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Biomedical subjects

J M Freyssinet

Publications and source records attributed to J M Freyssinet.

At least 37 records · Page 2Linked to original sources
PubMed1999

Annexin V delays apoptosis while exerting an external constraint preventing the release of CD4+ and PrPc+ membrane particles in a human T lymphocyte model.

Phosphatidylserine exposure in the exoplasmic leaflet of the plasma membrane is one of the early hallmarks of cells undergoing apoptosis. The shedding of membrane particles carrying Ags testifying to their tissue origin is another characteristic feature. Annexin V, a protein of as yet unknown specific physiologic function, presents a high Ca2+-dependent affinity for phosphatidylserine and forms two-dimensional arrays at the membrane surface. In this study, we report the delaying action of annexin V on apoptosis in the CEM human T cell line expressing CD4 and the normal cellular prion protein (PrPc), two Ags of particular relevance to cell degeneration and with different attachments to the membrane. The effect of annexin V was additive to that of z-Val-Ala-Asp-fluoromethyl ketone, a potent caspase inhibitor. Annexin V significantly reduced the degree of proteolytic activation of caspase-3, and totally blocked the release of CD4+ and PrPc+ membrane particles. z-Val-Ala-Asp-fluoromethyl ketone was a more powerful antagonist of caspase-3 processing, but prevented the shedding of CD4+ vesicles only partially and had no effect on that of PrPc+ ones. These results suggest that an external membrane constraint, such as that exerted by annexin V, has important consequences on the course of programmed cell death and on the dissemination of particular Ags. In vivo, annexin V had a significant protective effect against spleen weight loss in mice treated by an alkylating agent previously shown to induce lymphocyte apoptosis.

Animals↗
PubMed1999

Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia.

Paroxysmal nocturnal hemoglobinuria (PNH), frequently occurring during suppressed hematopoiesis including aplastic anemia (AA), is a clonal disorder associated with an increased incidence of thrombotic events. Complement-mediated hemolysis, impairment of the fibrinolytic system, or platelet activation are thought to be responsible for the associated thrombotic risk. We investigated here the elevation of membrane-derived procoagulant microparticles in the blood flow of such patients. Elevated levels of circulating microparticles were in fact detected in both de novo PNH patients and AA subjects with a PNH clone, but not in those with AA without a PNH clone. The cellular origin of the microparticles was determined in PNH samples; most stemmed from platelets. Glycophorin A+ particles were rarely detected. Therefore, platelet activation, resulting in the dissemination of procoagulant phospholipids in the blood flow, could be one of the main causes for the elevated thrombotic risk associated with PNH. These observations suggest that shed membrane particles can be considered a valuable biological parameter for the assessment of possible thrombotic complications in patients with PNH.

Anemia, Aplastic↗
PubMed1999

Detection of phosphatidylserine surface exposure on human erythrocytes using annexin V-ferrofluid.

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.

Annexin A5↗
PubMed1999

Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity.

BACKGROUND: The specific role of apoptosis in human atherosclerosis remains unknown. During apoptotic cell death, phosphatidylserine exposure on the cell surface confers a high tissue-factor (TF)-dependent procoagulant activity. METHODS AND RESULTS: In this study, we examined the role of apoptotic cell death in the promotion of plaque thrombogenicity. TF expression and its relation to apoptosis was analyzed in 16 human atherosclerotic plaques by the use of immunohistochemical techniques. The presence of shed membrane apoptotic microparticles was analyzed in extracts from 6 human atherosclerotic plaques and 3 underlying arterial walls. The microparticles were captured by annexin V and their amounts estimated with respect to their phospholipid content by use of a prothrombinase assay. The prothrombogenic potential of the microparticles was further assessed by the measurement of total and microparticle-dependent TF activity in the extracts. The cell origin of the microparticles was determined after capture by specific antibodies. We were able to detect marked TF expression in the plaques in close proximity to apoptotic cells and debris, suggesting a potential interaction between TF and the apoptotic cell surfaces. High levels of shed membrane apoptotic microparticles were detected in extracts from atherosclerotic plaques but not in the underlying arterial walls (29.5+/-3.7 nmol/L phosphatidylserine equivalent versus 1.3+/-0.4 nmol/L, respectively, P<0.02). The microparticles were mainly of monocytic and lymphocytic origin and retained 97+/-2% of total TF activity, indicating a direct causal relationship between shed membrane microparticles and procoagulant activity of plaque extracts. CONCLUSIONS: These results indicate that shed membrane microparticles with procoagulant potential are produced in human atherosclerotic plaques. Apoptosis could be a critical determinant of plaque thrombogenicity after plaque rupture.

Acute Disease↗
PubMed1998

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Amino Acid Sequence↗
PubMed1998

Impaired redistribution of aminophospholipids with distinctive cell shape change during Ca2+-induced activation of platelets from a patient with Scott syndrome.

