In vivo detection of apoptosis in an intracardiac tumor.
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Aged↗
Biomedical subjects
C P Reutelingsperger
Publications and source records attributed to C P Reutelingsperger.
Real-time imaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart.
We report a novel real-time imaging model to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, using fluorescent labeled annexin-V. Annexin-V binds to externalized phosphatidylserine (PS) of cells undergoing programmed cell death. With high-magnification (x100-160) real-time imaging, we visualized the binding of annexin-V to single cardiomyocytes. Kinetic studies at the single-cell level revealed that cardiomyocytes started to bind annexin-V within minutes after reperfusion, following an ischemic period of 30 minutes. The amount of bound annexin-V increased rapidly and reached a maximum within 20-25 minutes. Caspase inhibitors decreased the number of annexin-V-positive cardiomyocytes and slowed down the rate of PS exposure of cardiomyocytes that still bound annexin-V. This technology to study cell biology in the natural environment will enhance knowledge of intracellular signaling pathways relevant for cell-death regulation and strategies to manipulate these pathways for therapeutic effect.
Animals↗
Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation.
Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formation.
Animals↗
Patient dosimetry of intravenously administered 99mTc-annexin V.
UNLABELLED: Annexin V labeled with 99mTc is evaluated as a potential in vivo marker for tissue with increased apoptosis. Promising results in patients have been obtained with 99mTc-(n-1-imino-4-mercaptobutyl)-annexin V (99mTc-i-AnxV). Because information on biodistribution and radiation burden is desired for the application of any radiopharmaceutical, a dosimetric study of 99mTc-i-AnxV was undertaken. METHODS: Eight persons with normal kidney and liver functions were included in this study: six patients with myocardial infarction, one with Crohn's disease, and one healthy volunteer. Approximately 600 MBq 99mTc-i-AnxV were injected intravenously immediately before a dynamic study with a dual-head gamma camera in conjugate view mode. In the next 24 h, two to four whole-body scans were acquired. Patient thickness was determined from a transmission scan with a 57Co flood source. Organ uptake was estimated after correction for background, attenuation, and scatter, using a depth-independent buildup factor and an organ-size-dependent attenuation correction. Residence times were calculated from the dynamic and whole-body studies and used as input for the MIRDOSE 3.1 program to obtain organ-absorbed doses and effective dose. RESULTS: Activity strongly accumulated in the kidneys (21% +/- 6% of the injected dose at 4 h postinjection) and the liver (12.8% +/- 2.2%). Uptake in the target tissues (myocardium or colon) was limited and negligible from a dosimetric point of view. The biologic half-life of activity registered over the total body was 62 +/- 13 h. Of the excreted activity, approximately 75% went to the urine and 25% to the feces. The absorbed dose for the more strongly exposed organs was (in microGy/MBq): kidneys, 93 +/- 24; spleen, 22 +/- 6; liver, 17 +/- 2; testes, 15 +/- 3; thyroid, 10 +/- 6; urinary bladder wall, 7.5 +/- 2.6; and red bone marrow, 5.5 +/- 0.8. The effective dose was 9.7 +/- 1.0 microSv/MBq, corresponding to a total effective dose of 5.8 +/- 0.6 mSv for a nominally injected activity of 600 MBq. CONCLUSION: 99mTc-i-AnxV strongly accumulates in the kidneys and to a lesser degree in the liver. The associated effective dose per MBq is in the midrange of values found for routine 99mTc-labeled compounds. From a dosimetric point of view 99mTc-i-AnxV is therefore well suited for the study of apoptosis in patients.
Adult↗
Annexins: key regulators of haemostasis, thrombosis, and apoptosis.
Annexins form a family of proteins on the basis of structural and functional criteria. Their physiological relevance is still not fully understood, but is thought to involve phospholipid membrane associated processes. Recent data shed clues about their function in the regulation of thrombosis, haemostasis and apoptosis. These data arise from a multitude of physicochemical, cell biological, animal and clinical studies. This review presents this data and discusses them in the light of the novel understanding that apoptotic cells can contribute to thrombotic and haemostatic processes.
