Loss Of Phospholipid Asymmetry Research Articles

Potential fertilizer of spermatozoa in porcine epididymal tail post-orchiectomy.

The aim of this study was to determine the potential fertilizing of spermatozoa from the epididymal tail in different periods of time post-orchiectomy (P-OQ). Therefore, the study was approached in two stages. In the first stage, the orchiectomy was performed in 30 adult pigs. The testicles were stored at 5.00 ˚C in physiological saline solution for 5, 24, 48, 72, 96 and 120 hr. The spermatozoa were obtained by backflushing the vas deferens. The spermogram and fluorometric study were performed for each sample to evaluate the exposure of phosphatidyl-serine (PS) and acrosome reaction (AR). The second stage included the fertilization test, 16 prepubertal sows were selected, after synchronizing the oestrous cycle and the post-cervical artificial insemination was performed with the refrigerated sperm samples from each P-OQ time. The percentage of live sperm remained without significant changes until 96 hr P-OQ. An increase in the percentage of spermatozoa that showed a PS exposure was observed. The premature AR was evident after 72 hr. Considering that the artificial insemination was performed ensuring a minimum number of live sperms, no significant differences were observed in the number of embryos and corpora lutea. The results indicated that pig sperm collected from the epididymal tail P-OQ and stored for 5 and up to 72 hr at 5.00 ˚C had viable characteristics and maintained their fertilization ability. However, there was an increase in the loss of phospholipid asymmetry of the plasma membrane as time increased (72 and 96 hr), therefore, sperm viability was decreased.

Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 - a comparison with annexin V-FITC.

The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophore-stimulated T-lymphocytes further increases this fluorescence [Zweifach, 2000]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca2+-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss of phospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca2+ or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca2+-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca2+-release from intracellular stores and by Ca2+ and ionophore A23187 treatment showed that the increase in FM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca2+-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-014-0195-3 and is accessible for authorized users.

P4-175: Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer's disease

P4-175: Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer's disease

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

Investigation on the early events of apoptosis in senescent erythrocytes with special emphasis on intracellular free calcium and loss of phospholipid asymmetry in chronic renal failure

Background The pathophysiological link between increased blood concentrations of factors responsible for the derangement and erythrocyte membrane functions in chronic renal failure (CRF) patients are not thoroughly elucidated. We studied the erythrocyte characteristics and phospholipid asymmetry loss in CRF patients with different grades of uremia and also examined the involvement of intracellular free Ca 2+ in early events of apoptosis in uremic erythrocytes. Methods The studied population consisted of 90, age and sex matched control subjects (Group I) and 238 CRF cases divided into 3 groups (Group II, III and IV) according to urea concentrations and complexity of secondary complications. Erythrocyte membrane fluidity determined by binding of MC540. Intracellular free Ca 2+ concentration was determined by the 2-wavelength method by using fluorescent calcium-sensitive probe FURA-2AM. Measurement of erythrocyte phosphatidylserine exposure by flow cytometry using Annexin V-FITC. Results Cholesterol shedding increased with increasing severity of uremic complications. Erythrocytes from Group II show mild echinocyte or formation of spicules on the erythrocyte membrane surface whereas in Group III and IV they were echinocytic. Binding of MC540 was significantly higher with progression of uremic complications. Surface charge of uremic erythrocyte membrane was significantly reduced when compared with control subjects. Intracellular free Ca 2+ was positively correlated with binding of MC540 and surface hydrophobicity. The phosphatidylserine exposure of erythrocytes was significantly higher ( p < 0.001) in uremic patients when compared with controls. Conclusions Phosphatidylserine (PS) exposure erythrocytes were significantly increased in uremic patients when compared with controls. Uremic complications predisposes to membrane damages in erythrocytes.

