Immunofluorescent studies of tissue factor on U87MG cells: evidence for non-uniform distribution

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Abstract

U87MG cells (human glioblastoma) express tissue factor and shed membrane-derived vesicles enriched in procoagulant activity. Tissue factor antigen has been studied by flow cytometry, immunofluorescent microscopy and Western blotting. Flow cytometric analysis utilized monoclonal antibodies recognizing the tissue factor extracellular domain and the carboxyl-terminal nine amino acids. Studies with intact and permeabilized cells support the location of the carboxyl-terminal domain in the cytoplasm, as previously predicted from the protein sequence. Immunofluorescent microscopy revealed a heterogeneous staining pattern, indicating that tissue factor antigen may be clustered on the cell surface. Intense staining was occasionally observed in cytoplasmic extensions and membrane regions that appeared to be extruding from the cells. Western blot analysis of vesicles shed into the culture medium revealed a principal tissue factor band with mobility marginally slower than that of placental tissue factor. Both extracellular and cytoplasmic epitopes were present in this vesicular tissue factor.

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