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Biomedical subjects

M Lebret

Publications and source records attributed to M Lebret.

At least 37 records · Page 2Linked to original sources
PubMed1987

Relationship between cAMP and Ca2+ fluxes in human platelet membranes.

The effect of cAMP (which involved a 23 kDa protein phosphorylation) has been studied on the Ca2+ uptake and Ca2+ release from a human platelet membrane vesicle fraction. It was tested in the presence of the catalytic subunit of the cAMP-dependent protein kinase (C Sub). The addition of C Sub increased the steady state level of the Ca2+ uptake into the membrane vesicles. The effect was enhanced when tested in the absence of Ca2+ precipitating agent. The response was proportional to the dose of C Sub. Moreover, the effect varied with the Ca2+ concentration. The effect of C Sub has been tested on the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. A phosphorylated state of the 23 kDa protein appeared to be necessary. Indeed, a phosphorylation inhibition prevented the IP3 effect and the addition of C Sub increased the percentage of released Ca2+ (without modification of the time course). However, the C Sub dose-dependent response was not linear. The effect of cAMP on the two functions (Ca2+ uptake and Ca2+ release) appears to be different. Therefore, these results led us to suggest a more complex role of cAMP in the regulation of platelet Ca2+ concentration.

Blood Platelets↗
PubMed1987

Signal transduction in normal and pathological thrombin-stimulated human platelets.

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.

Blood Platelet Disorders↗
PubMed1987

Abnormal phosphoinositide metabolism and protein phosphorylation in platelets from a patient with the grey platelet syndrome.

Washed platelets isolated from one patient suffering from the inherited grey platelet syndrome were studied during thrombin-induced activation. The agonist-induced changes in (i) morphology, (ii) typical functional cell responses, (iii) membrane phospholipid metabolism and protein phosphorylation were studied and compared with the changes obtained with normal platelets. The morphology of the platelets as visualized by electron microscopy confirmed the almost total absence of intracellular alpha-granules and marked vacuolization. During thrombin stimulation the morphological changes were clearly delayed as compared to normal platelets, the granule centralization and aggregation occurred only 15 s after thrombin addition instead of 5 s in normal platelets. After 15 s, however, even though no alpha-granules were observed, a ring-like structure occurred centrally, indicating that they are not a prerequisite for this reaction. The whole release reaction, i.e. liberation of [14C]serotonin from dense granules and beta-N-acetylglucosaminidase activity from lysosomes, and the thromboxane synthesis were delayed and remained lower than in normal platelets. No thrombin-induced phosphatidyl 4,5-bisphosphate breakdown was measurable on 32P-prelabelled platelets although [32P]phosphatidate formation occurred normally. Phosphorylation time courses of myosin light chain (P20) and of protein P43 (mol wt 43,000) markedly differed from those of controls, being less than half of the normal during the first 15 s and remaining subnormal even after complete aggregation. These results suggest that in platelets devoid of alpha-granules a deficient transmembrane signalling system is likely responsible for the impaired physiological responses.

Blood Platelet Disorders↗
PubMed1987

Abnormal calcium transport into microsomes of grey platelet syndrome.

Calcium uptake into isolated membrane vesicles from two patients with a grey platelet syndrome has been investigated. An increase in calcium transport appears in both patients when compared to controls. Determination of the kinetic parameters of the calcium transport system gave similar apparent affinity for calcium and an increase in the calcium uptake velocity. This increase in calcium transport is correlated with the increase of the associated Ca2+ activated ATPase activity. The results would suggest a new relationship between the ultrastructural and functional abnormalities of the grey platelet syndrome.

Adenosine Triphosphatases↗
PubMed1986

Further characterization of wheat germ agglutinin interaction with human platelets: exposure of fibrinogen receptors.