Annexins↗
Biodistribution and dosimetry of 99mTc-BTAP-annexin-V in humans.
The purpose of this study was to determine the biodistribution and the associated radiation dose of technetium-99m 4,5-bis(thioacetamido)pentanoyl-annexin-V (99mTc-Apomate), a tracer proposed for the study of apoptosis. Eight patients (including two females) with normal kidney and liver functions were included in the study. An activity of 580 +/- 90 MBq of 99mTc-Apomate was injected intravenously, immediately followed by a dynamic study of 30 frames of 1 min each. At about 1 h, 4 h and 20 h p.i., whole-body scans were acquired. All activity distributions were measured using a dual-head gamma camera. Before injection of activity, a transmission scan with a cobalt-57 flood source had been performed to determine patient attenuation. Blood samples were taken every 10 min during the first hour after injection, and at about 4 and 20 h. Urine and faeces were collected during the first 20 h. Organ uptake was estimated after correction for body background activity, attenuation and scatter. Residence times were calculated from the dynamic and whole-body studies and used as input in the Mirdose 3.1 program to obtain organ doses and effective dose. It was found that radioactivity strongly accumulated in the kidneys and the liver [at 70 min p.i., 28% +/- 8% and 20% +/- 4% of the injected dose (ID), respectively]. Uptake in the target tissues (lymphomas or heart) was negligible from a dosimetric point of view. Extrapolating data from the first 20 h, one finds that approximately 73% of the ID will be excreted in the urine, and 27% in the faeces. The biological half-life of the activity in the total body was 16 +/- 7 h. Some organ doses +/- standard deviation (SD) in microGy/MBq were: kidneys 63 +/- 22, urinary bladder 20 +/- 6, spleen 15 +/- 3, liver 13 +/- 3, upper large intestine 12 +/- 6, lower large intestine 8 +/- 4, testes 6 +/- 2 and red bone marrow 4 +/- 0.7. The effective dose was 7.6 +/- 0.5 microSv/MBq, corresponding to a total effective dose of 4.6 +/- 0.3 mSv for a nominal injected activity of 600 MBq. In conclusion, 99mTc-Apomate has a high uptake in the kidneys and liver--in fact a factor of 1.3-1.6 higher than that found for the previously studied 99mTc-(n-1-imino-4-mercaptobutyl)-annexin-V. The biological half-life is shorter, however, but still long compared with the physical half-life of 99mTc. The faster appearance of activity in the intestines may preclude imaging of apoptosis in the abdomen. The effective dose is within the lower range of values reported for typical 99mTc compounds.
Adult↗
Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.
INTRODUCTION: Phosphatidylserine (PS) externalization is regarded as one of the earliest hallmarks of cells undergoing programmed cell death. We studied the use of labeled human recombinant annexin-V, a protein selectively binding to PS, to detect cardiomyocyte death in an in vivo mouse model of cardiac ischemia and reperfusion (I/R). METHODS AND RESULTS: I/R was induced in mouse hearts by ligation and subsequent release of a suture around the left anterior descending coronary artery. Annexin-V (25 mg/kg) fused to a marker molecule was injected intra-arterially 30 minutes before euthanasia. After 15 minutes of ischemia followed by 30 minutes of reperfusion, 1.4+/-1. 2% (mean+/-SD) of the cardiomyocytes in the area at risk were annexin-V positive (n=6). This increased to 11.4+/-1.9% after 15 minutes of ischemia followed by 90 minutes of reperfusion (n=7) and to 20.2+/-3.3% after 30 minutes of ischemia followed by 90 minutes of reperfusion (n=7). In control mice, including those injected with annexin-V at the binding site of PS, no annexin-V-positive cells were observed. DNA gel electrophoresis showed typical laddering starting after 15 minutes of ischemia followed by 30 minutes of reperfusion, suggesting activation of the cell death program. Intervention in the cell death program by pretreatment with a novel Na(+)-H(+) exchange inhibitor substantially decreased annexin-V-positive cardiomyocytes from 20.2% to 2.2% in mice after 30 minutes of ischemia followed by 90 minutes of reperfusion. CONCLUSIONS: These data suggest that labeled annexin-V is useful for in situ detection of cell death in an in vivo model of I/R in the heart and for the evaluation of cell death-blocking strategies.
