Molecular mechanism of dysfunctional factor VII associated with the homozygous missense mutation 331Gly to Ser

Summary

We have identified a Japanese homozygous FVII deficiency associated with the mutation G331S (c184 [in chymotrypsin numbering]), and have determined the mechanisms responsible for the dysfunctional FVII variant by expressing the mutant recombinant FVII protein. In addition, the recombinant proteins FVIIG331D, G331W and G331F were expressed. The purified recombinant FVII proteins ran as a single chain form on SDS-PAGE having a molecular mass of approximately 50Kda. The recombinant FVIIG331S expressed the level of the recombinant wild type FVII at 2.0%, and this mutant form was also similar to FVII in the patient’s plasma. However, the amidolytic activity of FVIIa using peptidyl substrate S-2288 differed little between the wild type and the four mutant FVII molecules. We suggest that the functional defect found in these mutants is not directly associated with peptidyl substrate recognition or catalysis. The Km values of FX and FIX for the mutant proteins were approximately 7.6– to 15-fold and 13– to 19-fold higher than those for the wild-type protein, respectively. Molecular modelling indicated that the side chain of the S331 mutant is oriented close to the side chain of D338 (c189) at the bottom of the specificity pocket of FVIIa. We show that the replacement of G331 with a serine likely results in a steric hindrance of macromolecular substrate binding, leading to a loss of FVIIa enzymatic activity.

Keywords

Dysfunctional factor VII variant - catalytic domain - substrate-binding region - chromogenic substrate

• References

• 1 Bajaj SP, Rapaport I S, Brown SF. Isolation and characterization of human factor VII. Activation of factor VII by factor Xa. J Biol Chem 1981; 256: 253-9.
• 2 Masys DR, Bajaj SP, Rapaport I S. Activation of human factor VII by activated factors IX and X. Blood 1982; 60: 1143-50.
• 3 Kisiel W, Fujikawa K, Davie EW. Activation of bovine factor VII (proconvertin) by factor XIIa (activated Hageman factor). Biochemistry 1977; 16: 4189-94.
• 4 Nakagaki T, Foster DC, Berkner KL et al. Initiation of the extrinsic pathway of blood coagulation: Evidence for the tissue factor dependent autoactivation of human coagulation factor VII. Biochemistry 1991; 30: 10819-24.
• 5 Wildgoose P, Kisiel W. Activation of human factor VII by factors IXa and Xa on human bladder carcinoma cells. Blood 1989; 73: 1888-95.
• 6 O'Hara PJ, Grant FJ, Haldeman BA. et al. Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation. Proc Natl Acad Sci USA 1987; 84: 5158-62.
• 7 Triplett DA, Brandt JT, Batard MA. et al. Hereditary factor VII deficiency: heterogeneity defined by combined functional and immunochemical analysis. Blood 1985; 66: 1284-7.
• 8 Tuddenham EG, Pemberton S, Cooper DN. Inherited factor VII deficiency: genetics and molecular pathology. Thromb Haemost 1995; 74: 313-21.
• 9 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.
• 10 Takamiya O. Assay of factor VII antigen by enzyme-linked immunosorbent assay (ELISA). Rinshobyori (Japanese) 1991; 39: 437-41.
• 11 Takamiya O, Okimoto Y. Severe factor VII deficiency with recurrent intracranial haemorrhages owing to double heterozygosity for a splice site mutation of an IVS4 and a novel nonsense mutation in exon 8 (Gln211-->Term). Br J Haematol 2001; 114: 369-74.
• 12 Takamiya O, Yoshioka A. Factor VII binding to tissue factor in plasma from warfarin-treated individuals. Thromb Res 1996; 81: 657-63.
• 13 Takamiya O, Kemball-Cook G, Martin DM. et al. Detection of missense mutations by single-strand conformational polymorphism (SSCP) analysis in five dysfunctional variants of coagulation factor VII. Hum Mol Genet 1993; 2: 1355-9.
• 14 Takamiya O, Seta M, Tanaka K. et al. Human factor VII deficiency caused by S339C mutation located adjacent to the specificity pocket of the catalytic domain. Clin Lab Haematol 2002; 24: 233-8.
• 15 Orita M, Suzuki Y, Sekiya T. et al. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5: 874-9.
• 16 Humphries S, Temple A, Lane A. et al. Low plasma levels of factor VIIc and antigen are more strongly associated with the 10 base pair promoter (-323) insertion than the glutamine 353 variant. Thromb Haemost 1996; 75: 567-72.
• 17 van't Hooft FM, Silveira A, Tornvall P. et al. Two common functional polymorphisms in the promoter region of the coagulation factor VII gene determining plasma factor VII activity and mass concentration. Blood 1999; 93: 3432-4.
• 18 Chaing SH, Wallmark A, Berntorp E. et al. A Nla III polymorphism within the human factor VII gene. Hum Genet 1994; 93: 722-3.
• 19 Marchetti G, Gemmati D, Patracchini P. et al. PCR detection of a repeat polymorphism within the F7 gene. Nucleic Acids Res 1991; 19: 4570
• 20 Green F, Kelleher C, Wilkes H. et al. A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals. Arterioscler Thromb 1991; 11: 540-6.
• 21 Takamiya O, Hino K. A patient homozygous for a Gly354Cys mutation in factor VII that results in severely impaired secretion of the molecule, but not complete deficiency. Br J Haematol 2004; 124: 336-42.
• 22 Millar DS, Kemball-Cook G, McVey JH. et al. Molecular analysis of the genotype-phenotype relationship in factor VII deficiency. Hum Genet 2000; 107: 327-42.
• 23 Etro D, Pinotti M, Wulff K. et al. The Gly331Ser mutation in factor VII in Europe and the Middle East. Haematologica 2003; 88: 1434-6.
• 24 Pinotti M, Etro D, Bindini D. et al. Residual factor VII activity and different hemorrhagic phenotypes in CRM(+) factor VII deficiencies (Gly331Ser and Gly283Ser). Blood 2002; 99: 1495-7.
• 25 Zheng DQ, Shurafa M, James HL. Factor VII G331D: a variant molecule involving replacement of a residue in the substrate-binding region of the catalytic domain. Blood Coagul Fibrinolysis 1996; 7: 93-6.
• 26 Banner DW, D'Arcy A, Chene C. et al. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996; 7 (380) 41-6.
• 27 Davidson CJ, Hirt RP, Lal K. et al. Molecular evolution of the vertebrate blood coagulation network. Thromb Haemost 2003; 89: 420-8.
• 28 Miyata T, Kojima T, Suzuki K. et al. Factor X Nagoya 1 and Nagoya 2: a CRM- factor X deficiency and a dysfunctional CRM+ factor X deficiency characterized by substitution of Arg306 by Cys and of Gly366 by Ser, respectively. Thromb Haemost 1998; 79: 486-90.
• 29 Liu JZ, Li X, Drost J, Thorland EC. et al. The human factor IX gene as germline mutagen test: Samples from mainland China have the putatively endogenous patter of mutation. Hum Mutat 2000; 16: 31-6.