Internalization of the anticoagulant thrombomodulin is constitutive and does not require a signal in the cytoplasmic domain

Endocytosis of the protein C activation cofactor thrombomodulin (TM) is thought to be induced by interaction of TM with its ligand, thrombin, or by the action of inflammatory cytokines on endothelial cells. To examine the internalization of TM in the absence of thrombin or cytokines we used two assays. The first was a two‐colour indirect immunofluorescence technique to simultaneously monitor cell surface and internal pools of TM. The second involved labelling a cell surface pool of TM and following its cellular redistribution over time. Using these techniques we demonstrated that in both TM‐transfected COS cells and in endothelial cells, TM internalizes constitutively. Removal of the cytoplasmic domain, which in most receptors contains the internalization signal, did not abolish TM internalization. These results suggest that endocytosis of TM does not occur via coated pits, and that internalization probably is not a significant cause of the endothelial TM loss associated with several pathological states.