Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complex into the subendothelial matrix

Internalization of the ternary vitronectin-thrombin-antithrombin (VN-TAT) complex by human umbilical vein endothelial cells was investigated. Radiolabeled VN-TAT was bound to the cell surface at 4 degrees C, and internalization was initiated by increasing the temperature to 37 degrees C. After 30 min about half of the VN-TAT complex disappeared from the cell surface and accumulated in the subendothelial matrix. Translocation of VN-TAT complex from the luminal to the basolateral side was confirmed by electron microscopic evaluation of cross-sections of endothelial cells incubated with gold-conjugated VN-TAT complex. Furthermore, cells cultured in VN-TAT deficient serum, incubated with purified VN-TAT, and subsequently assayed for fluorescent staining using a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of both antibodies in punctates. Punctates were randomly distributed in both the xy and xz plane of endothelial cells as evidenced by confocal laser scanning microscopy. Trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis showed that VN-TAT was not degraded during translocation and inhibition of the microfilament system reduced release of VN TAT to the matrix, indicating that transcytosis was responsible for translocation. These findings emphasize that VN-TAT complex is taken up by endothelial cells, not only leading to the removal of inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.