Animals↗
Visualisation of cell death in vivo in patients with acute myocardial infarction.
BACKGROUND: In-vivo visualisation and quantification of the extent and time-frame of cell death after acute myocardial infarction would be of great interest. We studied in-vivo cell death in the hearts of patients with an acute myocardial infarction using imaging with technetium-99m-labelled annexin-V-a protein that binds to cells undergoing apoptosis. METHODS: Seven patients with an acute myocardial infarction and one control were studied. All patients were treated by percutaneous transluminal coronary angioplasty (six primary and one rescue), resulting in thrombolysis in myocardial infarction (TIMI) III flow of the infarct-related artery. 2 h after reperfusion, 1 mg annexin-V labelled with 584 MBq Tc-99m was injected intravenously. Early (mean 3.4 h) and late (mean 20.5 h) single-photon-emission computed tomographic (SPECT) images of the heart were obtained. Routine myocardial resting-perfusion imaging was also done to verify infarct localisation. FINDINGS: In six of the seven patients, increased uptake of Tc-99m-labelled annexin-V was seen in the infarct area of the heart on early and late SPECT images. No increased uptake was seen in the heart outside the infarct area. All patients with increased Tc-99m-labelled annexin-V uptake in the infarct area showed a matching perfusion defect. In a control individual, no increased uptake in the heart was seen. INTERPRETATION: Increased uptake of Tc-99m-labelled annexin-V is present in the infarct area of patients with an acute myocardial infarction, suggesting that programmed cell death occurs in that area. The annexin-V imaging protocol might allow us to study the dynamics of reperfusion-induced cell death in the area at risk and may help to assess interventions that inhibit cell death in patients with an acute myocardial infarction.
Aged↗
S-nitroso-N-acetylpenicillamine and nitroprusside induce apoptosis in a neuronal cell line by the production of different reactive molecules.
CHP212 neuroblastoma cells were exposed to two different nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside. Apoptosis and necrosis were determined with flow cytometric analysis of annexin V binding and propodium iodide uptake. Both S-nitroso-N-acetylpenicillamine and sodium nitroprusside induced apoptosis, but with a different time dependency. Oxyhemoglobin (NO scavenger) attenuated the toxicity of S-nitroso-N-acetylpenicillamine, but had no effect on the toxicity of sodium nitroprusside. By contrast, deferoxamine (iron chelator) attenuated the toxicity of sodium nitroprusside, but had no effect on the toxicity of S-nitroso-N-acetylpenicillamine. Urate (ONOO(-) scavenger) did not influence the toxicity of either S-nitroso-N-acetylpenicillamine or sodium nitroprusside, but protected from SIN-1 (3-morpholinosydnonimine, ONOO(-) donor). It was shown that both dithiothreitol and ascorbic acid affected the toxicity of S-nitroso-N-acetylpenicillamine and sodium nitroprusside in opposite ways. In the presence of dithiothreitol, superoxide dismutase and catalase decreased the toxicity of sodium nitroprusside. In the presence of cells, but not in their absence, S-nitroso-N-acetylpenicillamine decomposed with a half-life of about 4 h as assessed by the production of nitrite and absorbance reduction at 335 nm. Sodium nitroprusside decomposed very slowly in the presence of cells as assessed by the production of ferrocyanide. It can be concluded that (1) slow and sustained release of NO from S-nitroso-N-acetylpenicillamine at the cell surface causes apoptosis in CHP212 cells, probably without the involvement of ONOO(-), (2) sodium nitroprusside causes apoptosis by the production of H(2)O(2) and/or iron, rather than NO, and probably has to be taken up by the cell for decomposition.
