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Thromb Haemost 2024; 124(05): 459-470
DOI: 10.1055/s-0043-1777133
Stroke, Systemic or Venous Thromboembolism

Ser252Asn Mutation Introduces a New N-Linked Glycosylation Site and Causes Type IIb Protein C Deficiency

Shijie Zhou    *
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Xi Wu   *
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Ying Song   *
2   Department of Clinical Hematology and osology, Shanghai Center of Clinical Laboratory, Shanghai, China
,
Lei Li
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Chunli Shi
3   Department of Molecular Biology, Shanghai Center of Clinical Laboratory, Shanghai, China
,
Zhe Lai
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Qiulan Ding
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Wenman Wu
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Jing Dai
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Xuefeng Wang
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Yeling Lu
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
› Author Affiliations Funding This study was supported by Shanghai Pujiang Program (21PJ1409800), Shanghai Natural Science Foundation Project (22ZR1439500), and the General Program of National Natural Science Foundation of China (81870107) to Y.L.; the General Program of National Natural Science Foundation of China (82070137) to X.W.; the General Program of National Natural Science Foundation of China (81970127) to J.D., and the project for Excellent Youth Scholars of Shanghai Public Health (GWV-10.2-YQ35) to X.W.
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Abstract

Background Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin–thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the “Paris” mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC).

Material and Methods We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation.

Results We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site (252NTT254) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC.

Conclusion The new N-glycosylation site (252NTT254) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

Keywords

protein C - mutation - anticoagulant - thrombosis - glycosylation

Authors' Contribution

S.Z. performed research and wrote the manuscript; X.W. and L.L. performed genetic analysis and coagulation assays with the subjects' plasma; Y.S., C.S., and Z.L. performed research; J.D. and W.W. collected and provided clinical data; X.W. and Q.D. supervised studies; and L.Y. designed experiments, analyzed data, supervised the project, and revised the manuscript. All authors approved the final version of this manuscript.


* These authors contributed equally to this work.




Publication History

Received: 27 May 2023

Accepted: 26 October 2023

Article published online:
27 November 2023

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