Thrombomodulin (p.Cys537Stop) is released from cells by an unusual membrane insertion/leakage mechanism.

The expression of thrombomodulin variant c.1611C>A (p.Cys537Stop) leads to the synthesis of a protein with no cytoplasmic tail and a transmembrane domain shortened by 3 amino acids (TM536). Little is known about the release mechanism and properties of TM536. Using umbilical vein endothelial cells and peripheral blood-derived endothelial colony-forming cells from a heterozygous carrier of TM536 variant as well as overexpression cell models, we demonstrate that TM536 is released from cells by an unusual mechanism. First, TM536 is inserted into the endoplasmic reticulum membrane, then, due to the low hydrophobicity of its intramembrane domain, it escapes from it and follows the conventional secretory pathway to be released into the extracellular compartment without the involvement of proteolysis. This particular secretion mechanism yields a soluble TM536 that is poorly modified by chondroitin sulfate glycosaminoglycan compared to conventionally secreted soluble forms of thrombomodulin, and therefore has a suboptimal capacity to mediate thrombin-dependent activation of protein C. We also show that TM536 cellular trafficking is altered, with retention in early secretory pathway and increased sensitivity to endoplasmic reticulum-associated degradation. As expected, activation of endoplasmic reticulum-associated degradation increases TM536 degradation and reduces its release. The expression of TM536 at the cell surface is low and its distribution in lipid raft-like membrane microdomains is altered, resulting in low thrombin-dependent PC activation on the cell surface.