Novel p.C252G and p.C280X mutations in the epidermal growth factor-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 epidermal growth factor-1 of the THBD gene, resulting in TM deficiency.

OBJECTIVES

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

METHODS

Mutant TM plasmids were expressed in human embryonic kidney 293T (HEK293T) cells, and recombinant proteins were employed to assess the impact of TM mutations on functional activity and spatial conformation.

RESULTS

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

CONCLUSION

We identified 2 novel TM variants (C252G and C280X) associated with disseminated intravascular coagulation-like thrombosis bleeding syndrome, providing the first genetic evidence of the critical role of Cys252-Cys265 in maintaining the structure and function of TM.