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Biomedical subjects

S Levy-Toledano

Publications and source records attributed to S Levy-Toledano.

At least 55 records · Page 3Linked to original sources
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

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients.

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.

Blood Platelets↗
PubMed1986

Platelet prothrombin converting activity in hereditary disorders of platelet function.

Prothrombinase activities of platelets have been measured in diluted platelet-rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense-body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and alpha-granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard-Soulier syndrome, however, have approximately 10-fold higher prothrombinase activities in the non-stimulated form than normal non-stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non-stimulated Bernard-Soulier platelets, earlier described by Perret et al (1983).

Bernard-Soulier Syndrome↗
PubMed1986

A role for glycoprotein IIb-IIIa complex in the binding of IgE to human platelets and platelet IgE-dependent cytotoxic functions.

A possible relationship between binding sites for Immunoglobulin E (IgE) on human platelets, involved in IgE-dependent cytotoxic functions of platelets against helminth parasites, and well-characterized platelet constituents involved in haemostasis, was investigated. We first explored the interaction with IgE of platelets from patients with rare inherited deficiencies of defined platelet constituents and functions: Glanzmann's thrombasthenia, Bernard-Soulier and grey platelet syndromes. We report that only type I and II thrombasthenic platelets, which lack the membrane glycoproteins (GP) IIb and IIIa, failed to bind IgE and to exhibit IgE-dependent effector functions. Since thrombasthenic monocytes, however, showed normal interaction with IgE, this defect appeared restricted to platelets. Polyclonal and monoclonal antibodies directed against GP IIb-IIIa complex, but not monoclonal antibody directed against GP Ib, inhibited the binding of IgE to normal platelets, and their IgE-dependent cytotoxicity. Taken together, these findings indicate a relation between the GP IIb-IIIa complex and the expression of IgE binding sites and IgE-dependent effector functions in human platelets.

Blood Platelets↗
PubMed1986

Thrombocytopenia with thrombocytopathy possibly related to abnormalities of intracellular Ca++ fluxes and followed by the development of leukaemia.

A patient is described who presented a thrombocytopenia with thrombocytopathy followed by the development of a leukaemia. The disorder was characterized by decreased aggregation in the presence of ADP, and a lack of aggregation in the presence of arachidonic acid, natural endoperoxide or collagen. In parallel, 14C-serotonin release was severely decreased or nil in response to these inducers. Thrombin induced a slightly decreased aggregation and a normal 14C-serotonin release. Thromboxane B2 (T X B2) synthesis was normal after stimulation by arachidonic acid, natural endoperoxide or thrombin showing a normal arachidonate metabolism. In addition, the mepacrine test showed no significant decrease of the number of dense bodies with an average of 4.6 per platelet (versus 5.4 +/- 0.8 sd in controls). Stimulation by ionophore A 23187 failed to induce aggregation, 14C-serotonin release, or T X B2 synthesis. Furthermore, in the presence of EDTA, A 23187 did not provoke activation as reflected by 14C-serotonin release or T X B2 synthesis. Thus, in this case of thrombocytopathy, the hypothesis of abnormal intracellular Ca++ fluxes responsible for the defective platelet release phenomenon, was suggested.

Adenosine Diphosphate↗
PubMed1985

Possible involvement of two proteins (phosphoprotein and CD9 (p24)) in regulation of platelet calcium fluxes.

The monoclonal antibody ALB6 directed against the leukocyte differentiation antigen CD9 (p24) increases the calcium incorporation into isolated platelet membrane vesicles enriched in internal membranes. The similarities of the effects of both the monoclonal antibody and the catalytic subunit of the cAMP-dependent protein kinase (C, subunit), which phosphorylates a protein of an apparent molecular mass of 23 kDa, led us to investigate the relationship between CD9 (p24) and the 23-kDa phosphoprotein (p23). ALB6IgG does not inhibit the C.subunit-induced phosphorylation of p23 and the immunoadsorption by ALB6IgG of p24 associated to membrane vesicles does not alter the phosphorylation pattern. Thus, proteins of similar molecular mass appear to be involved in calcium fluxes: one is recognized by the ALB6 antibody while the other can be phosphorylated by the C-subunit.

Antibodies, Monoclonal↗
PubMed1985

Platelet membrane molecular organization: relationship with membrane bound calcium.

The fatty acid spin label 5 nitroxide stearate has been used to determine the membrane organization changes induced by platelet aggregation. A decrease in order is observed with thrombin, even in the presence of EDTA, when aggregation is inhibited. Conversely, after aggregation by the calcium ionophore A23187 the rigidity of the phospholipids is not modified. These effects are discussed in relation to the release of membrane bound calcium induced by thrombin.

Blood Platelets↗
PubMed1985

Serotonin release and phosphoinositide breakdown in thrombin-induced activation of human platelets.

The release reaction and the metabolism of inositol lipids were studied in parallel in washed human platelets following the activation by low doses of thrombin. The breakdown of phosphatidylinositol 4,5-bisphosphate (PI-P2) was accompanied by the release of serotonin. These events preceded the breakdown of the other phosphoinositides and the release of alpha-granules and lysosome constituents, respectively. The secretion of serotonin probably is triggered by products of thrombin-induced activation of the phospholipase C directed against PI-P2.

Blood Platelets↗
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

Molecular membrane organization in normal and pathological platelets: changes in inositide metabolism and membrane fluidity.