It was previously shown that Wheat germ agglutinin, (WGA)-induced platelet activation occurred when only 17% of the lectin binding sites were occupied on the platelet surface and WGA caused the release of a platelet constituent which in turn participates in the observed effect. We now further define the platelet activation induced by WGA: the lectin induces a binding of fibrinogen to specific surface receptors. 125I-fibrinogen binding increases with the WGA concentration from 5 to 15 micrograms/ml. Binding occurs without addition of exogenous calcium; its analysis demonstrated 54,000 sites with a Ka = 0.8 X 10(6) M-1. Addition of 1 mM Ca2+ enhances the 125I-fibrinogen binding and reveals a second class of sites with higher affinity (9200 sites, Ka = 0.17 X 10(8) M-1). This 125I-fibrinogen binding is totally abolished by EDTA, ATP and arginine, and inhibited by 75% by CP/CPK; cyclooxygenase inhibitors and PGE1 also reduce the fibrinogen binding. Thus the WGA-induced fibrinogen binding is release-dependent and responsible for the aggregation process but not for the agglutinating effect of the lectin.

Alprostadil↗
PubMed1985

Mechanism of regulation of calcium concentration by platelet membranes.

Calcium is recognised as an important messenger in the platelet activation. Both the external and internal membranes are considered to play an important role in the Ca2+ homeostasis of the cell. The aim of this review is to try to understand the mechanisms of regulation of the cytoplasmic free Ca2+ concentration by both kinds of membranes and mainly by internal membranes.

Biological Transport, Active↗
PubMed1985

[Prenatal diagnosis of a cardiac tumor resembling Bourneville's tuberous sclerosis].

The antenatal diagnosis of a tumour of the heart after 32 weeks of amenorrhoea made us think Bourneville's tuberous sclerosis. This is a familial condition that had not been noted before and which was confirmed after the birth. The diagnosis and prognostic problems that arise when such tumours are discovered in the heart are considered.

Adult↗
PubMed1984

Interaction of wheat germ agglutinin with human platelets: a model for studying platelet response.

Binding of wheat germ agglutinin (WGA) to human platelets and their activation were studied. The binding of fluoresceinyl-substituted WGA (F-WGA) was saturable, specific, reversible and cooperative. The apparent association constant (Ka) was 2.4 X 10(6) M-1. Activation of platelets was measured by change in light transmission (LT) (aggregation) and by assays of 14C-serotonin and beta-thromboglobulin (secretion). The maximum platelet activation was obtained with 15 micrograms/ml WGA. At this concentration only 17% of all available binding sites were occupied. Increasing the WGA concentration diminished the change in LT and the secretion from alpha granules but not from dense bodies. The addition of EDTA partially reduced the platelet aggregation without any effect on the secretion. Remaining LT change was insensitive to all metabolic inhibitors tested (CP/CPK, arginine, indomethacin, PGE1, chlorpromazine, colchicine and cytochalasin B). The secretion measured in the presence of EDTA was only reduced by preincubation with PGE1 and CPZ. Succinyl-WGA which only binds glucoconjugates containing GlcNAc did not induce any platelet activation, whereas limulin, which binds to glucoconjugates containing NeuAc did induce platelet agglutination and secretion. These results indicate that: (1) the platelet aggregation and secretion induced by WGA occur with only partial occupancy of the membrane binding sites by the lectin; (2) the platelet LT change is due in part to the agglutinating properties of the lectin bound to the platelet surface, and in another part to the aggregation reaction mediated by a platelet constituent secreted from the storage sites in response to the binding of the lectin.

Alprostadil↗
PubMed1984

The Ca2+ uptake and the hydrolysis of various nucleotide triphosphates by human platelet membranes.

Several nucleotide triphosphates (NTPs) were tested as energy source for the Ca2+ uptake by human platelet membrane vesicles. The Ca2+ uptake by these membranes was driven by ATP, GTP, ITP, UTP and CTP. The steady-state level of accumulated Ca2+ was equal with the different NTPs. The highest uptake velocity was found with ATP, but about 40-80% of the velocity with ATP could be accomplished with the other nucleotides. The highest affinity was also found with ATP (Km apparent = 15 microM). The liberation of Pi from the various NTPs was measured simultaneously with the Ca2+ uptake. The coupling ratio (moles of Ca2+ taken up/moles of Pi liberated) varied from 0.4 for ATP to 2.3 for UTP and was almost independent of the NTP concentration. The enzyme activity with ATP as substrate is strongly dependent on the Ca2+ concentration in contrast to the activity with GTP, ITP, UTP or CTP.

