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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

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Owen WG, Esmon CT. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1981; 256 (11) 5532-5535
    CrossrefPubMedSearch in Google Scholar
  • 2 Mather T, Oganessyan V, Hof P. et al. The 2.8 A crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15 (24) 6822-6831
    CrossrefPubMedSearch in Google Scholar
  • 3 Griffin JH, Fernández JA, Gale AJ, Mosnier LO. Activated protein C. J Thromb Haemost 2007; 5 (Suppl. 01) 73-80
    CrossrefPubMedSearch in Google Scholar
  • 4 Grinnell BW, Walls JD, Gerlitz B. Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin. J Biol Chem 1991; 266 (15) 9778-9785
    CrossrefPubMedSearch in Google Scholar
  • 5 Fay PJ, Smudzin TM, Walker FJ. Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity. J Biol Chem 1991; 266 (30) 20139-20145
    CrossrefPubMedSearch in Google Scholar
  • 6 Heeb MJ, Griffin JH. Activated protein C-dependent and -independent anticoagulant activities of protein S have different structural requirements. Blood Cells Mol Dis 2002; 29 (02) 190-199
    CrossrefPubMedSearch in Google Scholar
  • 7 Rezaie AR, Mather T, Sussman F, Esmon CT. Mutation of Glu-80–>Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex. J Biol Chem 1994; 269 (05) 3151-3154
    CrossrefPubMedSearch in Google Scholar
  • 8 Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269 (42) 26486-26491
    CrossrefPubMedSearch in Google Scholar
  • 9 Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor signaling. J Thromb Haemost 2003; 1 (07) 1495-1503
    CrossrefPubMedSearch in Google Scholar
  • 10 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109 (08) 3161-3172
    CrossrefPubMedSearch in Google Scholar
  • 11 Griffin JH, Zlokovic BV, Mosnier LO. Activated protein C: biased for translation. Blood 2015; 125 (19) 2898-2907
    CrossrefPubMedSearch in Google Scholar
  • 12 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68 (05) 1370-1373
    CrossrefPubMedSearch in Google Scholar
  • 13 Khor B, Van Cott EM. Laboratory tests for protein C deficiency. Am J Hematol 2010; 85 (06) 440-442
    CrossrefPubMedSearch in Google Scholar
  • 14 Kottke-Marchant K, Comp P. Laboratory issues in diagnosing abnormalities of protein C, thrombomodulin, and endothelial cell protein C receptor. Arch Pathol Lab Med 2002; 126 (11) 1337-1348
    CrossrefPubMedSearch in Google Scholar
  • 15 Baker P, Platton S, Gibson C. et al; British Society for Haematology, Haemostasis and Thrombosis Task Force. Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis. Br J Haematol 2020; 191 (03) 347-362
    CrossrefPubMedSearch in Google Scholar
  • 16 Minford A, Brandão LR, Othman M. et al. Corrigendum to diagnosis and management of severe congenital protein C deficiency (SCPCD): Communication from the SSC of the ISTH [J Thromb Haemost. 2022 Jul;20(7):1735-1743]. [J Thromb Haemost. 2022 Jul;20(7):1735-1743] J Thromb Haemost 2023; 21 (04) 1069
    CrossrefPubMedSearch in Google Scholar
  • 17 Gandrille S, Alhenc-Gelas M, Gaussem P. et al. Five novel mutations located in exons III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity. Blood 1993; 82 (01) 159-168
    CrossrefPubMedSearch in Google Scholar
  • 18 Yang L, Manithody C, Rezaie AR. Contribution of basic residues of the 70-80-loop to heparin binding and anticoagulant function of activated protein C. Biochemistry 2002; 41 (19) 6149-6157
    CrossrefPubMedSearch in Google Scholar
  • 19 Chen C, Yang L, Villoutreix BO, Wang X, Ding Q, Rezaie AR. Gly74Ser mutation in protein C causes thrombosis due to a defect in protein S-dependent anticoagulant function. Thromb Haemost 2017; 117 (07) 1358-1369
    Thieme ConnectPubMedSearch in Google Scholar