The role of platelet plasma membrane in mediating cellular response to external stimuli was investigated by studying phosphatidylinositol turnover and structural physical molecular alterations. Phosphoinositide turnover was evaluated by phosphatidylinositol breakdown and phosphatidic acid (PA) synthesis. The membrane structure was investigated by measuring fatty acid chain organization, flexibility and movements using the spin label method. When platelets are stimulated by thrombin, a rapid phosphatidylinositol bisphosphate (PIP2) breakdown is observed, accompanied by an immediate PA synthesis. At the same time a membrane-bound calcium liberation within the cell is revealed by the decrease in chlortetracycline fluorescence, and the dense granule constituent serotonin is released from the cell. These events result in disorganization of the platelet membrane as estimated by the decrease in order parameter. All these thrombin-induced effects occur in the presence of EDTA, thus being independent of external calcium and aggregation. By contrast ionophore A 23187 does not induce any PIP2 breakdown nor structural disorganization and the dense granule release measured at 10 seconds is only very weak. These results were confirmed by studies on pathological platelets in which PIP2 breakdown was normal when release was normal, such as in Glanzman thrombasthenia, or did not occur when no granule release was measurable as in Gray-platelet syndrome. It is suggested that membrane-bound calcium has a structural rather than a functional role.

Blood Platelet Disorders↗
PubMed1984

Simultaneous isolation of two platelet membrane fractions: biochemical, immunological and functional characterization.

Simultaneous isolation of two platelet membrane subfractions was achieved by centrifugation on 40% sucrose from a 100.000 g crude membrane fraction. Characterization of both types of membranes was carried out by different biochemical and immunological markers. Using a surface label, 3H Concanavalin A (3HCon A), a marker enzyme, phosphodiesterase, and lipid analysis, one of the fraction has been identified as external or plasma membranes, the other consists of intracellular membranes. Further two specific antibodies directed against external membrane antigens (LeKa and IgG L) react almost exclusively with the external membranes. Finally both kinds of membranes were able to uptake calcium but the affinity for this cation was higher for the internal than for the external membranes. This suggests that both membranes are implicated in the regulation of the cytoplasmic calcium concentration and that the internal membranes (dense tubular system) play the major part in this regulation.

Antigens↗
PubMed1984

Further characterization of human platelet activation in the absence of aggregation: phosphorylations of specific proteins and relationship with platelet secretion.

Platelet aggregation and secretion have been described to be associated with phosphorylation reactions. Thrombasthenic and EDTA-treated control platelets undergo a normal serotonin release in the absence of aggregation. We now studied the phosphorylation of specific proteins associated with platelet secretion. In the presence of ionophore, significant increases occurred in the phosphorylation of two polypeptides of 43,000 and 20,000 molecular weight ( P43 and P20) in a concentration dependent manner, and this was accompanied by an increase in the 14C-5HT release. The 32P-labelling of P43 and P20 reaches a peak within 1 min of platelet activation and is followed by a rapid dephosphorylation over the next 2-10 min. While the P20 is identified as the myosin light chain, the identity and the function of the P43 remain unknown. Isoelectric focusing separates 4 proteins from P43 during two dimensional electrophoresis, but only one of them is phosphorylated by A 23187. Chlorpromazine (CPZ) and trifluoperazine (TFP) inhibit the P43 and P20 phosphorylation as well as the 14C-5HT release in a dose dependent manner. The inhibitory action of the drugs is more pronounced for P43 than for P20, especially when the reactions are carried out at 20 degrees C instead of at 37 degrees C, while the release reaction is still inhibited under these conditions. These results allow different hypotheses for the relationship of phosphorylation-secretion and indicate the importance of one of these proteins ( P43 ) for the release reaction.

Blood Platelets↗
PubMed1984

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase.

It is generally agreed that arachidonic acid (20: 4 omega 6) can stimulate platelet aggregation after conversion to prostaglandin G2 and H2 and thence to thromboxane A2. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca2+-free buffer. Their stimulation by C 20: 4 6 was followed by 14C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca2+ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [14C]-5HT release. Other C 20 polyunsaturated fatty acids could not substitute for C 20: 4 omega 6 in inducing aggregation, and Ca2+ was found to be a prerequisite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use of the lipoxygenase inhibitor BW 755 C did not prevent C 20: 4 omega 6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these conditions, C 20: 4 omega 6-induced platelet aggregation might be due to an increased membrane permeability to Ca2+ induced by this fatty acid in the absence of oxidation.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1983

Triphosphoinositide breakdown and dense body release as the earliest events in thrombin-induced activation of human platelets.

The activation by thrombin of human platelets prelabelled with 32P induced a 30-40% decrease in 32P-triphosphoinositides (TPI) in the first 10 sec; the decrease in the other 32P-labelled phosphoinositides occurred by 20-30 sec. At 10 sec., the intensity of these effects was maximum with 0.2-0.4 U/ml thrombin. Under these conditions, 53, 20 and 15% of the dense granule, alpha-granule and lysosome constituents, respectively were released and thromboxane B2 synthesis reached only 10% of its maximum. Together with experiments carried out with chlorpromazine - or PGE1 - treated platelets, our results suggest the existence of a close relationship between TPI-breakdown and dense body release which appear to be the earliest events resulting from the activation of human platelets by thrombin.

Blood Platelets↗
PubMed1983

Regulation of calcium accumulation and efflux from platelet vesicles. Possible role for cyclic-AMP-dependent phosphorylation and calmodulin.

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.

Adenosine Triphosphate↗