Aequorin↗