  • 20 Ding Q, Yang L, Dinarvand P, Wang X, Rezaie AR. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays. Blood 2015; 125 (15) 2428-2434
    CrossrefPubMedSearch in Google Scholar
  • 21 Smirnov MD, Esmon CT. Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994; 269 (02) 816-819
    CrossrefPubMedSearch in Google Scholar
  • 22 Lu Y, Giri H, Villoutreix BO, Ding Q, Wang X, Rezaie AR. Gly197Arg mutation in protein C causes recurrent thrombosis in a heterozygous carrier. J Thromb Haemost 2020; 18 (05) 1141-1153
    CrossrefPubMedSearch in Google Scholar
  • 23 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33 (01) 4-15
    CrossrefPubMedSearch in Google Scholar
  • 24 Walenga JM, Fasanella AR, Iqbal O. et al. Coagulation laboratory testing in patients treated with argatroban. Semin Thromb Hemost 1999; 25 (Suppl. 01) 61-66
    PubMedSearch in Google Scholar
  • 25 Funk DM. Coagulation assays and anticoagulant monitoring. Hematology (Am Soc Hematol Educ Program) 2012; 2012: 460-465
    CrossrefPubMedSearch in Google Scholar
  • 26 Ireland H, Bayston T, Thompson E. et al. Apparent heterozygous type II protein C deficiency caused by the factor V 506 Arg to Gln mutation. Thromb Haemost 1995; 73 (04) 731-732
    Thieme ConnectPubMedSearch in Google Scholar
  • 27 Baglin T, Gray E, Greaves M. et al; British Committee for Standards in Haematology. Clinical guidelines for testing for heritable thrombophilia. Br J Haematol 2010; 149 (02) 209-220
    CrossrefPubMedSearch in Google Scholar
  • 28 Bovill EG, Bauer KA, Dickerman JD, Callas P, West B. The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood 1989; 73 (03) 712-717
    CrossrefPubMedSearch in Google Scholar
  • 29 Dentali F, Gianni M. VTE recurrence in patients with inherited deficiencies of natural anticoagulants. Thromb Haemost 2009; 101 (01) 5-6
    Thieme ConnectPubMedSearch in Google Scholar
  • 30 Ho WK, Hankey GJ, Quinlan DJ, Eikelboom JW. Risk of recurrent venous thromboembolism in patients with common thrombophilia: a systematic review. Arch Intern Med 2006; 166 (07) 729-736
    CrossrefPubMedSearch in Google Scholar
  • 31 Seidel H, Haracska B, Naumann J, Westhofen P, Hass MS, Kruppenbacher JP. Laboratory limitations of excluding hereditary protein C deficiency by chromogenic assay: discrepancies of phenotype and genotype. Clin Appl Thromb Hemost 2020; 26: 1076029620912028
    CrossrefPubMedSearch in Google Scholar
  • 32 Simioni P, Kalafatis M, Millar DS. et al. Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma. Blood 1996; 88 (06) 2101-2108
    CrossrefPubMedSearch in Google Scholar
  • 33 Lu Y, Mehta-D'souza P, Biswas I. et al. Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis. Haematologica 2020; 105 (06) 1712-1722
    CrossrefPubMedSearch in Google Scholar
  • 34 Fisher CL, Greengard JS, Griffin JH. Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen. Protein Sci 1994; 3 (04) 588-599
    CrossrefPubMedSearch in Google Scholar
  • 35 Gerlitz B, Grinnell BW. Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin. J Biol Chem 1996; 271 (37) 22285-22288
    CrossrefPubMedSearch in Google Scholar
  • 36 Knobe KE, Berntsdotter A, Shen L, Morser J, Dahlbäck B, Villoutreix BO. Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling. Proteins 1999; 35 (02) 218-234
    CrossrefPubMedSearch in Google Scholar
  • 37 Gale AJ, Heeb MJ, Griffin JH. The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites. Blood 2000; 96 (02) 585-593
    CrossrefPubMedSearch in Google Scholar
  • 38 Friedrich U, Nicolaes GA, Villoutreix BO, Dahlbäck B. Secondary substrate-binding exosite in the serine protease domain of activated protein C important for cleavage at Arg-506 but not at Arg-306 in factor Va. J Biol Chem 2001; 276 (25) 23105-23108
    CrossrefPubMedSearch in Google Scholar