Ethyl acetate extract of Chinese medicinal herb Sarcandra glabra induces growth inhibition on human leukemic HL-60 cells, associated with cell cycle arrest and up-regulation of pro-apoptotic Bax/Bcl-2 ratio

Sarcandra glabra (Thunb.) Nakai, colloquially known as Caoshanhu, is a Chinese medicinal herb with reported anti-tumor, anti-inflammatory, anti-viral and non-specific immunoenhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are largely unknown and its mode of action has never been investigated. In this study, the anti-tumor property of ethyl acetate (EA) extract of S. glabra was investigated by determining its in vitro growth-inhibitory effects on a panel of human cancer cell lines of different histotypes. Growth inhibition of the EA extract on the cancer cells seemed to be selective, and the leukemic HL-60 was found to be the most responsive after 48 h of treatment (IC50=58 microg/ml). Flow cytometric studies further illustrated that the extract might interfere with DNA replication and thus arrested the cell cycle at S phase in the leukemic cells, followed by DNA fragmentation and loss of phospholipid asymmetry in the plasma membrane after 72 h of treatment. Concurrently, the pro-apoptotic Bax/Bcl-2 ratio was also up-regulated by more than 178% of the control level. All these findings suggested that the extract had initiated apoptosis to kill the leukemic cells. Results from this pioneer study help to establish a scientific foundation for future research and development of the bioactive ingredients in EA extract of S. glabra as efficacious anti-cancer agents.

Prolonged storage of red blood cells affects aminophospholipid translocase activity

Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet). Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined. PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling. This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.

Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis.

Modulation of phospholipid asymmetry in synaptosomal membranes by the lipid peroxidation products, 4-hydroxynonenal and acrolein: implications for Alzheimer's disease

Membrane lipid bilayer asymmetry is maintained by the ATP-dependent enzyme flippase. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine (PS) in the outer leaflet of the membrane. Two highly reactive products of lipid peroxidation, 4-hydroxynonenal (HNE) and acrolein, both elevated in Alzheimer's disease (AD) brain, have been shown to induce apoptosis and disrupt cellular ion homeostasis. These reactive aldehydes can structurally modify proteins by covalent interaction and inhibit enzyme function. Phospholipid asymmetry of PS is maintained by the ATP-requiring enzyme flippase. We have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by HNE and acrolein. Flippase activity depends on a critical cysteine residue, a possible site of covalent modification by HNE or acrolein. The present study demonstrates that these alkenals induce the appearance of PS on the outer bilayer lamellae and suggests that increases in intracellular Ca 2+ might not be the sole cause for loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in phospholipid asymmetry disruption. These results are discussed with potential relevance to neuronal loss in Alzheimer's disease brain.

Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes.

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios > or = 1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia.

Loss of Phospholipid Asymmetry and Surface Exposure of Phosphatidylserine Is Required for Phagocytosis of Apoptotic Cells by Macrophages and Fibroblasts

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.

Spermine Oxidation Leads to Necrosis with Plasma Membrane Phosphatidylserine Redistribution in Mouse Leukemia Cells

Oxidation by copper/quinone-containing serum amine oxidases (SAO) is a well-known cause of polyamine cytotoxicity. Spermine oxidation exerts potent immunosuppressive effects in animal cells, but the cell death mechanism involved remains unclear. We compared biochemical and morphological parameters of SAO-mediated cell death in L1210 mouse leukemia cells with normal or amplified ornithine decarboxylase gene expression with those observed during apoptosis induced by deregulated polyamine uptake or by okadaic acid. None of the characteristic features of apoptotic cell death (e.g., chromatin condensation, nuclear fragmentation, internucleosomal DNA cleavage, poly(ADP-ribose) polymerase cleavage) were observed during spermine oxidation-mediated cell death, which was clearly necrotic by morphological criteria. Inhibition of a wide spectrum of caspases did not prevent SAO-dependent cell death, whereas N-acetylcysteine completely abolished the cytotoxic effects of spermine oxidation. Catalase only delayed spermine oxidation-induced cell death without affecting its modality or preventing depletion of intracellular glutathione, suggesting that both H2O2 and aminoaldehydes generated by SAO-mediated spermine oxidation contribute to SAO-induced necrosis. Interestingly, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, usually a diagnostic feature of apoptosis, preceded necrotic cytolysis triggered by spermine oxidation. Thus, L1210 cell death caused by SAO-mediated spermine oxidation has all the attributes of primary necrosis, but is also accompanied by loss of phospholipid asymmetry, indicating that the latter phenomenon may not be unique to apoptosis. Phosphatidylserine exposure, a potent engulfment signal for phagocytes, might contribute to the immunosuppressive effects of plasma polyamines through a controlled and rapid necrotic process involving SAO.