Biological Transport, Active↗
PubMed1984

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase from human platelets.

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca2+ + Mg2+)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.

Adenosine Triphosphatases↗
PubMed1983

Evidence that abnormal platelet functions in human Chédiak-Higashi syndrome are the result of a lack of dense bodies.

The structure and functions of platelets from three patients with the Chédiak-Higashi syndrome were examined. Electron-microscopic observations revealed a large reduction in the number of serotonin-storage granules or dense bodies but otherwise normal ultrastructure and normal amounts of alpha-granules and catalase-positive granules. The number of mepacrine-labeled granules was also reduced. Platelets contained normal amounts of beta-thromboglobulin and Platelet Factor 4. The platelet release reaction studied with thrombin as the inducer was impaired. The serotonin uptake by the patients' platelets was low and not inhibited by reserpine, and its metabolism was increased. These findings clearly show that platelets from human Chédiak-Higashi syndrome are deficient in the storage pool of dense granule substances and suggest that this granule defect has an influence on the release mechanism of other granule constituents.

Blood Platelets↗
PubMed1982

Initial characterization of human platelet mepacrine-labelled granules isolated using a short metrizamide gradient.

A method for the purification of human platelet mepacrine-labelled granules is described. Characterization of these isolated granules allowed them to be identified as the serotonin storage organelles or dense bodies. Each step of the purification procedure has been controlled in order to obtain a minimum of leakage of the granule content during initial isolation of the platelets from the blood, the platelet washing procedures, and platelet lysis and the subcellular separation. A key step in the procedure was the centrifugation of the labelled granules across a short, discontinuous metrizamide gradient. The pellet of isolated mepacrine-fluorescent granules consisted almost entirely of granules with the typical appearance of dense bodies, as shown by electron microscopy, and was relatively free from membranes and other granule populations as evaluated by the presence of the different markers (tritiated lectin, beta-glucuronidase, monoamine oxidase, platelet factor 4). The method is simple, reproducible and allows the highest enrichment in dense bodies obtained hitherto with human platelets: x 177 in calcium and x 115 in [14C]serotonin after fractionation of [14C]serotonin-labelled whole platelets. Functional studies performed with the isolated granules showed that they rapidly accumulated [14C]serotonin.

Adenosine Triphosphate↗
PubMed1981

Further investigations on Bernard-Soulier platelet abnormalities.

Platelets from patients with the BSS make an interesting model for the study of 5-HT uptake, with their abnormal morphology including a defect in the plasma membrane GP I sialoglycoproteins (GP lb and glycocalicin), increased density in relation to an increased DB content, and increased size as a result of an abnormal shape change. The "apparent" kinetics of 5-HT incorporation by BSS platelets as measured with [3H]5-HT (0.2 to 2 micro M) were greatly modified: the VM was increased, but a high KM was needed. With a 5-HT concentration corresponding to the control platelet KM, twice as much 5-HT storage was taken up by BSS platelets as by normal platelets. The BSS platelets capacity for 5-HT storage as measure with [14C]5-HT (3 to 54 micro M) was increased up to to fivefold but normally inhibited b reserpine. The metabolism of platelet 5-HT was neither more rapid nor increased. The results, which further define the BSS platelet abnormalities, strongly suggest (1) a modification of the 5-HT transport process at the plasma membrane level and (2) an increased capacity of the platelets to accumulate 5-HT due to the increased DB content that parallels the increased platelet size. The possible relation between these two phenomena is discussed.

Blood Platelet Disorders↗
PubMed1979

Relationship between mepacrine-labelled dense body number, platelet capacity to accumulate 14C-5-HT and platelet density in the Bernard-Soulier and Hermansky-Pudlak syndromes.

