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Thromb Haemost 2017; 117(07): 1358-1369
DOI: 10.1160/TH17-01-0043
Coagulation and Fibrinolysis
Schattauer GmbH

Gly74Ser mutation in protein C causes thrombosis due to a defect in protein S-dependent anticoagulant function

Changming Chen
1   State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Likui Yang
2   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
,
Bruno O. Villoutreix
3   Inserm U973, Université Paris Diderot, Sorbonne Paris Cité, Molécules Thérapeutiques In Silico, Paris, France
,
Xuefeng Wang
4   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Qiulan Ding
4   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Alireza R. Rezaie
2   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
› Author Affiliations
Further Information

Publication History

Received: 19 January 2017

Accepted after minor revision: 25 March 2017

Publication Date:
28 November 2017 (online)

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Summary

Protein C is a vitamin K–dependent serine protease zymogen in plasma which upon activation by thrombin in complex with thrombomodulin (TM) down-regulates the clotting cascade by a feedback loop inhibition mechanism. Activated protein C (APC) exerts its anticoagulant function through protein S-dependent degradation of factors Va and VIIIa. We recently identified a venous thrombosis patient whose plasma level of protein C antigen is normal, but its anticoagulant activity is only 34% of the normal level. Genetic analysis revealed that the proband and her younger brother carry a novel heterozygous mutation c.346G>A, p.Gly74Ser (G74S) in PROC. Thrombin generation assay indicated that the TM-dependent anticoagulant activity of the proband’s plasma has been significantly impaired. We expressed protein C-G74S in mammalian cells and characterised its properties in established coagulation assays. We demonstrate that the protein C variant can be normally activated by the thrombin-TM complex and the resulting APC mutant also exhibits normal amidolytic and proteolytic activities toward both FVa and FVIIIa. However, it was discovered the protein S-dependent catalytic activity of APC variant toward both procoagulant cofactors has been significantly impaired. Protein S concentration-dependence of FVa degradation revealed that the capacity of APC variant to interact with the cofactor has been markedly impaired. The same results were obtained for inactivation of FVa-Leiden suggesting that the protein S-dependent activity of APC variant toward cleavage of Arg-306 site has been adversely affected. These results provide insight into the mechanism through which G74S substitution in APC causes thrombosis in the proband carrying this mutation.

Keywords

Protein C - protein S - factor Va - factor VIIIa - thrombosis

 

