Engineered annexin A5 variants have impaired cell entry for molecular imaging of apoptosis using pretargeting strategies.

Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell.

Annexin A5 uptake in ischemic myocardium: demonstration of reversible phosphatidylserine externalization and feasibility of radionuclide imaging.

Unlabelled: Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia.

Methods: Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using (99m)Tc-labeled AA5 and gamma-imaging were produced in rabbits.

Targeting phosphatidylserine in anti-cancer therapy.

Targeted delivery of cytotoxic agents limits the severe toxic side-effects of anti-cancer drugs on healthy tissues. Annexin A5 is a well explored probe to target phosphatidylserine (PS)-expressing cells in vivo. Our novel understanding of the cellular and molecular mechanism of annexin A5 as a cell-entry agent and the finding that PS is expressed on living tumour as well as endothelial cells in the tumour vasculature, will allow the development of lead compounds for anti-cancer therapy.

Extracellular annexin A5: functions of phosphatidylserine-binding and two-dimensional crystallization.

In normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. Annexin A5 has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice.

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions.

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains.

In vitro measurement of cell death with the annexin A5 affinity assay.

One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy.

Annexin A5 inhibits engulfment through internalization of PS-expressing cell membrane patches.

Apoptosis and subsequent clearance of apoptotic cells are important for the prevention of diseases. Therefore, it is essential to understand the mechanisms underlying the biology of phagocytic clearance of apoptotic cells. The best characterized "eat me" signal on the surface of apoptotic cells is phosphatidylserine (PS).

Annexin A5 down-regulates surface expression of tissue factor: a novel mechanism of regulating the membrane receptor repertoir.

Phosphatidylserine (PtdSer) is exposed on the external leaflet of the plasma membrane during apoptosis. The protein annexin A5 (anxA5) shows high affinity for PtdSer. When anxA5 binds to the PtdSer-expressing membranes during apoptosis, it crystallizes as an extended two-dimensional network and activates thereby a novel portal of cell entry that results in the internalization of the PtdSer-expressing membrane patches.

Cell surface-expressed phosphatidylserine and annexin A5 open a novel portal of cell entry.

Expression of phosphatidylserine (PtdSer) at the cell surface is part of the membrane dynamics of apoptosis. Expressed phosphatidylserine functions as an "eat me" flag toward phagocytes. Here, we report that the expressed phosphatidylserine forms part of a hitherto undescribed pinocytic pathway.

The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5.

We have demonstrated that imaging of programmed cell death (PCD) in patients is possible using 99mTc-Annexin A5. Because of the short half-life of the technetium label it is important to limit the time span between the preparation of 99mTc-Annexin A5 and its administration into the patient. Therefore methods of quality control that determine the biological active fraction in the 99mTc-Annexin A5 should be not only accurate and precise but also rapid.

Visualization of cell death in vivo with the annexin A5 imaging protocol.

Annexin A5 binds to phosphatidylserine (PS), which is one of the "eat me" signals at the surface of the apoptotic cell. This property has been the driving force for the research of annexin A5 as a probe to measure apoptosis in vitro and in vivo. A non-invasive imaging protocol using annexin A5 has been developed and applied successfully to measure programmed cell death programmed cell death (PCD) in patients.