Characterisation of blood coagulation factor XIT475I

John H. McVey; Kalpana Lal; Yasufumi Imanaka; Geoffrey Kemball-Cook; Paula H. B. Bolton-Maggs; Edward G. D. Tuddenham

doi:10.1160/TH05-01-0051

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 93(06): 1082-1088
DOI: 10.1160/TH05-01-0051

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Characterisation of blood coagulation factor XIT475I

John H. McVey

¹Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, London, U. K.

,

Kalpana Lal

¹Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, London, U. K.

²Manchester Haemophilia Comprehensive Care Centre, Department of Clinical Haematology, Manchester Royal Infirmary, Manchester, U. K.

^*K. Lal’s present address is Dept. of Biological Sciences, Imperial College London, U. K.; Present address Dr. Imanaka is Nara Medical College, Nara Medical University, Nara, Japan.

,

Yasufumi Imanaka

¹Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, London, U. K.

^*K. Lal’s present address is Dept. of Biological Sciences, Imperial College London, U. K.; Present address Dr. Imanaka is Nara Medical College, Nara Medical University, Nara, Japan.

,

Geoffrey Kemball-Cook

¹Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, London, U. K.

,

Paula H. B. Bolton-Maggs

²Manchester Haemophilia Comprehensive Care Centre, Department of Clinical Haematology, Manchester Royal Infirmary, Manchester, U. K.

,

Edward G. D. Tuddenham

¹Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, London, U. K.

› Author Affiliations

Abstract

Summary

PCR-SSCP and DNA sequence analysis of a factor XI (FXI) deficient patient (FXI:C 39 U/dL; FXI:Ag 27 U/dL) identified a C to T transition in exon 12 of the FXI gene (F11 c.1521C>T) that predicts the substitution of Thr475 by Ile (FXIT475I) within the serine protease domain of FXI. This mutation destroys a consensus sequence for N-linked glycosylation, N473-Y-T475, known to be utilized in vivo. The FXIT475I variant was generated by site-directed mutagenesis, together with other variants that could help explain the phenotype, and recombinant FXI variants were expressed in Chinese hamster ovary cells. FXI:Ag expression was analysed by Western blot analysis, ELISA and immunocyto-chemical staining. Wild-type FXI:Ag was secreted at high levels, however the mutant (FXIT475I) was secreted very poorly. Substitution of Thr475 by Ala, Pro, Lys or Arg (all of which abolish the glycosylation consensus sequence) also severely reduced the level of secreted FXI:Ag suggesting that glycosylation at Asn473 is required for folding or secretion. Concordant with this hypothesis the conservative substitution of Thr475 by Ser (which preserves the glycosylation consensus sequence) had no effect on FXI secretion. Thr/Ser475 is highly conserved in serine protease domains but the glycosylation site (Asn473) is not. Surprisingly, substitution of Asn473 by Ala (which removes the N-linked glycosylation site) had no effect on the levels of FXI:Ag secreted. In conclusion, although the FXI-T475I mutation destroys an N-linked glycosylation consensus sequence, the cause of failure to secrete FXI is not the loss of a glycosylation site but rather a direct effect of the substitution of this highly conserved residue.

