Background: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. Methods: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. Results: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content .2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. Conclusions: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology. Key terms: lactadherin; phosphatidylserine; apoptosis; leukemia; annexin V Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death (1-3) and marks those cells for clearance by macrophages (1). Transient exposure of PS on viable cells accompanies sperm capacitation (4), phagocytosis of apoptotic cells (5), myotube formation (6), and neutrophil (7) and platelet stimulation (8). On stimulated platelets, exposed PS supports procoagulant reactions, and on lymphocytes PS externalization appears mechanistically coupled to the function of ion channels and the multidrug resistance protein in lymphocytes (9). However, in spite of the essential relationship of PS exposure to cellular function, the topology and kinetics of PS exposure remain only partially characterized.PS exposure is frequently detected through binding of fluorescence-labeled annexin V (3). The extent to which annexin V binds to a membrane is a complex function related to the free annexin V concentration, membrane PS content, phosphatidylethanolamine content, and ambient Ca 11 concentration (10-15). Indeed, recent reports indicate that the cooperative interaction of annexin V on the plasma membrane drives a mechanism whereby invagination of the plasma membrane is followed by internal vesiculation (16).The complexity of annexin V binding and the internalization of annexin V by stressed cells leaves uncertainty about the extent to which annexin V fluorescence represents plasma membrane PS exposure in early apoptosis.Lactadherin is also a PS-binding protein and has a domain structure that includes two epithelial growth factorlike domains and two le...

S U M M A R Y Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical cancer cell line HeLa has been employed as a model system to compare the topographic distribution of PS with the two PS binding proteins as adherent cells enter the apoptotic program. HeLa cells were cultured on glass-bottom Petri dishes, and apoptosis was induced by staurosporine. Fluorescence-labeled lactadherin and/or annexin V were used to detect PS exposure by confocal microscopy. Both lactadherin and annexin V staining revealed PS localized to plasma membrane rim and blebs. In addition, lactadherin identified PS exposure on long filopodia-like extensions, whereas annexin V internalized in granule-like structures. All in all, the data further delineate the differences in PS binding patterns of lactadherin and annexin V.