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

Shijie Zhou; Xi Wu; Ying Song; Lei Li; Chunli Shi; Zhe Lai; Qiulan Ding; Wenman Wu; Jing Dai; Xuefeng Wang; Yeling Lu

doi:10.1055/s-0043-1777133

Thrombosis and Haemostasis, Table of Contents

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 ^*

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Xi Wu^*

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Ying Song^*

²Department of Clinical Hematology and osology, Shanghai Center of Clinical Laboratory, Shanghai, China

,

Lei Li

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Chunli Shi

³Department of Molecular Biology, Shanghai Center of Clinical Laboratory, Shanghai, China

,

Zhe Lai

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Qiulan Ding

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Wenman Wu

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Jing Dai

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Xuefeng Wang

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

,

Yeling Lu

¹Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

› Author Affiliations

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

Full Text

References

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