Annexin A5 Explained

Annexin A5 (or annexin V) is a cellular protein in the annexin group. In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane. The function of the protein is unknown; however, annexin A5 has been proposed to play a role in the inhibition of blood coagulation by competing for phosphatidylserine binding sites with prothrombin and also to inhibit the activity of phospholipase A1. These properties have been found by in vitro experiments.

Pathology

Antibodies directed against annexin A5 are found in patients with a disease called the antiphospholipid syndrome (APS), a thrombophilic disease associated with autoantibodies against phospholipid compounds.

Annexin A5 forms a shield around negatively charged phospholipid molecules. The formation of an annexin A5 shield blocks the entry of phospholipids into coagulation (clotting) reactions. In the antiphospholipid antibody syndrome, the formation of the shield is disrupted by antibodies. Without the shield, there is an increased quantity of phospholipid molecules on cell membranes, speeding up coagulation reactions and causing the blood-clotting characteristic of the antiphospholipid antibody syndrome.
Annexin A5 showed upregulation in papillary thyroid carcinoma.^[1]

Laboratory use

Annexin A5 is used as a non-quantitative probe to detect cells that have expressed phosphatidylserine (PS) on the cell surface, an event found in apoptosis as well as other forms of cell death.^{[2] [3] [4]} Platelets also expose PS and PE on their surface when activated, which serves as binding site for various coagulation factors.

The annexin A5 affinity assay typically uses a conjugate of annexin V and a fluorescent or enzymatic label, biotin or other tags, or a radioelement, in a suitable buffer (annexin V binding to aminophospholipids is Ca²⁺ dependent). The assay combines annexin V staining of PS and PE membrane events with the staining of DNA in the cell nucleus with propidium iodide (PI) or 7-Aminoactinomycin D (AAD-7), distinguishing viable cells from apoptotic cells and necrotic cells.^[5] Detection occurs by flow cytometry or a fluorescence microscope.

Interactions

Annexin A5 has been shown to interact with Kinase insert domain receptor^[6] and Integrin, beta 5.^[7]

