Reduced Activation of the Gla19Ala FX Variant via the Extrinsic Coagulation Pathway Results in Symptomatic CRM^red FX Deficiency

 

 Buy Article Permissions and Reprints

Summary

We characterized a symptomatic CRM^red factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation.

The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.

• References

• 1 James HL. Physiology and biochemistry of factor X. In: Haemostasis and Thrombosis. Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. eds. Edinburgh: Churchill Livingstone; 1994: 439-64.
  Google Scholar
• 2 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 29: 10363-70.
  PubMedGoogle Scholar
• 3 Butenas S, van’t Veer C, Mann KG. Evaluation of the initiation phase of blood coagulation using ultrasensitive assays for serine proteases. J Biol Chem 1997; 272: 21527-33.
  CrossrefPubMedGoogle Scholar
• 4 Furie BC, Furie B. Coagulant protein of Russell’s viper venom. Methods Enzymol 1976; 45: 191-205.
  PubMedGoogle Scholar
• 5 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surfacedependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
  PubMedGoogle Scholar
• 6 Sunnerhagen M, Forsen S, Hoffren AM, Drakenberg T, Teleman O, Stenflo J. Structure of the Ca2+-free Gla domain sheds light on membrane binding of blood coagulation proteins. Nat Struct Biol 1995; 02: 504-9.
  CrossrefPubMedGoogle Scholar
• 7 Furie B, Bouchard BA, Furie BC. Vitamin K-dependent biosynthesis of )’-carboxyglutamic acid. Blood 1999; 93: 1798-808.
  PubMedGoogle Scholar
• 8 Falls LA, Furie BC, Jacobs M, Furie B, Rigby AC. The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes. J Biol Chem 2001; 276: 23895-902.
  CrossrefPubMedGoogle Scholar
• 9 Fung MR, Colin WH, MacGillivray RTA. Characterization of an almost full-length cDNA coding for human blood coagulation factor X. Proc Natl Acad Sci USA 1985; 82: 3591-5.
  CrossrefPubMedGoogle Scholar
• 10 Huang Q, Neuenschwander PF, Rezaie AR, Morrissey JH. Substrate recognition by tissue factor-factor VIIa. J Biol Chem 1996; 271: 21752-7.
  CrossrefPubMedGoogle Scholar
• 11 Ruf W, Shobe J, Rao SM, Dickinson CD, Olson A, Edgington TS. Importance of factor VIIa Gla domain residue Arg-36 for recognition of the macromolecular substrate factor X Gla-domain. Biochemistry 1999; 38: 1957-66.
  CrossrefPubMedGoogle Scholar
• 12 Kirchhofer D, Lipari MT, Moran P, Eigenbrot C, Kelley RF. The tissue factor region that interacts with substrates factor IX and factor X. Biochemistry 2000; 39: 7380-7.
  CrossrefPubMedGoogle Scholar
• 13 Kirchhofer D, Eigenbrot C, Lipari MT, Moran P, Peek M, Kelley RF. The tissue factor region that interacts with factor Xa in the activation of factor VII. Biochemistry 2001; 40: 675-82.
  CrossrefPubMedGoogle Scholar
• 14 Hedner U, Davie EW. Introduction to hemostasis and the vitamin Kdependent coagulation factors. In: The Metabolic Basis of Inherited Disease. Scriver CR, Beaudet AL, Sly WS, Valle D. eds. New York: Mc-Graw-Hill; 1989: 2107-27.
  Google Scholar
• 15 Peyvandi F, Mannucci PM, Lak M, Abdoullahi M, Zeinali S, Sharifian R, Perry D. Congenital factor X deficiency: spectrum of bleeding symptoms in 32 Iranian patients. Br J Haematol 1998; 102: 626-8.
  CrossrefPubMedGoogle Scholar
• 16 Bernardi F, Marchetti G, Patracchini P, Volinia S, Gemmati D, Simioni P, Girolami A. Partial gene deletion in a family with factor X deficiency. Blood 1989; 73: 2123-7.
  PubMedGoogle Scholar
• 17 Cooper DN, Millar DS, Wacey A, Pemberton S, Tuddenham EGD. Inherited Factor X deficiency: molecular genetics and pathophysiology. Thromb Haemost 1997; 78: 161-72.
  Article in Thieme ConnectPubMedGoogle Scholar