1-Methyl-4-phenylpyridinium↗
In situ detection of apoptosis in dental and periodontal tissues of the adult mouse using annexin-V-biotin.
An early event in apoptosis is exposure of phosphatidylserine, an aminophospholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space. Annexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylserine, which can be applied to detect apoptosis. We injected biotin-labelled Anx-V intravenously in adult mice and examined the tissue distribution of Anx-V-labelled cells in dental and periodontal tissues using ABC-peroxidase histochemistry. In the continuously erupting incisors, strong and frequent immunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts. Frequency of staining in odontoblasts and pulp cells was low but increased slightly at older stages of dentinogenesis. Labelling was also seen in phagocytic or phagocytic-like cells in the enamel organ and pulp. A positive staining was furthermore found in fibroblasts of the periodontal ligament in continuously erupting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pressure cooking. We conclude that Anx-V-biotin labels dental cells in early stages of cell death and indirectly cells that have ingested labelled apoptotic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturation stage ameloblasts. Thus, labelling with Anx-V is a useful marker for studying cell death and the dynamics of clearance of apoptotic cells during tooth development.
Animals↗
Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling.
Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.
Animals↗
Markers of apoptosis in cardiovascular tissues: focus on Annexin V.
In the last decade, apoptosis (or programmed cell death) has become appreciated as an important process in the development of the cardiovascular system. Moreover, apoptosis contributes to the adaptation of the system to the environment. We are at the beginning of understanding its relevance to cardiovascular physiology and pathology. This understanding forms the key to implement apoptosis in diagnosis and therapy of cardiovascular diseases. New avenues for pharmacological intervention are expected to arise from the synergy of our knowledge about the molecular mechanisms of apoptosis, and how apoptosis integrates in the complex environment of the cardiovascular tissue. The latter strongly depends on techniques to measure apoptosis. Currently, we are facing a relative paucity in available techniques, covering both specificity and sensitivity, and furthermore allowing quantitative analysis, preferably in combination with morphology. This field, however, is rapidly evolving and is fed by the expanding knowledge about the molecular mechanisms of apoptosis. In this paper we will briefly review the available techniques to detect and/or quantify apoptosis. These methods are based on the analysis of cellular morphology, either by light- or electron microscopy, DNA fragmentation (TdT-mediated X-dUTP nick end labeling or in situ nick end labeling), or cytoplasmic and membrane changes. Furthermore, the advantages and limitations of these techniques for their use in cardiovascular research will be outlined. In the text we will refer to available reviews and protocols which discuss the techniques in more detail. The main part of this article will, however, focus on a recently introduced technique, the Annexin V-based apoptosis detection assay. The principle, characteristics, pro's and contra's of this new apoptosis detection assay will be discussed.
Animals↗
A new principle for rapid immunoassay of proteins based on in situ precipitate-enhanced ellipsometry.
A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.
Annexin A5↗
Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death.
The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca2+ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin V adhesion and by Propidium Iodide exclusion. Transition of the mitochondrial membrane potential was monitored by the occurrence of decay of DiOC6 fluorescence. Intracellular pH shifts were monitored by changes in the ratio of fluorescence at 575 nm and at 635 nm of SNARF-1-AM. Fluctuations in intracellular Ca2+ concentration were established by changes in Fura red quenching. The Jurkat cells were sensitive to anti-Fas treatment, while HSB-2 cells were not. HSB-2 cells appeared more sensitive to radiation damage than Jurkat cells. In all experiments the transition of mitochondrial membrane potential occurred first, almost immediately followed by PS exposure. Fluctuations in intracellular Ca2+ concentration occurred later and were less outspoken. A decrease in intracellular pH occurred not earlier than 24 hours after anti-Fas treatment. Chelation of intracellular Ca2+ concentration with BAPTA-AM had no effect on the time sequence of cell death related events.
