Functional assessment of genetic variants in thrombomodulin detected in patients with bleeding and thrombosis

• Rare THBD VUS are detected in patients with inherited bleeding and thrombotic disorders.

• Thrombomodulin L433P has an impaired effect on the natural anticoagulant pathway by decreased protein C and TAFI activation.

Thrombomodulin (TM) expressed on endothelial cells regulates coagulation. Specific nonsense variants in the TM gene, THBD, result in high soluble TM levels causing rare bleeding disorder. In contrast, though THBD variants have been associated with venous thromboembolism, this association remains controversial. A multigene panel was used to diagnose 601 patients with inherited bleeding or thrombotic disorders. This resulted in the identification of 8 THBD variants for six patients with a thrombotic (C175S, A282P, L433P, P501L, G502R, P508L) and two patients with a bleeding (P260A, T478I) phenotype. These were all classified as variants of uncertain significance, and we here aimed to assess their functional role in coagulation. For this purpose, soluble and cell membrane-bound recombinant TM were produced in Expi293FTM cells. L433P TM showed a marked decrease in the inhibition of thrombin generation and complete inhibition of protein C and TAFI activation. Soluble C175S TM showed decreased inhibition of thrombin generation and protein C activation, while no effect was observed for membrane-bound TM. For the other TM variants, no effect on thrombin generation nor protein C or TAFI activation could be observed. Surface plasmon resonance analysis showed absent thrombin-TM binding in the presence of L433P, as this residue is located at their interaction site. In conclusion, our study shows functional effects of L433P TM and potentially also C175S TM that are compatible with an increased thrombosis risk. THBD variants are rare but can be relevant to both bleeding and thrombosis. Functional assays for these variants are critical to understand their role.