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology.

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

Platelet Aging In Vivo Is Associated with Loss of Membrane Phospholipid Asymmetry

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. The loss of membrane phospholipid (PL) asymmetry, with phosphatidylserine (PS) exposure appears to be an important signal for the ingestion by macrophages of apoptotic nucleated cells and it has also been suggested as a signal for the removal of aged erythrocytes. Accordingly, it seems possible that the clearance of normal aged platelets from circulation might be triggered by PS exposure. To investigate this, we determined PS exposure in human aging platelets taking advantage of the relationship between platelet density and platelet age and in dog platelets in a model of platelet aging in vivo. PS exposure was determined in two experimental conditions: 1) human platelet density subpopulations obtained by centrifugation in arabinogalactan gradients; 2) circulating canine platelets during decline in platelet count after suppression of thrombopoiesis following estradiol injection. PS exposure was determined by flow cytometry after labeling the cells with FITC-conjugated annexin V. The proportion of human platelets with exposed PS was significantly higher in high density (HD) platelets compared to low density (LD) platelets (11.3 +/- 8.0% vs 5.2 +/- 3.7%; p <0.05, respectively). In dogs, the proportion of cells with exposed PS rises dramatically with age, from 3.1 +/- 0.4% before to 17.7 +/- 12.3% ten days after estradiol injection. These findings suggest that platelet aging is associated with loss of phospholipid asymmetry and PS exposure on the outer leaflet of cell membrane, which may play an important role in the recognition and subsequent removal of senescent platelets.

Surface exposure of phosphatidylserine during apoptosis of rat thymocytes precedes nuclear changes

Cell surface exposure of phosphatidylserine (PS) during apoptosis serves recognition and removal of the dying cell by phagocytes. Loss of phospholipid asymmetry and PS exposure is investigated by immuno-cytochemistry and related to morphological changes. Loss of membrane asymmetry was determined on dexamethasone-treated rat thymocytes using the PS specific probe annexin V. Thymocytes incubated in the presence of dexamethasone were studied in time series during the execution of the apoptotic program. Thymocytes first start to expose PS at their cell surface. At this initial stage the barrier function of the plasma membrane remains intact. At a later stage the plasma membrane becomes leaky for compounds like propidium iodide and subsequently the cell disintegrates into apoptotic bodies. Microscopical evaluation of dexamethasone-treated thymocytes showed that the cells with an apoptotic morphology all bound annexin V. The cells with a normal viable morphology lacked annexin V binding except for those cells that started to shed small vesicles. These vesicles were positive for annexin V, indicating a local disturbance of the phospholipid asymmetry. The local exposure of PS is considered to be a very early event of apoptosis, preceding the full sequence of morphological changes at the ultrastructural level.

Activation of the alternative pathway of complement by calcium-loaded erythrocytes resulting from loss of membrane phospholipid asymmetry

The aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed on the outer membrane leaflet of deoxygenated and irreversibly sickled erythrocytes and senescent normal cells. PS exposure on erythrocytes results in the expression of procoagulant activity for the conversion of prothrombin to thrombin. Because liposomes or vesicles composed of aminophospholipids can activate the alternative pathway of complement, the possibility that increased exposure of PS and PE on intact erythrocytes would also make them capable of activating the alternative pathway was examined. Loss of normal membrane phospholipid asymmetry was induced by incubation of erythrocytes with calcium (Ca 2+) and the calcium ionophore A23187. PS exposure on 60% of erythrocytes was confirmed by binding of fluorescein isothiocyanate-conjugated annexin V. Expression of procoagulant activity, measured with the Russell's viper venom clothing assay, was significantly increased on the Ca 2+/A23187-treated erythrocytes. In addition, the erythrocytes became capable of activating the alternative pathway of complement, as judged by an increase in cell-bound C3b after incubation with serum and a decrease in alternative pathway hemolytic activity of the serum. The effect could be reversed by incubation of the Ca 2+/A23187-treated erythrocytes under conditions that induced recovery of normal membrane phospholipid asymmetry. In contrast, tetrathionate-treated erythrocytes showed no increase in binding of annexin V and no procoagulant activity and failed to activate the alternative pathway of complement. These findings demonstrate that loss of phospholipid asymmetry in erythrocytes not only results in expression of procoagulant activity but also renders the cells capable of activating the alternative pathway of complement.