We have investigated phospholipid redistribution, membrane vesicle shedding, shape change, and granule release following A23187 activation of platelets from a patient with Scott syndrome, characterized by impaired transmembrane migration of phosphatidylserine (PS) accompanied by haemorrhagic complications, and two of her children. Electron spin resonance spectroscopy measurement of phospholipids redistribution showed that the internalization of PS was unaffected by the disorder but, after activation, PS exposure was significantly reduced in platelets from the homozygous-type patient. Vesicle shedding was also reduced in these platelets. However, the slow redistribution of phosphatidylcholine was similar to that observed in normal platelets. When treated with calpeptin, platelets from the homozygous-type patient, unlike normal or heterozygous Scott syndrome platelets, showed a smoothly rounded shape without filopods after activation. Following A23187 activation of normal platelets, filopod formation was consecutive to the re-exposition of aminophospholipids on the outer leaflet of the plasma membrane, and the existence of a floppase (outward aminoPLs translocase) has been suggested. In homozygous Scott syndrome platelets the deficiency in PS re-exposition, the absence of filopod formation, and low vesicle shedding are correlated with each other, and argue in favour of a disruption of the proposed floppase activity.

Biological Transport↗
PubMed1997

Another link between phospholipid transmembrane migration and ABC transporter gene family, inferred from a rare inherited disorder of phosphatidylserine externalization.

The mechanisms involved in the maintenance or loss of the asymmetric distribution of phospholipids in the cell plasma membrane remain mysterious. In the yeast Saccharomyces cerevisiae, the transmembrane migration of certain phospholipids is controlled by transcription regulators of various ATP-binding cassette (ABC) transporters. The P-glycoprotein membrane transporters encoded by the multidrug resistance (MDR) genes, members of the ABC protein family, act as lipid translocases in mammalian cells. We report here the lack of expression of MDR genes in lymphoblasts derived from the B cells of a patient with an inherited Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications. From microsatellite analysis of 7q21.1 and functional assessment, the most likely explanation accounting for Scott phenotype is a mutation in an unlinked gene coding for a regulatory protein necessary for the expression of MDR genes. Because phosphatidylserine externalization is also one of the hallmarks of cells undergoing apoptosis, these observations are suggestive of a relationship between basic processes such as multidrug transport, apoptosis and procoagulant phospholipid exposure.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

Conformational adaptation of annexin V upon binding to liposomes: a time-resolved fluorescence study.

The fluorescence intensity decay of the single tryptophan residue, Trp-187, of free annexin V is described by the sum of three lifetime components (5.4, 1.3, and 0.4 ns), which may be correlated to three ground-state classes of Trp conformers. The two major classes (44 and 48%) are embedded in the protein matrix. When annexin V binds to calcium and liposomes made of dioleoylphosphatidylcholine and dioleoylphosphatidylserine, similar results are obtained whatever the (10-200) lipid ratio. The Trp fluorescence decay is fitted with only two components (6.9-7.2 and 2.0-2.2 ns). Decay-associated spectra reveal that the longest lifetime of bound annexin V can be related to Trp residues (60%) located in a partially polar environment, which could correspond to the protein-membrane interface. The shortest lifetime is attributed to Trp residues (40%) which reside in a hydrophobic surrounding: these Trp residues would penetrate into the phospholipid membrane and contribute to the stabilization of the 2D-array of annexin V molecules.

Annexin A5↗
PubMed1997

The significance of shed membrane particles during programmed cell death in vitro, and in vivo, in HIV-1 infection.

The plasma membrane remodeling, including the early transverse redistribution of phosphatidylserine, is a general feature occurring in cells in which a death program has been induced. In most cases, studies of this kind have focused mainly on cells. In this study, we report a clear correlation between the degree of apoptosis induced by a variety of agents in several types of cultured cells and the amount of shed membrane microparticles captured in the corresponding supernatants by insolubilized annexin V, a protein showing a strong affinity for phosphatidylserine. Such particles carry membrane antigens specific of the cells they stem from, and through which capture is also feasible. Homologous circulating microparticles were captured in peripheral blood from individuals with HIV-1 infection. A substantial proportion bore CD4 antigen. In some cases, CD4+ particles could be detected even in the absence of circulating CD4+ T cells, testifying to the presence of such resident cells in lymphoid tissues. These results suggest that shed membrane particles are one of the hallmarks of programmed cell death, of particular interest when the corresponding cells are hardly accessible.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages.

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.

Animals↗
PubMed1997

Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics.

The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium-ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine-labelled proteins in platelets treated with thrombin or a thrombin + collagen mixture and in particular the phosphorylation of a 40 kD band were severely reduced. Furthermore, inhibition of thiol-containing enzymes. including tyrosine-phosphatases, by N-ethyl maleimide did not lead to aminophospholipid exposure in the patient's platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.

Aged↗
PubMed1997

The significance of human monocyte thrombomodulin during membrane vesiculation and after stimulation by lipopolysaccharide.