We have used the mepacrine-labelling procedure to measure the dense body (serotonin storage organelle) content of the platelets of 2 hereditary disorders where abnormalities in dense body number were suspected. The platelets were incubated with mepacrine and examined by fluorescence microscopy. A mean number of 5.4 +/- 0.8 (SD) dense bodies per platelet was calculated from the data obtained using platelets isolated from 40 normal human subjects. In contrast the platelets of 2 patients with the Bernard-Soulier syndrome contained an average of 14 and 17 labelled granules. This increase was associated with a much greater capacity of the platelets to accumulate 14C-5-HT. The opposite result was obtained using the platelets from 2 patients with the Hermansky-Pudlak syndrome which contained few granules labelled by mepacrine and took up less 14C-5-HT than normal human platelets. Centrifugation of the patients' platelets on discontinuous sucrose gradients showed that the platelets of the 2 Bernard-Soulier patients were much denser than normal whereas a high proportion of low density platelets was observed in the Hermansky-Pudlak syndrome. These results further define the platelet abnormalities in the two syndromes and suggest that dense body number may be one of the factors governing platelet density.

Adolescent↗
PubMed1978

Studies on a new variant of the Hermansky-Pudlak syndrome: qualitative, ultrastructural, and functional abnormalities of the platelet-dense bodies associated with a phospholipase A defect.

The structure and functions of platelets from a patient in whom albinism and hemorrhagic diathesis were associated have been investigated. Electron microscope studies showed a large reduction in the number of dense bodies and this was confirmed by an examination of fluorescent platelets loaded with mepacrine. The rare dense bodies were much bigger than normally observed; their density was diminished and was localized in a peripheral ring. Other platelet constituents were found to be normal. Platelet peroxidase activity was normal in the canaliculi of the dense tubular system; catalase-positive granules were also present. Serotonin uptake by the patient's platelets was much decreased and reserpine, a potent inhibitor of serotonin accumulation by normal human platelets, did not further decrease this incorporation. The uptake of free 14 C-arachidonic acid by the platelets was greatly diminished, as was its thrombin-induced liberation from phosphatidyl-choline and phosphatidyl inositol. Moreover, platelet phospholipase A1 activity was much reduced and phospholipase A2 activity was undetectable.

Adult↗
PubMed1976

Two simple methods for the quantitative evaluation of platelet adhesion to collagen.

14C serotonine labelled platetel rich plasma (PRP) incubated with fibrillar collagen is either gel filtered through a Sepharose 2B column (Sepharose test) or centrifugated on a 23% Ficoll layer (Ficoll test). In the Sepharose test, the adhesion is quantified by the determination of the per cent yield of free platelets eluted from the column and the release by a quantitative evaluation of the 14C serotonin. In the Ficoll test, the released seroton is measured from the distribution of radioactivity in the different layers (supernatant plasma, platelets adherent to collagen, Ficoll, platelet pellet) of the tube after centrifugation. Both methods are rapid and quite reproducible.

Carbon Radioisotopes↗
PubMedDate not supplied

An ellipticine derivative (oxazolopyridocarbazolium) 3' linked to tetrathymidylate stacks intramolecularly with the nearest thymine at low concentration and head-to-tail intermolecularly at high concentration.

The solution conformation of a tetrathymidylate linked through an ester bond to an ellipticine derivative oxazolopyridocarbazolium (OPC) at the 3' position was investigated using one- and two-dimensional nmr experiments. Since the total electric charge of the OPC ring may influence self-association, we first determined the pKa of the oxazole cyclic acidic function. Nuclear Overhauser effect spectroscopy experiments showed that, at low concentration, the OPC stacks intramolecularly with the nearest thymine at the 3' end. At highest concentration, however, the OPC rings are self-associated. The stacking constant was calculated using 1H chemical shift dilution experiment. The conformational model suggested by P-nmr was tested by molecular mechanics computations.

Alkaloids↗