16-256 Sigma-AldrichAnti-Phosphatidylserine Antibody, clone 1H6, Alexa Fluor® 488

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid that is synthesized by prokaryotic and eukaryotic cells. In this review we focus on genes and enzymes that are involved in PS biosynthesis in bacteria, yeast, plants and mammalian cells and discuss the available information on the regulation of PS biosynthesis in these organisms. The enzymes that synthesize PS are restricted to endoplasmic reticulum membranes in yeast and mammalian cells, yet PS is widely distributed throughout other organelle membranes. Thus, mechanisms of inter-organelle movement of PS, particularly the transport of PS from its site of synthesis to the site of PS decarboxylation in mitochondria, are considered. PS is normally asymmetrically distributed across the membrane bilayer, thus the mechanisms of transbilayer translocation of PS, particularly across the plasma membrane, are also discussed. The exposure of PS on the outside surface of cells is widely believed to play a key role in the removal of apoptotic cells and in initiation of the blood clotting cascade. PS is also the precursor of phosphatidylethanolamine that is made by PS decarboxylase in bacteria, yeast and mammalian cells. Furthermore, PS is required as a cofactor for several important enzymes, such as protein kinase C and Raf-1 kinase, that are involved in signaling pathways.

Phosphatidylserine on the dying cell surface helps identify apoptotic cells to phagocytes, which then engulf them. A candidate phagocyte receptor for phosphatidylserine was identified using phage display, but the phenotypes of knockout mice lacking this presumptive receptor, as well as the location of the protein within cells, cast doubt on the assignment of this protein as the phosphatidylserine receptor.

CD43 down-regulation during the apoptosis of PMN (polymorphonuclear cells) is not caused by proteolysis or internalization. Could it be released with bleb-derived membrane vesicles? Membrane blebbing was followed by microscopy on PMN 'synchronized' by an overnight incubation at 15 degrees C before their spontaneous apoptosis at 37 degrees C. Released vesicles were quantified by flow cytometry. Membrane blebbing, release of bleb-derived membrane vesicles, decrease of CD43/CD16 expression and phosphatidylserine externalization occurred simultaneously. However, caspase and PKC inhibition prevented annexin binding but not blebbing, vesicle release or CD43 expression decrease; myosin light chain kinase inhibition prevented cell blebbing and vesicle release but had no effect on CD43/CD16 down-regulation or annexin V binding. By electron microscopy, CD43 appeared poorly expressed on membrane blebs and concentrated at bleb 'necks'. In conclusion, CD43 down-regulation is not caused by cell blebbing. Cell blebbing, phospholipid 'flip-flop' and CD43/CD16 down-regulation are independent membrane events.

During the time that erythrocytes (RBC) spend in the circulation, a series of progressive events take place that lead to their removal and determine their apparent aging and limited survival. In addition, a fraction of RBC precursors will be removed during erythropoiesis by apoptotic processes, often described as "ineffective erythropoiesis". Both will determine the survival of erythroid cells and play an important role in red cell pathology, including hemoglobinopathies and red cell membrane disorders. The loss of phospholipid asymmetry, and the exposure of phosphatidylserine (PS) on the surface of plasma membranes may be a general trigger by which cells, including aging RBC and apoptotic cells, are removed. Oxidant stress and inactivation of the system that maintains phospholipid asymmetry play a central role in the events that will lead to PS exposure, death and removal.

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.

In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.