Annexin A5 affinity assay

Annexin A5 is a protein that binds in a calcium dependent manner to phosphatidylserine containing membrane surfaces.

Cell surface expression of phosphatidylserine during cell death

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Apoptosis is a form of programmed cell death which is used by the body to remove unwanted, damaged or senescent cells from tissues. Removal of apoptotic cells is carried out via phagocytosis by white blood cells such as macrophages or dendritic cells. Phagocytic white blood cells recognize apoptotic cells by their exposure of negatively charged phospholipids(phosphatidylserine) on the cell surface. In normal cells the negative phospholipids reside on the inner side of the cellular membrane and the outer surface of the membrane is occupied by phospholipids which do not have a charge. After cells have made the decision to commit suicide the negatively charged phospholipids are transported to the outer cell surface by a hypothetical protein known as scramblase. Phagocytic white blood cells express a receptor which can detect the negatively charged phospholipids. After detection the apoptotic cells are removed.

Detection of cell death with annexin A5

Healthy individual apoptotic cells are rapidly removed by phagocytes. However in pathological processes the removal of apoptotic cells may be delayed or even absent. Dying cells in tissue can be detected with annexin A5. Labeling of annexin A5 with fluorescent or radioactive molecules makes it possible to detect binding of labeled annexin A5 to the cell surface of apoptotic cells. After binding to the phospholipid surface, annexin A5 assembles into a cluster known as a trimer. This trimer consists of three annexin A5 molecules which are bound to each other via non-covalent protein-protein interactions. The formation of annexin A5 trimers results in the formation of a two-dimensional crystal lattice on the phospholipid membrane. This clustering of annexin A5 on the membrane greatly increases the intensity of annexin A5 when labeled with a fluorescent or radioactive probe. Two-dimensional crystal formation is believed to cause internalization of annexin A5 through a novel process of endocytosis if it occurs on cells which are in the early phase of executing cell death [Kenis et al. J Biol Chem 279 (2004) 52623-9]. Internalization amplifies additionally the intensity of the annexin A5 stained cell.

Annexin A5 has been successively used to detect apoptotic cells in vitro (cells in a culture tube) and in vivo (in laboratory mice and in patients in the clinic). Pathological processes in which apoptosis occurs include inflammation, ischemia damage of the heart caused by myocardial infarction, apoptotic white blood cells and smooth muscle cells present in atherosclerotic plaques of blood vessels, transplanted organs in the donor patient which are rejected by the immune system or tumor cells which are exposed to cytostatic drugs during chemotherapy.

The non-invasive detection of diseased tissue with, for example, radioactively labeled annexin A5 is the goal of a recently developed line of research known as Molecular Imaging.

Molecular Imaging of cell death using radioactive annexin A5 can become of clinical significance to diagnose vulnerability of atherosclerotic plaques (unstable atherosclerosis) [Kietselaer et al. N Engl J Med 350 (2004) 1472-3], heart failure [Kietselaer et al. J Nucl Med 48 (2007) 562-7] transplant rejection [Narula et al. Nat Med 7 (2001) 1347-52], and to monitor efficacy of anti-cancer therapy [Rottey et al. J Nucl Med 47 (2006) 1813-8; Haas et al. Int J Radiat Oncol Biol Phys 59 (2004) 782-7].

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