Thrombomodulin Gly430Ser mutation attenuates the affinity with thrombin and contributes to thrombosis development

Thrombomodulin (TM) is essential in maintaining vascular homeostasis. Its anticoagulant function is mainly mediated through the formation of thrombomodulin-thrombin complex, which could accelerate the conversion of protein C (PC) to activated protein C (APC). We identified 3 patients who carried the mutation c.1288G > A, p.G430S in THBD and suffered from recurrent thrombosis. The objective of this study is to elucidate the molecular basis of thrombosis underlying the TM Gly430Ser mutation. We expressed the wild-type and Gly430Ser mutant TM in both full-length and soluble fragment forms in mammalian cells. The cofactor capacity of TM was evaluated by measuring the thrombin-dependent generation of APC and activated TAFI in a time- and concentration-dependent manner. The binding affinity between TM and thrombin was determined by surface plasmon resonance (SPR). Furthermore, the overall impact of TM was evaluated through thrombin generation test (TGT) and APTT prolongation test. The cofactor function of TM-G430S in promoting thrombin-mediated activation of PC and TAFI was substantially compromised. SPR analysis revealed that the binding affinity of TM-G430S for thrombin was significantly impaired, with only about 10% of WT level, as reflected by the increased affinity constant. Consistent with this defect, TGT revealed that TM-G430S exhibited increased peak height and ETP at all concentrations tested and confirmed its impaired capacity to suppress thrombin generation. The ability of TM-G430S to prolong APTT was impaired as well. The Gly430Ser mutation in TM compromises the thrombin dependent activation of PC and TAFI through impaired thrombin binding, underlying the hypercoagulable state and the increased risk of thrombosis observed in patients carrying this mutation.