Novel p.C252G and p.C280X mutations in EGF-1 domain of thrombomodulin lead to a thrombosis-bleeding syndrome.

Background: Thrombomodulin (TM) binds thrombin forming a thrombin-TM complex, thereby regulating the balance between coagulation and fibrinolysis. We found a proband carrying compound heterozygous mutations (c.754T>G, p.C252G and c.840C>A, p.C280X) in EGF-1 of THBD gene resulting into TM deficiency.

Objectives: To explore the molecular mechanism underlying the anticoagulant defect caused by C252G and C280X mutations.

Results: A 5-year-old Chinese boy with lifelong large ecchymosis due to TM deficiency showed marked hypofibrinogenemia with significant increased fibrinogen/fibrin degradation products, D-dimer, thrombin-antithrombin complex, and plasmin-α2 plasmin inhibitor complex, indicating a disseminated intravascular coagulation (DIC)-like microcirculatory thrombosis-bleeding syndrome. The proband carried heterozygous mutations (C252G and C280X) in THBD gene. In HEK293T cells transfected with TM-wild-type (WT), TM-C252G and TM-C280X cDNAs, both TM-WT and TM-C252G were comparably expressed on cell surface, while TM-C280X was not expressed due to the substitution of c.840C with A leading to a stop codon formation. The recombinant TM (rTM) carrying C252G mutation in both the extracellular domain (rTM-EC/C252G) and the membrane-penetrating type exhibited attenuated inhibitory effect on thrombin generation compared to rTM-WT. Moreover, rTM-EC/C252G exhibited lower activation of thrombin catalytic protein C and thrombin activatable fibrinolysis inhibitor (TAFI) than rTM-EC/WT, which may enhance coagulation and fibrinolysis. Protein conformation heatmap analysis revealed that rTM-EC/C252G mutant induced TM structural changes with >25% relative uptake differences compared to rTM-EC/WT.

Conclusion: We identified two novel TM variants (C252G and C280X) associated with DIC-like thrombosis-bleeding syndrome, providing the first genetic evidence for the critical role of Cys252-Cys265 in maintaining the structure and function of TM.