Intracellular Localization Of F.VIII R:Ag In Platelet And Endothelial Cells Using Peroxidase Fab Conjugates In Electron Microscopy
In previous studies (Jeanneau et al Thromb. Haemost.1977, 38, 42) specific rabbit antibodies against human F.VIII R:Ag isolated by immuno absorption on insolubilized polymers from monospecific rabbit antisera against the F.VIII/ VW factor molecule and coupled to peroxidase showed the labelling of platelet and endothelial cell membranes but did not allow to visualize intra cellular localization of this antigen. Preparation of the peroxidase coupled Fab fragments of the same specific antibodies showed the intra cellular localization of F.VIII R:Ag in platelet and endothelial cells. Fab antibody fragments were prepared using papain digestion. Fab and Fc fragments were separated from the non digested antibody by filtration through Sephedex G 100. The Fab fragments were then separated from the Fc using CM 52 carboxymethyl cellulose.In human washed platelets, staining was observed on the plasma membrane, the canalicular system and some granules. After thrombin activation the release of granules containing F.VIII R:Ag better visualized in the canalicular system. In patients with storage pool disease only some platelets showed normal SCS labelling, however no stained granules were observed.Native human endothelial cells were obtained from umbilical cord veins, washed and resuspended in culture medium prior to incubation with peroxidase Fab conjugates. After fixation in glutaraldehyde and exposure to diaminobenzidine, the peroxidase staining was seen on the plasma membrane and in a large number of vacuoles. In some endothelial cells the Golgi apparatus appeared labelled demonstrating evidence of F.VIII R:Ag synthesis by endothelial cells.