Methods Utilized in the Study of Apoptosis

The growing awareness of the frequency and importance of physiological cell death (PCD) has resulted from two related developments, an increasing understanding of the biochemistry of PCD and a utilization of that understanding to develop sensitive methods for detecting PCD. The realization that this form of cell death is generally accompanied by internucleosomal DNA cleavage led to a variety of techniques for detecting this process, including DNA electrophoresis, flow cytometry, filtration assays, and in situ nick end labeling. Recent studies have demonstrated that PCD is accompanied by loss of phospholipid asymmetry across the plasma membrane as well as activation of various proteases, particularly members of the interleukin-1/3 converting enzyme (ICE) family. This chapter briefly describes the biochemical basis for each of these techniques and then attempts to summarize the strengths and weaknesses of each approach. In this regard Inhibitors of proteases (and other enzymes) are potentially useful tools when employed properly. Because the selectivity of these inhibitors is not fully known, however, experiments must be carefully designed, suitably controlled, and cautiously interpreted. Moreover, multiple alternative techniques (including antisense approaches and genetic knockouts) must be employed to confirm or refute conclusions suggested by inhibitor studies.

Detection of Altered Membrane Phospholipid Asymmetry in Subpopulations of Human Red Blood Cells Using Fluorescently Labeled Annexin V

The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.

Secretory phospholipase A 2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells

Nonpancreatic secretory phospholipase A 2 (sPLA 2) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA 2 hydrolyzed phospholipids in membrane microvesicles shed from Ca 2+-loaded erythrocytes as well as from platelets and from whole blood cells challenged with inflammatory stimuli. sPLA 2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet aggregation. Finally, lysophospholipid-containing vesicles and sPLA 2 were detected in inflammatory fluids in relative proportions identical to those used in vitro. We conclude that upon loss of phospholipid asymmetry, cell-derived microvesicles provide a preferential substrate for sPLA 2. SPH hydrolysis, which is provoked by various cytokines, regulates sPLA 2 activity, and the novel lipid mediator LPA can be generated by this pathway.

Loss of phospholipid asymmetry in human platelet plasma membrane after 1-12 days of storage. An ESR study.

We used paramagnetic analogs of endogenous phospholipids to study modification of phospholipid distribution in platelet plasma membranes during aging. Asymmetrical distributions and translocation kinetics were very different for spin-labeled phosphatidylserine and spin-labeled phosphatidylcholine in fresh platelet plasma membranes. In freshly prepared platelets and up to day 7, spin-labeled phosphatidylserine very rapidly penetrated to the inner leaflet of the platelet plasma membrane. However, spin-labeled phosphatidylcholine was mainly retained on the external leaflet. From day 7 to day 9, the two translocation kinetics became identical with symmetrical distribution of both spin-labeled phospholipids at equilibrium. Inhibition of translocase activity and modification of membrane stability accounted for these transformations. The rapid re-exposition of spin-labeled phosphatidylserine after stimulation by the calcium ionophore A23187, measured in fresh platelet concentrates, persisted up to day 9 but disappeared between day 10 and day 12. From day 7 to day 9, a strong cytoskeleton proteolysis and marked decrease in intracellular ATP were observed. Moreover, complete suppression of beta-N-acetyl glucosaminidase secretion and vesicle formation after A23187 stimulation of aged platelets indicated that platelets could no longer be activated beyond day 9. Taken together, these results showed that during in vitro aging there are metabolic and membrane modifications in platelet similar to those described for platelet activation. In addition, all of the observed events occurred simultaneously between day 7 and day 9. These results highlight the importance of maintaining plasma membrane asymmetry to increase the hemostatic effectiveness of transfused platelet concentrates.