A missense mutation in lectin domain of thrombomodulin causing functional deficiency
Introduction
Thrombomodulin (TM) is an important regulator in various physiological processes, including coagulation, fibrinolysis and inflammation. It is encoded by THBD [MIM188040], and is mainly expressed on the membrane of endothelial cells.1,2 Since its discovery in 1981 by Esmon and Owen,3 the structure and regulatory function of TM has been well explored. Its extracellular structure consists of a N-terminal C-type lectin-like domain, six epidermal growth factor (EGF)-like modules and a serine/threonine (Ser/Thr) rich region.4,5 This structure also exists in plasma as functionally active soluble thrombomodulin (sTM), lacking the single transmembrane stretch and cytoplasmic region.6 When bound to thrombin with EGF-like modules 5 and 6, TM significantly changes the substrate specificity of thrombin, turning the key factor recognizing fibrinogen in coagulation cascade an anticoagulant and anti-fibrinolytic enzyme.
The anticoagulant property of TM is mainly mediated by directing thrombin toward protein C (PC) activation. Activated protein C (APC) by thrombin-thrombomodulin complex proteolytically inactivates activated coagulation factors Ⅴ and Ⅷ and limits further thrombin generation.7 APC also mediates anti-inflammatory and cytoprotective effects by binding to endothelial protein C receptor.8 It has been identified that EGF-like domain 4–6 plays a vital role when TM interacts with PC.9 When it comes to thrombin-activatable fibrinolysis inhibitor (TAFI) activation, EGF modules 3–6 are required.10 Activated TAFI (TAFIa) removes the carboxyterminal lysine residues from the surface of fibrin, which is essential in binding of plasminogen and tissue-type plasminogen activator (tPA) to fibrin. Other than downregulating fibrinolysis, TAFIa also inactivates activated complements C3 and C5.11 The thrombin-mediated activation of PC and TAFI is amplified by more than 3 orders of magnitude with the help of TM.12 TM also promotes the proteolytic cleavage of high mobility group box 1 (HMGB1) by thrombin.13 HMGB1 is a chromosomal protein that induces cell signaling by binding to toll-like receptors 2, 4 and receptor for advanced glycation end products .14 The lectin domain of TM helps thrombin to release the N-terminus from HMGB1, leading to a loss of pro-inflammatory activity.15
It has been widely reported that single nucleotide polymorphisms in THBD are related to thrombophilic tendency and thrombotic microangiopathy. Nevertheless, mutations in different domains of TM may lead to very different results in functional and clinical consequences.16 So far there has been few reports on mutation of TM in lectin domain related to thrombotic disease.
In our previous work regarding the genetic background for venous thrombosis in the Chinese population, several variants in THBD were reported.17 Among them was a heterozygous c.376G>T (p.Asp126Tyr) mutation located in the C-type lectin domain found in a 10-year-old boy who suffered from recurrent thrombosis in both venous and artery. The very same mutation was then identified in other 2 patients diagnosed with deep-vein thrombosis in our following study, which led us to the hypothesis that this mutation in lectin domain of TM tended to cause thromboembolic diseases in these probands. Therefore, we explored whether functional deficiencies of TM were caused by this c.376G>T (p.Asp126Tyr) mutation and here are the results.
Section snippets
Reagents and mice
Human thrombin, hippuryl-L-arginine (Hip-Arg), cyanuric chloride, 1, 4-dioxane were purchased from Sigma Aldrich (St Louis, MO, USA). L-a-phosphatidylcholine, L-a-phosphatidylserine, L-a-phosphatidylethanolamine, dioleoyl (PC/PS/PE) vesicles were from Avanti Polar Lipids (AL, USA). TAFI and tPA were from Sino Biological (Beijing, China). Human protein C was from HYPHEN BioMed (Neuville-sur-Oise, France). Bovine native HMGB1 was purchased from Chondrex (Redmond). Pyro-Glu-Pro-Arg-pNA•HCl
The genetic analysis, sTM level and clinical characterizations of the probands
Together we detected the heterozygous c.376G>T mutation of THBD in 3 patients, from whom deficiencies in antithrombin, PC or protein S was ruled out. The sTM antigen level in the 3 probands' plasma was detected by ELISA. The result together with some clinical characterizations of the heterozygotes was shown in Table 1. They all suffered from recurrent thromboembolic diseases, and thrombotic events were also reported in the second patient's father and sister. The first patient was found to be a
Discussion
As a multidomain transmembrane glycoprotein mainly distributed on vascular endothelial cell surface, TM has crucial roles in modulating coagulation, fibrinolysis and inflammation. Our results, together with those of previous investigations, revealed that mutations in TM might lead to functional defects in these aspects. It has been proved in animal studies that ablation of TM gene leads to intrauterine death at early stage and significant deficiency in TM function highly elevates the
Conclusions
In summary, we investigated the sTM level reduction and functional deficiencies of TM caused by the Asp126Tyr mutation in lectin domain. This may help us better understand the physiological roles of TM in regulating coagulation, fibrinolysis and inflammation, and the importance of lectin domain in TM carrying out these modulating functions. This work also provides supportive evidence on the relation between TM mutation and thrombotic tendency, which may present new prospects for diagnosis and
Authors' Contributions
Conception and design of work: T.L. and H.Y. Acquisition and analysis of data: M.J., T.Y. and F.Y. Drafting and revision: M.J., C.Z. L.W. and H.B. All authors have approved the submitted version of the manuscript.
Ethics approval and consent to participate
The experimental protocol was established in accordance with the guidelines of the Helsinki Declaration. The study was approved by the Wuhan Union Hospital Ethics Committee (approved number: [2019] IECS514). Written informed consents were obtained from the patients and their family members. All animal studies were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology.
Availability of data and materials
All data included in this study are available upon request by contract with the corresponding author.
Acknowledgments
All the authors have read the journal's policy on conflicts of interest and the authors declare that they have no competing interests.
This work was supported by the Program for HUST Academic Frontier Youth Team (No. 2018QYTD14) and National Natural Science Foundation of China (No.81900144).
All the authors have read the journal's authorship agreement. We are grateful for the technical assistance from Wu Tingting and Huang Zhongpei, and the valuable suggestions from Zuo Yuyue and Zhong Yanqi.
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Cited by (1)
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Ma Jiewen and Tao Yanyi are co-first authors.