Journal Article↗
Spatiotemporal distribution of dying neurons during early mouse development.
Apoptosis is a critical cellular event during several stages of neuronal development. Recently, we have shown that biotinylated annexin V detects apoptosis in vivo in various cell lineages of a wide range of species by binding to phosphatidylserines that are exposed at the outer leaflet of the plasma membrane. In the present study, we tested the specificity by which annexin V binds apoptotic neurons, and subsequently investigated developmental cell death in the central and peripheral nervous system of early mouse embryos at both the cellular and histological level, and compared the phagocytic clearance of apoptotic neurons with that of apoptotic mesodermal cells. Our data indicate: (i) that biotinylated annexin V can be used as a sensitive marker that detects apoptotic neurons, including their extensions at an early stage during development; (ii) that apoptosis plays an important part during early morphogenesis of the central nervous system, and during early quantitative matching of brain-derived neurotrophic factor and neurotrophic factor 3 responsive postmitotic large clear neurons in the peripheral ganglia with their projection areas; and (iii) that apoptotic neurons are removed by a process that differs from classical phagocytosis of non-neuronal tissues.
Animals↗
Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis.
Cells under oxidative stress induced by peroxides undergo functional and morphological changes, which often resemble those observed during apoptosis. Peroxides, however, also cause the oxidation of intracellular reduced glutathione (GSH). We investigated the relation between these peroxide-induced effects by using human umbilical vein endothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 and ECV304. With HUVEC, tert-butyl hydroperoxide (tBH) or hydrogen peroxide application in the presence of serum induced, in a dose-dependent way, reorganization of the actin cytoskeleton, membrane blebbing, and nuclear condensation. These processes were accompanied by transient oxidation of GSH. With ECRF24 cells, this treatment resulted in less blebbing and a shorter period of GSH oxidation. However, repeated tBH addition increased the number of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVEC and ECRF24 cells, but not in ECV304 cells. Neither membrane blebbing nor nuclear condensation in any of these cell types was due to apoptosis, as evidenced by the absence of surface expression of phosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although high tBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells, peroxide-induced cytoskeletal reorganization and bleb formation correlate with the degree of GSH oxidation but do not represent an early stage of the apoptotic process.
Apoptosis↗
Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.
Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI.
Binding Sites↗
Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer.
Annexin V belongs to the family of calcium-dependent phospholipid binding proteins and binds almost solely to phosphatidylserine (PS). When annexin V is used to detect loss of membrane asymmetry in cellular systems, the binding properties under physiological conditions are of importance. Most biochemical studies use optimized binding conditions, conditions that are often far from physiological. For the interpretation of flow cytometric studies that use fluorescent annexin V to probe PS exposure, it is important to know what mixture of lipid species exposed in the outer leaflet of a membrane can evoke a positive annexin V signal. The lipid species is important in this respect as well as the concentration that just evokes a positive signal (detection level). Furthermore, the influence of the composition of the lipid matrix (cholesterol content, other phospholipid species) was investigated, as well as the influence of the calcium concentration on annexin V binding. In this study, we report on the binding of annexin V to phospholipid bilayers (adsorbed to glass beads) as measured by flow cytometry at physiological conditions. Annexin V binding was found to increase rapidly, with increasing PS concentrations up to a certain level (attained at 6 mol% PS). Further increase of the PS concentration resulted only in a slight increase of annexin V binding. Calcium concentrations below 3 mM were found to reduce the sensitivity of the binding assay. Phosphatidylethanolamine incorporated in the phospholipid bilayer resulted in a lower threshold for the binding assay, whereas sphingomyelin had no influence on the binding of annexin V and cholesterol reduces binding of annexin V to lipid bilayers. These data may help in the interpretation of results obtained from binding of annexin V to cell membranes (e.g., involved in apoptosis).
Annexin A5↗