Van Laer Christine, Lavend'homme Renaud, Baert Sarissa, De Wispelaere Koenraad, Thys Chantal, Kint Cyrielle, Noppen Sam, Peerlinck Kathelijne, Van Geet Chris, Schols Dominique, Vanassche Thomas, Labarque Veerle, Verhamme Peter, Jacquemin Marc, Freson Kathleen
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium.
Blood. 2025 Apr 24;145(17):1929-1942. doi: 10.1182/blood.2024026454.
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 disorders. In contrast, although 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 6 patients with a thrombotic (C175S, A282P, L433P, P501L, G502R, and P508L) and 2 patients with a bleeding (P260A and 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 Expi293F cells. L433P TM showed a marked decrease in the inhibition of thrombin generation and complete inhibition of protein C and thrombin activatable fibrinolysis inhibitor (TAFI) activation. Soluble C175S TM showed decreased inhibition of thrombin generation and protein C activation, whereas no effect was observed for cell membrane-bound recombinant TM. For the other TM variants, no effect on thrombin generation, protein C, or TAFI activation could be observed. Surface plasmon resonance analysis showed no thrombin-TM binding in the presence of L433P because this residue is located at their interaction site. In conclusion, our study shows the functional effects of L433P TM and potentially C175S TM, which 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 roles.
内皮细胞上表达的血栓调节蛋白（TM）可调节凝血。血栓调节蛋白基因（THBD）中的特定无义变体导致可溶性TM水平升高，引发罕见的出血性疾病。相比之下，尽管THBD变体与静脉血栓栓塞有关，但这种关联仍存在争议。使用多基因检测板对601例遗传性出血或血栓形成疾病患者进行诊断。这使得6例血栓形成表型患者（C175S、A282P、L433P、P501L、G502R和P508L）和2例出血表型患者（P260A和T478I）鉴定出8种THBD变体。这些均被归类为意义未明的变体，我们在此旨在评估它们在凝血中的功能作用。为此，在Expi293F细胞中产生了可溶性和细胞膜结合的重组TM。L433P TM在抑制凝血酶生成以及完全抑制蛋白C和凝血酶激活的纤维蛋白溶解抑制剂（TAFI）激活方面表现出显著降低。可溶性C175S TM在抑制凝血酶生成和蛋白C激活方面表现出降低，而对于细胞膜结合的重组TM未观察到影响。对于其他TM变体，未观察到对凝血酶生成、蛋白C或TAFI激活的影响。表面等离子体共振分析显示在存在L433P的情况下没有凝血酶-TM结合，因为该残基位于它们的相互作用位点。总之，我们的研究显示了L433P TM以及潜在的C175S TM的功能作用，这与血栓形成风险增加相符。THBD变体很少见，但可能与出血和血栓形成均相关。对这些变体进行功能测定对于理解它们的作用至关重要。