  • References

  • 1 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 29-35.
    PubMedGoogle Scholar
  • 2 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-956.
    CrossrefPubMedGoogle Scholar
  • 3 Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. FASEB J 1992; 6: 2561-2567.
    CrossrefPubMedGoogle Scholar
  • 4 Foster D, Davie EW. Characterization of a cDNA coding for human protein C. Proc Natl Acad Sci USA 1984; 81: 4766-4770.
    CrossrefPubMedGoogle Scholar
  • 5 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-1651.
    PubMedGoogle 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: 190-199.
    CrossrefPubMedGoogle Scholar
  • 7 Norstrom EA, Steen M, Tran S. et al. Importance of protein S and phospholipid for activated protein C-mediated cleavage in factor Va. J Biol Chem 2003; 278: 24904-24911.
    CrossrefPubMedGoogle 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: 26486-26491.
    PubMedGoogle Scholar
  • 9 Mosnier LO, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
    CrossrefPubMedGoogle Scholar
  • 10 Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor signaling. J Thromb Haemost 2003; 1: 1495-1503.
    CrossrefPubMedGoogle Scholar
  • 11 Joyce DE, Grinnell BW. Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-kB. Crit Care Med 2002; 30: S288-S293.
    CrossrefPubMedGoogle Scholar
  • 12 Regan LM, Mollica JS, Rezaie AR. et al. The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C. J Biol Chem 1997; 272: 26279-26284.
    CrossrefPubMedGoogle Scholar
  • 13 Ahnström J, Andersson HM, Canis K. et al. Activated protein C cofactor function of protein S: a novel role for a ?-carboxyglutamic acid residue. Blood 2011; 117: 6685-6693.
    CrossrefPubMedGoogle Scholar
  • 14 Villoutreix BO, Teleman O, Dahlbäck B. A theoretical model for the Gla-TSR-EGF-1 region of the anticoagulant cofactor protein S: from biostructural pathology to species-specific cofactor activity. J Comput Aided Mol Des 1997; 11: 293-304.
    CrossrefPubMedGoogle Scholar
  • 15 He X, Shen L, Dahlbäck B. Expression and functional characterization of chimeras between human and bovine vitamin-K-dependent protein-S-defining modules important for the species specificity of the activated protein C cofactor activity. Eur J Biochem 1995; 227: 433-440.
    CrossrefPubMedGoogle Scholar
  • 16 Hackeng TM, Yegneswaran S, Johnson AE. et al. Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S. Biochem J 2000; 349: 757-764.
    CrossrefPubMedGoogle Scholar
  • 17 Mather T, Oganessyan V, Hof P. et al. The 2.8 Å crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15: 6822-6831.
    PubMedGoogle Scholar
  • 18 Preston RJ, Ajzner E, Razzari C. et al. Multifunctional specificity of the protein C/activated protein C Gla domain. J Biol Chem 2006; 281: 28850-28857.
    CrossrefPubMedGoogle Scholar
  • 19 Dahlbäck B. The protein C anticoagulant system: inherited defects as basis for venous thrombosis. Thromb Res 1995; 77: 1-43.
    CrossrefPubMedGoogle Scholar
  • 20 Griffin JH, Evatt B, Zimmerman TS. et al. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373.
    CrossrefPubMedGoogle Scholar
  • 21 Jalbert LR, Rosen ED, Moons L. et al. Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice. J Clin Invest 1998; 102: 1481-1488.
    CrossrefPubMedGoogle Scholar
  • 22 Reitsma PH, Bernardi F, Doig RG. et al. Protein C deficiency: a database of mutations, 1995 update. On behalf of the Subcommittee on Plasma Coagulation Inhibitors of the Scientific and Standardization Committee of the ISTH. Thromb Haemost 1995; 73: 876-889.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 Mackie I, Cooper P, Lawrie A, Kitchen S, Gray E, Laffan M. British Committee for Standards in Haematology. Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis. Int J Lab Hematol 2013; 35: 1-13.
    PubMedGoogle Scholar
  • 24 Ding Q, Yang L, Dinarvand P. et al. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays. Blood 2015; 125: 2428-2434.
    CrossrefPubMedGoogle Scholar
  • 25 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
    CrossrefPubMedGoogle Scholar
  • 26 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: 6149-6157.
    CrossrefPubMedGoogle Scholar
  • 27 Ding Q, Yang L, Hassanian SM. et al. Expression and functional characterization of natural R147W and K150del variants of protein C in the Chinese population. Thromb Haemost 2013; 109: 614-624.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 28 Soriano-Garcia M, Padmanabhan K, de Vos AM. et al. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry 1992; 31: 2554-2566.
    CrossrefPubMedGoogle Scholar
  • 29 Villoutreix BO, Covell DG, Blom AM. et al. Screening the molecular surface of human anticoagulant protein C: a search for interaction sites. J Comput Aided Mol Des 2001; 15: 13-27.
    CrossrefPubMedGoogle Scholar
  • 30 Banner DW, D’Arcy A, Chène C. et al. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996; 380: 41-46.
    CrossrefPubMedGoogle Scholar
  • 31 Kozakov D, Grove LE, Hall DR. et al. The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins. Nat Protoc 2015; 10: 733-755.
    CrossrefPubMedGoogle Scholar
  • 32 Shen L, Dahlbäck B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 1994; 269: 18735-18738.
    PubMedGoogle Scholar
  • 33 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-1008.
    CrossrefPubMedGoogle Scholar
  • 34 Bertina RM, Koeleman BP, Koster T. et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67.
    CrossrefPubMedGoogle Scholar
  • 35 Kalafatis M, Mann KG. Role of the membrane in the inactivation of factor Va by activated protein C. J Biol Chem 1993; 268: 27246-27257.
    PubMedGoogle Scholar
  • 36 Nicolaes GA, Tans G, Thomassen MC. et al. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and Factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 21158-21166.
    CrossrefPubMedGoogle Scholar
  • 37 Rosing J, Hoekema L, Nicolaes GA. et al. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-27858.
    CrossrefPubMedGoogle Scholar
  • 38 Ohlin AK, Landes G, Bourdon P. et al. Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity. J Biol Chem 1988; 263: 19240-19248.
    PubMedGoogle Scholar
  • 39 Laimer J, Hiebl-Flach J, Lengauer D. et al. MAESTROweb: a web server for structure-based protein stability prediction. Bioinformatics 2016; 32: 1414-1416.
    CrossrefPubMedGoogle Scholar
  • 40 Yin S, Ding F, Dokholyan NV. Eris: an automated estimator of protein stability. Nat Methods 2007; 4: 466-467.
    CrossrefPubMedGoogle Scholar
  • 41 David A, Razali R, Wass MN. et al. Protein-protein interaction sites are hot spots for disease-associated nonsynonymous SNPs. Hum Mutat 2012; 33: 359-363.
    CrossrefPubMedGoogle Scholar
  • 42 Villoutreix BO, Lagorce D, Labbé CM. et al. One hundred thousand mouse clicks down the road: selected online resources supporting drug discovery collected over a decade. Drug Discov Today 2013; 18: 1081-1089.
    CrossrefPubMedGoogle Scholar
  • 43 Villoutreix BO, Kuenemann MA, Poyet JL. et al. Drug-Like Protein-Protein Interaction Modulators: Challenges and Opportunities for Drug Discovery and Chemical Biology. Mol Inform 2014; 33: 414-437.
    CrossrefPubMedGoogle Scholar
  • 44 Saller F, Villoutreix BO, Amelot A. et al. The gamma-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding. Blood 2005; 105: 122-130.
    CrossrefPubMedGoogle Scholar
  • 45 Giri TK, Villoutreix BO, Wallqvist A. et al. Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling. Thromb Haemost 1998; 80: 798-804.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 46 He X, Shen L, Villoutreix BO, Dahlbäck B. Amino acid residues in thrombin-sensitive region and first epidermal growth factor domain of vitamin K-dependent protein S determining specificity of the activated protein C cofactor function. J Biol Chem 1998; 273: 27449-27458.
    CrossrefPubMedGoogle Scholar
  • 47 Andersson HM, Arantes MJ, Crawley JT. et al. Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain. Blood 2010; 115: 4878-4885.
    CrossrefPubMedGoogle Scholar
  • 48 Boerger LM, Morris PC, Thurnau GR. et al. Oral contraceptives and gender affect protein S status. Blood 1987; 69: 692-694.
    PubMedGoogle Scholar
  • 49 Malm J, Laurell M, Dahlbäck B. Changes in the plasma levels of vitamin K-dependent proteins C and S and of C4b-binding protein during pregnancy and oral contraception. Br J Haematol 1988; 68: 437-443.
    CrossrefPubMedGoogle Scholar