Factor VIII-related antigen. A pericellular matrix component of cultured human endothelial cells.
We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of of endothelial cells as well. In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with Mr 220-240; 180; 160 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.
D000941:Antigens / Q000032:analysis*; D002448:Cell Adhesion; D002474:Cell-Free System; D002478:Cells, Cultured; D004727:Endothelium / Q000166:cytology*; D005109:Extracellular Matrix / Q000032:analysis / Q000276:immunology*; D005169:Factor VIII / Q000032:analysis / Q000276:immunology*; D005353:Fibronectins / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D003012:Microbial Collagenase; D011347:Procollagen / Q000032:analysis; D014841:von Willebrand Factor
D000941:Antigens; D005353:Fibronectins; D011347:Procollagen; D014841:von Willebrand Factor; D005169:Factor VIII; D003012:Microbial Collagenase