Localization of factor VIII/von Willebrand factor antigen by immunoelectron microscopy in human endothelial cells using Fab fragments coupled to peroxidase.

Abstract

Factor VIII/von Willebrand factor antigen (VIII R:Ag) has been localized in human endothelial cells by an immunoperoxidase method and electron microscopy. Specific antibodies against VIII R:Ag were isolated from monospecific rabbit antisera by an immunoadsorbant. Fab fragments were prepared and coupled to peroxidase. Native human endothelial cells from umbilical cord veins were incubated with the Fab conjugates. Peroxidase labeling was seen on the plasma membrane, in vesicles, and in the Golgi apparatus. In order to eliminate nonspecific antibody internalization, the same procedure was reproduced on fixed endothelial cells and similar localization was observed. Staining was only found when specific VIII R:Ag Fab conjugates were used. All the controls were devoid of labeling, thus the peroxidase reaction product localized in the Golgi cisternae is specific for VIII R:Ag and suggests VIII R:Ag synthesis in endothelial cells.