Protein C Sapporo (protein C Glu 25 → Lys): A heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor

Summary

Interaction of the γ-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G toA transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25→Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25→Lys mutation induces type IIb protein C deficiency in individuals.

Keywords

Protein C deficiency - type IIb deficiency - protein C Sapporo - Gla domain (γ-carboxyglutamic acid domain) - EPCR (endothelial protein C receptor)

• References

• 1 Dahlback B. The protein C anticoagulant system: inherited defects as basis for venous thrombosis. Thromb Res 1995; 77: 1-43.
• 2 Esmon CT. The endothelial cell protein C receptor. Thromb Haemost 2000; 83: 639-43.
• 3 Esmon CT. Protein C, protein S and thrombomodulin. Hemostasis and Thrombosis: Basic Principles and Clinical Practice.. Lippincott: Williams & Wilkins; 2001: 335-53.
• 4 Plutzky J, Hoskins JA, Long GL. et al. Evolution and organization of the human protein C gene. Proc Natl Acad Sci USA 1986; 83: 546-50.
• 5 Zhang L, Jhingan A, Castellino FJ. Role of individual γ-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity. Blood 1992; 80: 942-52.
• 6 Colpitts TL, Prorok M, Castellino FJ. Binding of calcium to individual γ-carboxyglutamic acid residues of human protein C. Biochemistry 1995; 34: 2424-30.
• 7 Griffin JH, Evatt B, Zimmerman TS. et al. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-3.
• 8 Branson HE, Katz J, Marble R. et al. Inherited protein C deficiency and coumarin-responsive chronic relapsing purpura fulminans in a newborn infant. Lancet 1983; 2: 1165-8.
• 9 Marlar RA, Adcock DM, Madden RM. Hereditary dysfunctional protein C molecules (type II): assay characterization and proposed classification. Thromb Haemost 1990; 63: 375-9.
• 10 Greengard JS, Fisher CL, Villoutreix B. et al. Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: patterns revealed by three-dimensional molecular modelling of mutations of the protease domain. Proteins 1994; 18: 367-80.
• 11 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: 876-89.
• 12 Gaussem P, Gandrille S, Duchemin J. et al. Influence of six mutations of the protein C gene on the Gla domain conformation and calcium affinity. Thromb Haemost 1994; 71: 748-54.
• 13 Faioni EM, Hermida J, Rovida E. et al. Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity. Br J Haematol 2000; 108: 265-71.
• 14 Alhenc-Gelas M, Gandrille S, Aubry ML. et al. Thirty-three novel mutations in the protein C gene. French INSERM network on molecular abnormalities responsible for protein C and protein S. Thromb Haemost 2000; 83: 86-92.
• 15 Wakabayashi K, Sakata Y, Aoki N. Conformationspecific monoclonal antibodies to the calcium-induced structure of protein C. J Biol Chem 1986; 261: 11097-105.
• 16 Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-7.
• 17 Watanabe K, Shibuya A, Ishii E. et al. Identification of simultaneous mutation of fibrinogen α chain and protein C genes in a Japanese kindred. Br J Haematol 2003; 120: 101-8.
• 18 Miyata T, Zheng YZ, Sakata T. et al. Three missense mutations in the protein C heavy chain causing type I and type II protein C deficiency. Thromb Haemost 1994; 71: 32-7.
• 19 Ido M, Ohiwa M, Hayashi T. et al. A compound heterozygous protein C deficiency with a single nucleotide G deletion encoding Gly-381 and an amino acid substitution of Lys for Gla-26. Thromb Haemost 1993; 70: 636-41.
• 20 Ebina Y, Yamada H, Kato EH. et al. Thromboprophylaxis with low molecular weight heparin in thrombophilia- complicated pregnancy. J Obstet Gynaecol Res 2002; 28: 251-7.
• 21 Kraaijenhagen RA, in’t Anker PS, Koopman MM. et al. High plasma concentration of factor VIIIc is a major risk factor for venous thromboembolism. Thromb Haemost 2000; 83: 5-9.
• 22 van Hylckama Vlieg A, van der Linden IK, Bertina RM. et al. High levels of factor IX increase the risk of venous thrombosis. Blood 2000; 95: 3678-82.
• 23 Meijers JC, Tekelenburg WL, Bouma BN. et al. High levels of coagulation factor XI as a risk factor for venous thrombosis. N Engl J Med 2000; 342: 696-701.
