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-T AT) complex by human umbilical vein endothelial cells was investigated . Radiolabeled VN-TAT was bound to the cell surface at 4 degrees C, an d internalization was initiated by increasing the temperature to 37 de grees C. After 30 min about half of the VN-TAT complex disappeared fro m the cell surface and accumulated in the subendothelial matrix. Trans location of VN-TAT complex from the luminal to the basolateral side wa s confirmed by electron microscopic evaluation of cross-sections of en dothelial cells incubated with gold-conjugated VN-TAT complex. Further more, cells cultured in VN-TAT deficient serum, incubated with purifie d VN-TAT, and subsequently assayed for fluorescent staining using a mo noclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of bo th antibodies in punctates. Punctates were randomly distributed in bot h the xy and xz plane of endothelial cells as evidenced by confocal la ser scanning microscopy. Trichloroacetic acid precipitation and SDS-po lyacrylamide gel electrophoresis showed that VN-TAT was not degraded d uring translocation and inhibition of the microfilament system reduced release of VN TAT to the matrix, indicating that transcytosis was res ponsible for translocation. These findings emphasize that VN-TAT compl ex is taken up by endothelial cells, not only leading to the removal o f inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.