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<p>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 &gt; A, p.G430S in <i>THBD</i> 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.</p> Graphic abstract <p>The schematic illustrates the molecular pathogenetic basis of Gly430Ser mutation in thrombomodulin. Genetic screening was performed as part of our routine diagnosis on patients with thrombosis, among whom three unrelated thrombotic probands were identified with G430S mutation in TM in our study. According to our structural predictions, this mutant leads to an unstable molecular structure while binding to thrombin. SPR further verified a tenfold decreased affinity of TM-G430S to thrombin in comparison of the KD value of TM-WT (K<sub>D</sub> = 3.4*10<sup>−8</sup>&#xa0;M) and TM-G430S(K<sub>D</sub> = 2.55*10<sup>−7</sup>&#xa0;M). The attenuated affinity to thrombin of G430S mutation resulted in severely impaired cofactor ability for activating PC and TAFI, which disrupted the balance between coagulation system and fibrinolysis system. What’s more, the significantly decreased potency of G430S to inhibit thrombin generation and to prolong APTT revealed a hypercoagulable state. Eventually, these integrated results increased the risk of thrombosis development in the patients.</p> <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年7月28日

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  • Nifei Chen,
  • Zhe Lai,
  • Junwei Yuan,
  • Xi Wu,
  • Fang Li,
  • Qiulan Ding,
  • Wenman Wu,
  • Ying Song,
  • Xuefeng Wang,
  • Jing Dai,
  • Xiaobo Hu,
  • Yeling Lu

摘要

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.

Graphic abstract

The schematic illustrates the molecular pathogenetic basis of Gly430Ser mutation in thrombomodulin. Genetic screening was performed as part of our routine diagnosis on patients with thrombosis, among whom three unrelated thrombotic probands were identified with G430S mutation in TM in our study. According to our structural predictions, this mutant leads to an unstable molecular structure while binding to thrombin. SPR further verified a tenfold decreased affinity of TM-G430S to thrombin in comparison of the KD value of TM-WT (KD = 3.4*10−8 M) and TM-G430S(KD = 2.55*10−7 M). The attenuated affinity to thrombin of G430S mutation resulted in severely impaired cofactor ability for activating PC and TAFI, which disrupted the balance between coagulation system and fibrinolysis system. What’s more, the significantly decreased potency of G430S to inhibit thrombin generation and to prolong APTT revealed a hypercoagulable state. Eventually, these integrated results increased the risk of thrombosis development in the patients.