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Query: UNIPROT:P08758 (annexin V)
 9,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphatidylserine (PS) exposure serves as a procoagulant stimulus and a signal for phagocytic clearance of apoptotic cells. In order to measure PS exposure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leukocytes were identified by CD45 and side-scatter gating, and platelets by CD6 1 and side-scatter gating. The absolute number of annexin V molecules bound per cell was determined from an independent calibration procedure. Normal populations had the following levels of annexin V binding (in molecules per cell): lymphocytes, 0.53 x 10(3) neutrophils, 1.75 x 10(3) monocytes, 2.45 x 10(3) platelets, 0.14 x 10(3). These levels represent </= 0.1% of the values obtained after maximal stimulation of PS exposure with calcium ionophore, confirming that virtually all PS is intracellular in normal circulating leukocytes and platelets. Pretreatment of whole-blood samples with ammonium chloride to lyse erythrocytes caused a 9- to 300-fold increase in annexin V binding to leukocytes, indicating that analysis of unlysed whole-blood samples is essential to avoid artifactual increases in annexin V binding to leukocytes. Comparison of annexin V with two other markers of platelet activation, CD62P and the activation-dependent epitope of glycoprotein IIb/IIIa detected by the PAC I antibody, indicated that platelets from normal donors showed the least amount of activation with the annexin V marker. Whole-blood flow cytometry with annexin V can reliably measure the state of PS exposure in platelets and leukocytes, and the results confirm that these cell
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PMID:Measurement of phosphatidylserine exposure in leukocytes and platelets by whole-blood flow cytometry with annexin V. 1074 22

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.
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PMID:Phosphatidylserine-dependent adhesion of T cells to endothelial cells. 1083 84

Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.
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PMID:Apoptosis in T-lymphocyte subsets in human immunodeficiency virus-infected children measured immediately ex vivo and following in vitro activation. 1113 98

Annexin V is a 36-kDa protein which, it has been suggested, is a factor in protecting the vascular endothelium from attack by antibodies to other phospholipid-binding proteins. Competition between annexin V and beta2-glycoprotein I (beta2GPI) for phospholipid surfaces is complicated by empirical observations regarding alterations in binding to anionic phospholipid, primarily phosphatidylserine. In order to elucidate the effect of phospholipid composition and divalent cations (Ca(+2) and Mg(+2)) on annexin V binding to phospholipid, we used biotinylated annexin V and peroxidase-conjugated avidin D to probe the binding of annexin V to phospholipid-coated wells of polystyrene microtiter plates. Binding of annexin V to anionic phospholipid is Ca(+2)-dependent and, in its absence, annexin V was found to bind most avidly to 100% phosphatidylcholine in a saturable manner, followed by decreasing percentages of phosphatidylcholine. Ca(+2) was found to inhibit phosphatidylcholine binding and promote the binding of phospholipid mixtures containing phosphatidylserine. Phosphatidylserine (100%) did not bind annexin V as strongly as mixtures of 50% and 75% phosphatidylserine. The effect with Ca(+2) suggests saturation of Ca(+2)-binding sites on annexin V, reached under our experimental conditions at approximately 1 mM. Under the same conditions, Mg(+2) slightly enhanced the binding of all of the phospholipid compositions studied. Ca(+2)-dependent binding of annexin V was competitively inhibited by Mg(+2); 5 mM Mg(+2) reduced binding significantly (p < 0.0001 by ANOVA, p < 0.05 for post hoc test of 5 mM vs 0 mM). These data suggest that the translocation of membrane phospholipid under the dynamics of ion transport in vascular endothelium may alter annexin V binding.
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PMID:The influence of lipid composition and divalent cations on annexin V binding to phospholipid mixtures. 1131 66

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean (Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A(2), and (31)P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A(2) assay. (31)P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.
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PMID:Transmembrane topography of plasma membrane constituents in mung bean (Vigna radiata L.) hypocotyl cells. I. Transmembrane distribution of phospholipids. 1142 92

