Why patients with THBD c.1611C>A (p.Cys537X) nonsense mutation have high levels of soluble thrombomodulin?

BACKGROUND: Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail. OBJECTIVE: We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation. METHODS: Complementary DNA of TM wild type (TM-WT) was incorporated into a pcDNA3.1 vector for transient transfection in COS-1 cells. Mutagenesis was performed to create the c.1611C<A (TM(1-536)) mutant and 4 other TM mutants (TM(1-515), TM(1-525), TM(1-533) and TM(1-537)) with a transmembrane domain having different lengths. The effect of shear stress, metalloprotease inhibitor, certain proteases and reducing agents were tested on TM shedding. RESULTS: Western blot and immunofluorescent analysis showed that TM(1-536) was produced and a certain amount of TM(1-536) was anchored on the cell membrane. A significantly higher levels of soluble TM was observed in the TM(1-536) cell medium in comparison with TM-WT (56.3 +/- 5.2 vs 8.8 +/- 1.6 ng/mL, respectively, p = 0.001). The shedding of TM(1-536) was 75% decreased in cells cultured in the presence of a metalloprotease inhibitor. No difference was observed between TM(1-536) and TM-WT shedding after cell exposure to cathepsin G, elastase, several reducing agents and high shear stress (5000 s(-1)). Significantly higher levels of soluble TM were observed in the cell media of TM(1-533), TM(1-525), TM(1-515) in comparison with TM-WT (p < 0.05). CONCLUSION: The mechanism responsible for TM shedding is complex and is not completely understood: higher sensitivity of the TM(1-536) to the proteolysis by metalloproteases and a defect of synthesis due to the decreased size of the transmembrane domain might explain the high levels of soluble TM in plasma of the carriers.