• 24 Kyrle PA, Eichinger S. Deep vein thrombosis. Lancet 2005; 365: 1163-74.
• 25 Bertina RM, Koeleman BP, Koster T. et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
• 26 Poort SR, Rosendaal FR, Reitsma PH. et al. A common genetic variation in the 3’-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996; 88: 3698-703.
• 27 den Heijer M, Rosendaal FR, Blom HJ. et al. Hyperhomocysteinemia and venous thrombosis: a metaanalysis. Thromb Haemost 1998; 80: 874-7.
• 28 Liaw PC, Neuenschwander PF, Smirnov MD. et al. Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function. J Biol Chem 2000; 275: 5447-52.
• 29 Saposnik B, Reny JL, Gaussem P. et al. A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004; 103: 1311-8.
• 30 Uitte de Willige S, Van Marion V, Rosendaal FR. et al. Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis. J Thromb Haemost 2004; 2: 1305-10.
• 31 Medina P, Navarro S, Estelles A. et al. Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk. Thromb Haemost 2004; 91: 905-11.
• 32 Preston RJ, Villegas-Mendez A, Sun YH. et al. Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation. FEBS J 2005; 272: 97-108.
• 33 Simioni P, Morboeuf O, Tognin G. et al. Soluble endothelial protein C receptor (sEPCR) levels and venous thromboembolism in carriers of two dysfunctional protein C variants. Thromb Res. in press.
• 34 Stearns-Kurosawa DJ, Swindle K, D’Angelo A. et al. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood 2002; 99: 526-30.
• 35 Triplett DA, Sandquist DS, Musgrave KA. Clinical application of a functional assay for protein C. Hematol Pathol 1987; 1: 239-47.
• 36 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: 1337-48.
• 37 Ireland H, Thompson E, Lane DA. Gene mutations in 21 unrelated cases of phenotypic heterozygous protein C deficiency and thrombosis. Protein C Study Group. Thromb Haemost 1996; 76: 867-73.
• 38 McDonald JF, Shah AM, Schwalbe RA. et al. Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site. Biochemistry 1997; 36: 5120-7.
• 39 Aiach M, Gandrille S. Molecular basis for protein C hereditary deficiency. Haemostasis. 1996 04 9-19.
• 40 Reitsma PH. Protein C deficiency: from gene defects to disease. Thromb Haemost 1997; 78: 344-50.
• 41 Simioni P, Kalafatis M, Tormene D. et al. Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma: characterization of the dysfunctional protein C Padua3 (protein C(R-1L/propeptide)). Thromb Haemost 2001; 86: 1017-22.
• 42 Persson E. Ca2+ binding to proteins containing γ-carboxyglutamic acid residues. Methods Mol Biol 2002; 172: 81-95.
• 43 Furie B, Bouchard BA, Furie BC. Vitamin K-dependent biosynthesis of γ-carboxyglutamic acid. Blood 1999; 93: 1798-808.
• 44 Tartary M, Vidaud D, Piao Y. et al. Detection of a molecular defect in 40 of 44 patients with haemophilia B by PCR and denaturing gradient gel electrophoresis. Br J Haematol 1993; 84: 662-9.
• 45 Nagaizumi K, Inaba H, Suzuki T. et al. Two double heterozygous mutations in the F7 gene show different manifestations. Br J Haematol 2002; 119: 1052-8.
• 46 Nobauer-Huhmann IM, Holler W, Krinninger B. et al. Factor X Frankfurt I: molecular and functional characterization of a hereditary factor X deficiency (Gla+25 to Lys). Blood Coagul Fibrinolysis 1998; 9: 143-52.
• 47 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS. et al. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-6.
• 48 Fukudome K, Ye X, Tsuneyoshi N. et al. Activation mechanism of anticoagulant protein C in large blood vessels involving the endothelial cell protein C receptor. J Exp Med 1998; 187: 1029-35.
• 49 Taylor Jr. FB, Peer GT, Lockhart MS. et al. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-8.
• 50 Regan LM, Mollica JS, Rezaie AR. et al. The interaction between the endothelial cell protein C receptor and protein C is dictated by the γ-carboxyglutamic acid domain of protein C. J Biol Chem 1997; 272: 26279-84.
• 51 Oganesyan V, Oganesyan N, Terzyan S. et al. The crystal structure of the endothelial protein C receptor and a bound phospholipid. J Biol Chem 2002; 277: 24851-4.