Anemia is common in dialysis patients. Change in phospholipids asymmetry in red blood cells (RBCs) may affect the removal of RBCs from the circulation and thus shorten the lifespan of RBCs. In the present study, we investigated phospholipids asymmetry in RBCs in uremic patients and its relationship with anemia. We studied 34 continuous ambulatory peritoneal dialysis (CAPD) patients (age: 51 +/- 15 years), 73 hemodialysis (HD) patients (age: 48 +/- 12 years), 8 pre-dialysis renal-failure patients (age: 42 +/- 21 years), and 16 healthy controls (age: 32 +/- 9 years). All patients were clinically stable. Phospholipids asymmetry as measured by phosphatidylserine exposure was determined by a flow-cytometric annexin V-binding assay. Hemoglobin levels were 93 +/- 20 g/L, 83 +/- 17 g/L, 78 +/- 21 g/L, and 145.8 +/- 12.5 g/L for CAPD patients, pre-dialysis patients, HD patients, and healthy controls respectively. Phosphatidylserine exposure in RBCs was significantly higher in uremic patients as compared with healthy controls, especially in HD patients--whose values were significantly higher than values seen in CAPD patients and pre-dialysis patients. No significant difference was seen in RBC phosphatidylserine exposure between pre-dialysis patients and CAPD patients. Cells positive for annexin V binding were 1.58%, 1.40%, 2.11%, and 0.71% for CAPD patients, pre-dialysis patients, HD patients, and healthy controls respectively. Significant reverse correlations were seen between annexin V and hemoglobin (r = -0.381, p < 0.001), and between annexin V and hematocrit (r = -0.355, p < 0.001). Our results suggest that (1) anemia is common in our uremic patients, especially in HD patients; and (2) anemia in uremic patients may be partly related to the loss of phospholipids asymmetry in RBCs.
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PMID:Loss of phospholipids asymmetry in red blood cells contributes to anemia in uremic patients. 1151 Feb 98

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes. which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (deltapsi) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial deltapsi was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791+/-98 vs 567+/-102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial deltapsi collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial deltapsi (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5+/-1.7 vs 4.7+/-1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial deltapsi alteration. The results show that platelet aging in vivo is associated with a decrease in mitochondrial deltapsi and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.
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PMID:Platelet aging in vivo is associated with activation of apoptotic pathways: studies in a model of suppressed thrombopoiesis in dogs. 1203 96

Small pulmonary arterial thromboses can occur following splenectomy of patients with haemoglobin E/beta-thalassaemia (Hb E/beta-thal). We compared plasma markers of coagulation activation in vivo and red blood cell (RBC) markers of procoagulant activity in 15 Hb E/beta-thal patients who were not splenectomized (NS), 15 who had been splenectomized (S), and 15 normal controls (NC). Levels of plasma thrombin-antithrombin III complex (TAT) were significantly higher in the S group than in either the NS or the NC groups, and levels of prothrombin fragment 1.2 (F 1.2) were significantly higher in the S than in the NC group. Diluted Russell's viper venom clotting times were significantly shorter when RBCs from group S patients were added to the assay compared with RBCs from the NC group. Phosphatidylserine (PS) expression (% of annexin V-positive RBCs) on the outer leaflet of RBC membrane of both 'larger'- and 'smaller'-sized RBCs was significantly higher for the S than the NC group. The RBC PS expression of the S and the NS groups, respectively, accounted for 25 x 3% (P = 0 x 174) and 6.3% (P = 0 x 675) of the variation in plasma TAT levels. Our findings indicated that, when compared with NC, splenectomized patients with Hb E/beta-thal were in a chronic low-grade hypercoagulable state associated with increased numbers of circulating PS exposed RBCs. This condition may have a role in the risk of these patients for pulmonary arterial thromboses.
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PMID:Relationship between hypercoagulable state and erythrocyte phosphatidylserine exposure in splenectomized haemoglobin E/beta-thalassaemic patients. 1218 Oct 63

Phosphatidylserine (PS) is a membrane phospholipid which in intact cells is exclusively localized in the inner leaflet of the lipid bilayer. However, once cells undergo apoptosis or oxidative stress, PS molecules are exposed on the external surface of the cells and this contributes to their adherence to macrophages or endothelial cells. PS exposure on Plasmodium falciparum-infected red cells was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS. Involvement of exposed PS in the adherence of malaria-infected red cells to endothelial cells was examined by in vitro cytoadherence assays. Infected cells exposed PS on their surface as the intracellular parasites matured to trophozoite and schizont stages. Adherence of malaria-infected cells to CD36, CD36-expressing Chinese hamster ovary cells, thrombospondin, and C32 amelanotic melanoma cells was inhibited by annexin V, whereas ICAM-1- and chondroitin sulfate A-mediated binding was not. Further, PS liposomes and glycerophosphorylserine, but not phosphatidylcholine liposomes and glycerophosphorylcholine, inhibited the binding of infected cells to CD36 and thrombospondin. In conclusion, these results demonstrate that PS exposed on the surface of malaria-infected red cells contributes, in part, to the adherence of P. falciparum-parasitized red cells to CD36 and thrombospondin.
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PMID:Cytoadherence of malaria-infected red blood cells involves exposure of phosphatidylserine. 1243 74

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.
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PMID:Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold. 1270 40


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