Calcium-Independent Detection and Isolation of Phosphatidylserine-Exposing Cells and Microparticles Using Recombinant Del-1

Abstract 4224

Del-1 is an extracellular matrix protein that is highly expressed during the embryonic period. It comprises, from N- to C-terminus, three epidermal growth factor (EGF) domains (containing an RGD motif in the second EGF domain) and two factor VIII-homologous C domains (C1C2 domain). After the embryonic period, hypoxia and vascular injury can increase Del-1 expression by endothelial cells and macrophages, promoting angiogenesis and phagocytosis of phosphatidylserine (PS)-exposing apoptotic bodies. This is possibly due to the capacity of Del-1 to bridge the interaction between apoptotic bodies and phagocytes. Del-1 binds to PS-exposing cells and membrane fragments through its C1C2 domains and to integrins of phagocytic cells through its RGD sequence, enhancing phagocytosis. We expressed in mammalian cells a Del-1 fragment composed of the C1 and C2 domains containing two human protein C tags and a biotin tag at the C-terminus. The recombinant fragment was purifed using monomeric avidin resin. The recombinant Del-1 C1C2 bound PS specifically on a commercial lipid array (PIP-strip, Echelon), a finding confirmed in a 96-well assay using immobilized lipids. The binding of C1C2 Del-1 to phosphatidylserine (PS) was consistent and stable when compared to the binding of commercially available annexin-V (biotinylated and PE-conjugated). We evaluated by flow cytometry the ability of Del-1 C1C2 to bind membrane-exposed PS. Apoptotic THP-1 cells were incubated with Del-1 C1C2 in the absence of calcium or with annexin V (2.5 mM calcium was present in all the buffers and through all washing steps). Protein binding was detected using a monoclonal anti-biotin-PE conjugated antibody. Neither the percentage of PS-positive cells nor the mean fluorescence intensity (MFI) was statistically different between Del-1 C1C2 and annexin V when samples were diluted with buffer (instead of washed) before flow cytometric analysis. However, both the percentage of PS-positive cells and MFI decreased by approximately 50% in the annexin V samples when the cells were washed after incubation. Cell-derived microparticles from outdated RBC units were also used to compare the utility of C1C2 Del-1 vs commercial PE-conjugated annexin V for the detection of microparticles by flow cytometry. We found a 1.4-fold average increase in the quantity of microparticles detected with C1C2 Del-1 when compared to annexin V. In addition, cell-derived microparticles can be isolated and concentrated from the Del-1 C1C2–treated sample using magnetic beads coupled to anti-protein C antibody. In conclusion, the C1C2 domain of Del-1 represents a powerful and sensitive tool for the detection and isolation of PS-exposing cells and microparticles. The calcium-independence of this reagent for PS simplifies the detection of apoptotic cells and microparticles in plasma. Moreover, the addition of multiple tags at the C-terminus allows the capture, labeling and enrichment of PS-specific microparticles by bridging C1C2-coated microparticles to magnetic beads. C1C2 Del-1 thus represents a powerful new tool for analysis in the fields of microparticle biology, thrombosis, and immunology.