Di-Annexin, a Novel Tool To Identify and Protect Phosphatidylserine Exposing Cells.

Phosphatidylserine (PS) is found exclusively in the inner monolayer of plasma membranes. During programmed cell death this well-preserved asymmetry of phopholipid distribution is lost, and PS is exposed on the outer surface. PS exposure is also found in subpopulations of red blood cells (RBC) in disease states such as sickle cell disease (SCD). PS exposure leads to increased cell-cell interactions as well as recognition and removal by macrophages. PS serves as a binding site for factors involved in blood coagulation and provides a target for secretory phospholipase A2 (sPLA2) mediated membrane degradation. Measurement of PS-exposing cells (PSRBC) relies on the use of proteins that bind to PS and can be fluorescently labeled and visualized by flow-cytometry. The human protein annexin V (AV) has a high affinity for PS and has been shown to interfere with the physiologic processes induced by the exposure of PS. To improve detection of PS exposing cells, and develop a probe that may be used in vivo, we generated plasmids encoding a dimer of human AV (DAV), and expressed the protein in E.coli. We show that DAV has a 5–8 fold higher sensitivity than AV in binding to PSRBC, and effectively identifies PSRBC in SCD blood samples. Moreover, DAV protects PSRBC from sPLA2-induced hemolysis. While normal RBC show only 2% hemolysis after incubation with 100 ng/ml sPLA2 for one hour, sPLA2 induces more than 90% hemolysis in PSRBC. DAV, when added to PSRBC before incubation with sPLA2, protects more than 90% of the PSRBC from hemolysis. Our data show that DAV effectively competes with factor Va for PS binding and inhibits thrombin formation in the presence of the pro-thrombinase complex. At low concentrations, DAV was significantly more efficient in inhibiting thrombin formation than AV (P<0.001). The increased size of DAV gives the molecule a significantly increased survival time in the murine circulation compared to AV, and allows visualization of PS exposing membranes in vivo. Together this data shows that DAV is a superior probe to identify PS-exposing membranes, protects PSRBC from sPLA2-induced hemolysis, and interferes with cellular hemostasis. The use of DAV will allow more precise determination of PS exposure by flow-cytometry as compared to currently available commercial probes. Moreover, it will allow the measurement of PS exposing membranes in vivo, and may ultimately be proven to be useful as a therapeutic agent in SCD pathology by masking PS exposing cell membranes.