Further reading

• Cederholm A, Frostegård J . Annexin A5 as a novel player in prevention of atherothrombosis in SLE and in the general population . Annals of the New York Academy of Sciences . 1108 . 1 . 96–103 . Jun 2007 . 17893975 . 10.1196/annals.1422.011 . 2007NYASA1108...96C . 29296971 .
• Schlaepfer DD, Jones J, Haigler HT . Inhibition of protein kinase C by annexin V . Biochemistry . 31 . 6 . 1886–91 . Feb 1992 . 1310621 . 10.1021/bi00121a043 .
• Huber R, Berendes R, Burger A, Schneider M, Karshikov A, Luecke H, Römisch J, Paques E . Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins . Journal of Molecular Biology . 223 . 3 . 683–704 . Feb 1992 . 1311770 . 10.1016/0022-2836(92)90984-R .
• Kirsch T, Pfäffle M . Selective binding of anchorin CII (annexin V) to type II and X collagen and to chondrocalcin (C-propeptide of type II collagen). Implications for anchoring function between matrix vesicles and matrix proteins . FEBS Letters . 310 . 2 . 143–7 . Sep 1992 . 1397263 . 10.1016/0014-5793(92)81316-E . 9498732 . free .
• Dawson SJ, White LA . Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin . The Journal of Infection . 24 . 3 . 317–20 . May 1992 . 1602151 . 10.1016/S0163-4453(05)80037-4 .
• Tait JF, Frankenberry DA, Shiang R, Murray JC, Adler DA, Disteche CM . Chromosomal localization of the human gene for annexin V (placental anticoagulant protein I) to 4q26----q28 . Cytogenetics and Cell Genetics . 57 . 4 . 187–92 . 1992 . 1683830 . 10.1159/000133143 .
• Huber R, Römisch J, Paques EP . The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes . The EMBO Journal . 9 . 12 . 3867–74 . Dec 1990 . 2147412 . 552154 . 10.1002/j.1460-2075.1990.tb07605.x.
• Huber R, Schneider M, Mayr I, Römisch J, Paques EP . The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity . FEBS Letters . 275 . 1–2 . 15–21 . Nov 1990 . 2148156 . 10.1016/0014-5793(90)81428-Q . 8975064 . free .
• Maurer-Fogy I, Reutelingsperger CP, Pieters J, Bodo G, Stratowa C, Hauptmann R . Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein . European Journal of Biochemistry . 174 . 4 . 585–92 . Jul 1988 . 2455636 . 10.1111/j.1432-1033.1988.tb14139.x . free .
• Rothhut B, Coméra C, Cortial S, Haumont PY, Diep Le KH, Cavadore JC, Conard J, Russo-Marie F, Lederer F . A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation . The Biochemical Journal . 263 . 3 . 929–35 . Nov 1989 . 2532007 . 1133519 . 10.1042/bj2630929.
• Schlaepfer DD, Mehlman T, Burgess WH, Haigler HT . Structural and functional characterization of endonexin II, a calcium- and phospholipid-binding protein . Proceedings of the National Academy of Sciences of the United States of America . 84 . 17 . 6078–82 . Sep 1987 . 2957692 . 299011 . 10.1073/pnas.84.17.6078 . free . 1987PNAS...84.6078S .
• Funakoshi T, Heimark RL, Hendrickson LE, McMullen BA, Fujikawa K . Human placental anticoagulant protein: isolation and characterization . Biochemistry . 26 . 17 . 5572–8 . Aug 1987 . 2960376 . 10.1021/bi00391a053 .
• Iwasaki A, Suda M, Nakao H, Nagoya T, Saino Y, Arai K, Mizoguchi T, Sato F, Yoshizaki H, Hirata M . Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein . Journal of Biochemistry . 102 . 5 . 1261–73 . Nov 1987 . 2963810 . 10.1093/oxfordjournals.jbchem.a122165.
• Funakoshi T, Hendrickson LE, McMullen BA, Fujikawa K . Primary structure of human placental anticoagulant protein . Biochemistry . 26 . 25 . 8087–92 . Dec 1987 . 2964863 . 10.1021/bi00399a011 .
• Kaplan R, Jaye M, Burgess WH, Schlaepfer DD, Haigler HT . Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein . The Journal of Biological Chemistry . 263 . 17 . 8037–43 . Jun 1988 . 10.1016/S0021-9258(18)68438-8 . 2967291 . free .
• Grundmann U, Abel KJ, Bohn H, Löbermann H, Lottspeich F, Küpper H . Characterization of cDNA encoding human placental anticoagulant protein (PP4): homology with the lipocortin family . Proceedings of the National Academy of Sciences of the United States of America . 85 . 11 . 3708–12 . Jun 1988 . 2967495 . 280287 . 10.1073/pnas.85.11.3708 . free . 1988PNAS...85.3708G .
• Pepinsky RB, Tizard R, Mattaliano RJ, Sinclair LK, Miller GT, Browning JL, Chow EP, Burne C, Huang KS, Pratt D . Five distinct calcium and phospholipid binding proteins share homology with lipocortin I . The Journal of Biological Chemistry . 263 . 22 . 10799–811 . Aug 1988 . 10.1016/S0021-9258(18)38041-4 . 2968983 . free .
• Ahn NG, Teller DC, Bienkowski MJ, McMullen BA, Lipkin EW, de Haën C . Sedimentation equilibrium analysis of five lipocortin-related phospholipase A2 inhibitors from human placenta. Evidence against a mechanistically relevant association between enzyme and inhibitor . The Journal of Biological Chemistry . 263 . 35 . 18657–63 . Dec 1988 . 10.1016/S0021-9258(18)37335-6 . 2974032 . free .
• Demange P, Voges D, Benz J, Liemann S, Göttig P, Berendes R, Burger A, Huber R . Annexin V: the key to understanding ion selectivity and voltage regulation? . Trends in Biochemical Sciences . 19 . 7 . 272–6 . Jul 1994 . 7519374 . 10.1016/0968-0004(94)90002-7 .
• Fernández MP, Morgan RO, Fernández MR, Carcedo MT . The gene encoding human annexin V has a TATA-less promoter with a high G+C content . Gene . 149 . 2 . 253–60 . Nov 1994 . 7958998 . 10.1016/0378-1119(94)90157-0 .

Notes and References

1. Sofiadis A, Becker S, Hellman U, Hultin-Rosenberg L, Dinets A, Hulchiy M, Zedenius J, Wallin G, Foukakis T, Höög A, Auer G, Lehtiö J, Larsson C . Proteomic profiling of follicular and papillary thyroid tumors . European Journal of Endocrinology. 166 . 4 . 657–67 . Apr 2012 . 22275472 . 10.1530/EJE-11-0856 . 3315832.
2. Meers P and Mealy T . 1994. Phospholipid determinants for annexin V binding sites and the role of tryptophan. Biochemistry. 33. 19. 5829–37. 10.1021/bi00185a022. 8180211.
3. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH . Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis . Blood . 84 . 5 . 1415–20 . Sep 1994 . 10.1182/blood.V84.5.1415.1415 . 8068938 .
4. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C . A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V . Journal of Immunological Methods . 184 . 1 . 39–51 . Jul 1995 . 7622868 . 10.1016/0022-1759(95)00072-I .
5. http://www.interchim.fr/ft/B/BH4140.pdf Annexin-FP488 fluorescent staining protocol
6. Wen Y, Edelman JL, Kang T, Sachs G . Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1 . Biochemical and Biophysical Research Communications . 258 . 3 . 713–21 . May 1999 . 10329451 . 10.1006/bbrc.1999.0678 .
7. Cardó-Vila M, Arap W, Pasqualini R . Alpha v beta 5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V . Molecular Cell . 11 . 5 . 1151–62 . May 2003 . 12769841 . 10.1016/S1097-2765(03)00138-2 . free .