Thrombomodulin acts as an essential membrane cofactor of thrombin in the triggering of the natural anticoagulant protein C pathway responsible for the inactivation of procoagulant cofactors VIIIa and Va. Because monocytes play a critical role in the coupling between infection/inflammation and thrombosis, the fate of monocyte thrombomodulin was assessed at the cell plasma membrane and on derived microparticles. A significant basal level of thrombomodulin activity was measured on unstimulated monocytes and microparticles. Lipopolysaccharide treatment resulted in increased thrombomodulin activity on monocytes (approximately 40%) and microparticles (approximately 80%), whereas tissue factor and prothrombinase activities were strongly expressed on both. Flow cytometry detection of thrombomodulin antigen confirmed its presence on unstimulated monocytes and microparticles. A decrease (approximately 30%) in thrombomodulin labelling was noticed on stimulated monocytes. Labelling of microparticles shed from stimulated and unstimulated monocytes remained unchanged, only an increased proportion of microparticles (approximately 20%) was observed. The absence of early down-regulation of thrombomodulin following monocyte stimulation suggests that it fulfils an important regulatory function of membrane-associated procoagulant activities. This would be of particular significance at the surface of microparticles having the ability to diffuse and concentrate by adherence at inflammatory sites.

Cell Membrane↗
PubMed1996

Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells.

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7beta-hydroxycholesterol (7beta-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7beta-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may results from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

Annexin A5↗
PubMed1996

Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications, is an inherited disorder.

An as yet single family with a bleeding history is shown to present the characteristic lack of membrane expression of procoagulant phospholipids observed in Scott syndrome. Low prothrombin consumption in the serum of the propositus, a 71-year-old woman, and two of her children was the sole abnormal hemostasis parameter. The degree of exposure of procoagulant phospholipids, chiefly phosphatidylserine, was reduced in stimulated platelets, erythrocytes and Epstein-Barr virus-infected B lymphocytes. The data are compatible with homozygous status of the propositus and heterozygous status of her children. Scott syndrome appears to be transmitted as an autosomal recessive trait reflecting the deletion or mutation of a putative outward phosphatidylserine translocase. The detailed knowledge of this transporter could have an impact in membrane physiology.

Aged↗
PubMed1996

Detection of bilayer phospholipid-binding antibodies using flow cytometry.

Antiphospholipid antibodies (APL) are usually detected using solid-phase immunoassays, where cardiolipin is the most common capture antigen. Phosholipids are believed to adopt a monolayer organization when coated onto polystyrene after evaporation of the solvent. However, bilayer phospholipids are probably those evidenced as microparticles or cell fragments circulating in vivo under various pathological circumstances. The surface density of monolayer phospholipids on polystyrene is six times lower than that of bilayer phospholipids. In order to assess the influence of phospholipid organization on the detection of APL, we prepared glass microspheres coated with bilayer phospholipids (cardiolipin, phosphatidylcholine, cholesterol). Such lipospheres enabled us to study the binding of antibodies in 1:100 diluted plasma samples from patients with anticardiolipin antibodies of IgG isotype previously diagnosed by ELISA. Among the 39 plasma samples analysed by flow cytometry, 17 showed positive IgG binding to lipospheres. Only four additional samples became positive when adding 20 micrograms/ml apolipoprotein H. The specificity of the binding was demonstrated by complete reversibility with 1.4 microM annexin V and with a large excess of liposomes of the same composition. The absence of correlation between liposphere and ELISA results suggests that different subgroups of antibodies are detected depending on the method. The detection of APL using bilayer phospholipids is an original assay and may represent a more physiopathological approach to the specificity of APL.

Aged↗
PubMed1996

Physiopathological significance of catalytic phospholipids in the generation of thrombin.

Thrombin generation is the culminating event of the coagulation cascade. It is initiated after the expression of tissue factor by endothelial cells and monocytes exposed to thrombogenic stimuli. Anionic phospholipids, chiefly phosphatidylserine, are necessary for the optimal activity of tissue factor and completion of the clotting process. They display a catalytic potential by allowing the formation of the characteristic enzyme complexes at the membrane surface. Platelets are viewed as the main source of procoagulant phospholipid referred to as platelet factor 3. The plasma membrane of resting cells presents an asymmetrical distribution of phospholipids, aminophospholipids being sequestered in the inner leaflet. Procoagulant phospholipids become available at the outer surface after cell stimulation. The collapse of the membrane asymmetry is thought to promote a phospholipid scrambling accompanied by the shedding of microparticles. The plasma membranes of such vesicles bear irreversibly externalized procoagulant phosphatidylserine and contain glycoproteins that testify to their tissue origin. Hence, microparticles could disseminate a dual procoagulant and adhesive potential. Thrombin autoamplification is exerted through feedback activation loops involving either coagulation factors or platelets. This article details the mechanisms by which procoagulant phospholipids promote the generation of an excess of thrombin. A new pharmacological approach of thrombosis is presented, based on the control of the exposure of procoagulant phospholipids and membrane microparticle shedding.

Animals↗