Internalization of antithrombin III by cultured human endothelial cells and its subcellular localization

The presence of antithrombin III was demonstrated in cultured human endothelial cells derived from the umbilical cord by using immunofluorescence, immunoelectron microscopy studies, and an enzyme-linked immunosorbent assay (ELISA) specific for antithrombin III. Immunofluorescence studies indicated the presence of antithrombin III in granule-like structures in the endothelial cell. Immunoelectron microscopy studies performed with ultrathin cryosections of endothelial cells showed a colocalization of antithrombin III and a lysosomal marker protein in low electron dense organelles, indicating a lysosomal localization of antithrombin III. By using the ELISA, 77 +/- 40 ng (n = 8) antithrombin III was quantitated in 10(6) endothelial cells. Immunoprecipitation studies performed with metabolically labeled cultured human endothelial cells indicated that antithrombin III was not synthesized by the cells. Endothelial cells cultured in antithrombin III-depleted human serum did not contain antithrombin III, as was measured by ELISA. Internalization studies performed with radiolabeled purified antithrombin III and antithrombin III-thrombin complexes indicated that endothelial cells internalize antithrombin III when it is complexed to thrombin. Antithrombin III alone was not internalized by the endothelial cells.