Keywords

Factor XI - deficiency - mutation - glycosylation

Full Text

References

References
1 Davie EW, Fujikawa K, Kisiel w. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-70.
2 Bouma BN, Griffin JH, Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII. J Biol Chem 1977; 252: 6432-7.
3 Oliver JA, Monroe DM, Roberts HR. et al. Thrombin activates factor XI on activated platelets in the absence of factor XII. Arterioscler Thromb Vasc Biol 1999; 19: 170-7.
4 Gailani D, Broze GJJ, Factor XI. et al. activation in a revised model of blood coagulation. Science 1991; 253: 909-12.
5 Brunnee T, La Porta C, Reddigari SR. et al. Activation of factor XI in plasma is dependent on factor XII [see comments]. Blood 1993; 81: 580-6.
6 Asakai R, Davie EW, Chung DW. Organization of the gene for human factor XI. Biochemistry 1987; 26: 7221-8.
7 Kato A, Asakai R, Davie EW. et al. Factor XI gene (F11) is located on the distal end of the long arm of human chromosome 4. Cytogenet Cell Genet 1989; 52: 77-8.
8 Seligsohn U. et al. High gene frequency of factor XI (PTA) deficiency in Ashkenazi Jews. Blood 1978; 51: 1223-8.
9 Asakai R, Chung DW, Ratnoff OD. et al. Factor XI (plasma thromboplastin antecedent) deficiency in Ashkenazi Jews is a bleeding disorder that can result from three types of point mutations. Proc Natl Acad Sci U S A 1989; 86: 7667-71.
10 Peretz H, Zivelin A, Usher S. et al. A 14-bp deletion (codon 554 del AAGgtaacagagtg) at exon 14/intron N junction of the coagulation factor XI gene disrupts splicing and causes severe factor XI deficiency. Hum Mutat 1996; 8: 77-8.
11 Hancock JF, Wieland K, Pugh RE. et al. A molecular genetic study of factor XI deficiency. Blood 1991; 77: 1942-8.
12 O'connell NM, Saunders RE, Lee CA. et al. Structural interpretation of 42 mutations causing factor XI deficiency using homology modeling. J Thromb Haemost 2005; 3: 127-38.
13 Meijers JC, Davie EW, Chung DW. et al. Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency. Blood 1992; 79: 1435-40.
14 Pugh RE, Mcvey JH, Tuddenham EGD. et al. Six point mutations that cause factor XI deficiency. Blood 1995; 85: 1509-6.
15 Kunkel LM, Smith KD, Boyer SH. et al. Analysis of human Y chromosome-specific reiterated DNA in chromosome variants. Proc Natl Acad Sci U S A 1977; 74: 1245-9.
16 Imanaka Y, Lal K, Nishimura T. et al. Identification of two novel mutations in non-Jewish factor XI deficiency. Br J Haematol 1995; 90: 916-20.
17 Hayashi K. PCR-SSCP: A simple and sensitive method for the detection of mutations in genomic DNA. PCR Meth Appl 1991; 1: 34-8.
18 Fujikawa K, Chung DW, Hendrickson LE. et al. Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein. Biochemistry 1986; 25: 2417-24.
19 Kemball-Cook G, Garner I, Imanaka Y. et al. Highlevel production of human blood coagulation factors VII and XI using a new mammalian expression vector. Gene 1994; 139: 275-9.
20 Ohkubo Y, O'Brien DP, Kanehiro T. et al. Characterization of a panel of monoclonal antibodies to human coagulation factor XI and detection of factor XI in Hep G2 cell conditioned medium. Thromb Haemost 1990; 63: 417-23.
21 Kirkwood TB, Snape TJ. Biometric principles in clotting and clot lysis assays. Clin Lab Haematol 1980; 2: 155-67.
22 Mcmullen BA, Fujikawa K, Davie EW. et al. Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule. Biochemistry 1991; 30: 2050-6.
23 Martincic D, Zimmerman SA, Ware RE. et al. Identification of mutations and polymorphisms in the factor XI genes of an African American family by dideoxyfingerprinting. Blood 1998; 92: 3309-17.
24 Helenius D, Aebi M. Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 2004; 73: 1019-49.
25 Guan JL, Machamer CE, Rose JK. Glycosylation allows cell-surface transport of an anchored secretory protein. Cell 1985; 42: 489-96.
26 Larsen GR, Metzger M, Henson K. et al. Pharmacokinetic and distribution analysis of variant forms of tissue-type plasminogen activator with prolonged clearance in rat. Blood 1989; 73: 1842-50.
27 Ni H, Blajchman MA, Ananthanarayanan VS. et al. Mutation of any site of N-linked glycosylation accelerates the in vivo clearance of recombinant rabbit antithrombin. Thromb Res 2000; 99: 407-15.
28 Cousin P, Dechaud H, Grenot C. et al. Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood. J Steroid Biochem Mol Biol 1999; 70: 115-21.
29 Greer J. Comparative modeling methods: application to the family of the mammalian serine proteases. Proteins 1990; 7: 317-34.