First printed in R&D Systems' 1996 Catalog.
Annexin V is a member of a calcium and phospholipid binding family of proteins with vascular anticoagulant activity. Various synomyms for Annexin V exist: placental protein 4 (PP4), placental anticoagulant protein I (PAP I), calphobindin I (CPB-I), calcium dependent phospholipid binding protein 33 (CaBP33), vascular anticoagulant protein alpha (VACa), anchorin CII, lipocortin-V, endonexin II, and thromboplastin inhibitor. Largely found on the cytosolic face of plasma membranes, this molecule has high affinity for phospholipids in the presence of physiological concentrations of calcium.
The gene for Annexin V (ANXV) is located on human chromosome 4q26-q28 and spans a region of DNA 28 kb in length containing 13 exons and 12 introns.1 The Annexin V mature molecule is a 320 amino-acid residue, 35-36 kDa protein2 The protein is folded into a planar cyclic arrangement of four repeats with each repeat composed of 5 alpha-helical segments.3
The potent anticoagulant activity of Annexin V is derived from its inhibitory effect on prothrombin activation4 and its ability to effectively prevent thrombus formation under normal venous and arterial blood flow conditions.5 Annexin V has additionally been demonstrated to inhibit phospholipase A2 activity.6 This latter property appears to endow Annexin V with antiinflammatory activity due its ability to prevent the release of arachidonic acid by phospholipase A2. Annexin V is also an inhibitor of protein kinase C.7 Lastly, Annexin V has been reported to bind the hepatitis B surface antigen and therefore may play a role in the hepatitis virus infection process.8 Annexin V has a wide tissue distribution, having been detected in cardiac myocytes, vascular endothelium9, chondrocytes, osteoblasts10, glial cells, astrocytes, oligodendrocytes, Schwann cells, skeletal muscles11, optic nerve, hepatocytes and bronchi.12
Apoptosis is a programmed cell death mechanism that many organisms utilize to selectively eliminate cells which show either deleterious reactivities to the host or which have not received a full complement of activation or survival signals. Although comparable in their final outcome, necrosis and apoptosis are distinctly different processes. Fundamental differences between the two death-generating mechanisms are evident in the triggers necessary to initiate the two events. Necrosis is usually the result of an accumulation of toxic reagents within cells while apoptosis can be triggered by various environmental stimuli which lead to the activation of an endogenous endonuclease activity.13 At the cell membrane level, disruption of internal and external membranes is a normal consequence of necrosis. Alternatively, during programmed cell death, loss of cell membrane integrity is a very late event usually preceded by the destructive action of endogenous cellular enzymes.14
The ability to exclude viable dyes such as trypan blue, propidium iodide (PI) or 7-aminoactinomycin D (7AAD) is a property of cells that have an intact plasma membrane. Cells in the early phases of apoptosis fall into this category. On the other hand, necrotic cells have lost membrane integrity and therefore easily stain with the above viable dyes. Various other fluorescent dyes are available as tools to distinguish between live and dead cells (e.g., Hoechst 33342, rhodamine 123 and acridine orange). Of all of these fluorochromes, PI and 7AAD appear to be the most useful and convenient for flow cytometry applications.
The utility of Annexin V in flow cytometry applications is derived from its selective affinity for negatively charged phospholipids. Under defined salt and calcium concentrations, Annexin V is predisposed to bind phosphatidylserine (PS) over most other phospholipid species.4 The Kd for the binding of Annexin V to phospholipids has been estimated at 5 x 10-10 M.4 This is the basis for the use of Annexin V as a tool to monitor cell membrane anomalies.
Use of fluorochromes in flow cytometric analysis for the detection and determination of the frequency of apoptotic cells within a defined population has been clearly demonstrated.13,15,16 Although apoptotic cells have other properties that lend themselves to flow cytometric identification, most of these characteristics become subjective and therefore require interpretative aid. Under this category are changes in cell morphology resulting in altered light scattering properties, total cellular protein and total cellular RNA content.
The observation that apoptotic cells acquire subtle, yet detectable, plasma membrane changes was key to identifying the use of Annexin V as an indicator of these cell membrane changes.17 Changes in cell membrane composition appear to be crucial in identifying cells destined for removal. Phagocytes involved in the clearance of apoptotic cells have been demonstrated to use multiple mechanisms to recognize target cells. For example, use of the alpha v beta 3 vitronectin receptor integrin, recognition of thrombospondin, membrane carbohydrate changes, changes in cell membrane net surface charge, and changes in membrane phospholipid asymmetry have all been described as recognition elements for phagocytic cells.18 Noteworthy is the finding that an increase in outer membrane phosphatidylserine composition appears to be a consistent finding in apoptotic thymocytes, the CTLL-2 cell line, and lymphocytes.17
Although cell microscopy and DNA electrophoretic analysis remain as reliable methods to determine whether cells are undergoing apoptosis, they do not easily lend themselves to rapid multiparameter analysis at the single cell level. Normal blood cell membranes exhibit significant phospholipid asymmetry, with phosphatidyl choline and sphingomyelin largely found on the cell's exterior.19 On the inner side of the lipid bilayer, phosphatidyl serine and phosphatidyl ethanolamine are the two major lipid components. Apoptotic cells undergo numerous physiological changes including alterations in their membrane asymmetry.17,20 This in turn leads to exposure of the cell's inner phospholipids to the exterior. These are thought to be early events during the apoptotic process that culminate in cell death. Annexin V can selectively bind to cells with a compromised membrane phospholipid asymmetry and this property has been exploited to identify populations of cells undergoing apoptosis.21-23 Furthermore, apoptotic cells have reduced DNA stainability with fluorochromes24 and this is thought to give rise to the sub-G1 or A0 peak when performing DNA cell cycle analysis. The reduced DNA stainability with DNA binding dyes has been reasoned to likely be a result of both the reduced accessibility of the dye for the DNA and a progressive loss of cleaved DNA from cells.13
A useful property of Annexin V in the staining of apoptotic cells is derived from the fact that it can bind many sites on cell surfaces and therefore result in a very intense signal. The number of binding sites for Annexin V has been reported as 6 - 24 x 106/cell in tumor cells25 and 8.8 x 106/cell for endothelial cells.26 Normal human red cells may have as few as 276 binding sites27, while resting platelets have 5000 binding sites.28 Studies with phosphatidylserine-containing liposomes found that the stoichiometry of Annexin V binding to PS ranges between 4 and 8 Annexin V molecules per one PS molecule.29,30 This may also reflect the fact that Annexin V has a tendency to form trimers.31
The binding of Annexin V to phospholipids is very rapid, extremely dependent on the presence of Ca2+, and reversible in the presence of the ion chelator EDTA. The utility of combining the staining properties of Annexin V and PI are exemplified in reports demonstrating that the Annexin V-staining population, which simultaneously excludes PI molecules from entering the cell, are undergoing the classical DNA fragmentation pattern found in apoptotic cells.22,23 In contrast, cells that are undergoing necrosis stain with Annexin V and also stain with PI. Thus, these two populations of dying cells can be distinguished.
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