• 18 Millar DS, Elliston L, Deex P, Krawczak M, Wacey AI, Reynaud J, Nieuwenhuis HK, Bolton-Maggs P, Mannucci PM, Reverter JC, Cachia P, Pasi KJ, Layton DM, Cooper DN. Molecular analysis of the genotypephenotype relationship in factor X deficiency. Hum Genet 2000; 106: 249-57.
  CrossrefPubMedGoogle Scholar
• 19 Bernardi F, Castaman G, Redaelli R, Pinotti M, Lunghi B, Rodeghiero F, Marchetti G. Topologically equivalent mutations causing dysfunctional coagulation factors VII (294Ala→Val) and X (334Ser→Pro). Hum Mol Genet 1994; 03: 1175-7.
  CrossrefPubMedGoogle Scholar
• 20 Bernardi F, Castaman G, Pinotti M, Ferraresi P, Di Iasio MG, Lunghi B, Rodeghiero F, Marchetti G. Mutation pattern in clinically asymptomatic coagulation factor VII deficiency. Hum Mutat 1996; 08: 108-15.
  CrossrefPubMedGoogle Scholar
• 21 Millar DS, Kemball-Cook G, McVey JH, Tuddenham EGD, Mumford AD, Attock GB, Reverter JC, Lanir N, Parapia LA, Reynaud J, Meili E, von Felton A, Martinowitz U, Prangnell DR, Krawczak M, Cooper DN. Molecular analysis of the genotype-phenotype relationship in factor VII deficiency. Hum Genet 2000; 107: 327-42.
  CrossrefPubMedGoogle Scholar
• 22 Watzke HH, Lechner K, Roberts HR, Reddy SV, Welsch DJ, Friedman P, Mahr G, Jagadeeswaran P, Monroe DM, High KA. Molecular defect (Gla+14Lys) and its functional consequences in a hereditary factor X deficiency (Factor X “Voralberg”). J Biol Chem 1990; 265: 11982-9.
  PubMedGoogle Scholar
• 23 Racchi M, Watzke HH, High KA, Lively MO. Human coagulation factor X deficiency caused by a mutant signal peptide that blocks cleavage by signal peptidase but not targeting and translocation to the endoplasmic reticulum. J Biol Chem 1993; 268: 5735-40.
  PubMedGoogle Scholar
• 24 Bezeaud A, Miyata T, Helley D, Zeng YZ, Kato H, Aillaud MF, Juhan-Vague I, Guillin MC. Functional consequence of the Ser334→Pro mutation in a human factor X variant. Eur J Biochem 1995; 234: 140-7.
  CrossrefPubMedGoogle Scholar
• 25 Rudolph AE, Mullane MP, Porche-Sorbet R, Tsuda S, Miletich JP. Factor X St Louis II. Identification of a glycine substitution at residue 7 and characterization of the recombinant protein. J Biol Chem 1996; 271: 28601-6.
  CrossrefPubMedGoogle Scholar
• 26 Kim DJ, Girolami A, James HL. Characterization of recombinant human coagulation factor X Friuli. Thromb Haemost 1996; 75: 313-7.
  Article in Thieme ConnectPubMedGoogle Scholar
• 27 Nobauer-Huhmann IM, Holler W, Krinninger B, Turecek PL, Richter G, Scharrer I, Forberg E, Watzke HH. Factor X Frankfurt I: molecular and functional characterization of a hereditary factor X deficiency (Gla+25 to Lys). Blood Coagul Fibrinolysis 1998; 09: 143-52.
  CrossrefPubMedGoogle Scholar
• 28 Forberg E, Huhmann I, Jimenez-Boj E, Watzke HH. The impact of Glu102Lys on the factor X function in a patient with a doubly homozygous factor X deficiency (Gla14Lys and Glu102Lys). Thromb Haemost 2000; 83: 234-8.
  Article in Thieme ConnectPubMedGoogle Scholar
• 29 Iijima K, Murakami M, Kimura O, Murakami F, Shimomura T, Ikawa S. A dysfunctional factor X (factor X Kurayoshi) with a substitution of Arg139 for Ser at the carboxyl-terminus of the light chain. Thromb Res 2000; 101: 311-6.
  PubMedGoogle Scholar
• 30 Simioni P, Vianello F, Kalafatis M, Barzon L, Ladogana S, Paolucci P, Carotenuto M, Dal Bello F, Palu G, Girolami A. A dysfunctional factor X (factor X San Giovanni Rotondo) present at homozygous and double heterozygous level: identification of a novel microdeletion (delC556) and missense mutation (Lys408Asn) in the factor X gene A study of an Italian family. Thromb Res 2001; 101: 219-30.
  CrossrefPubMedGoogle Scholar
• 31 Larson PJ, Camire RM, Wong D, Fasano NC, Monroe DM, Tracy PB, High KA. Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles. Biochemistry 1998; 37: 5029-38.
  CrossrefPubMedGoogle Scholar
• 32 Marchetti G, Castaman G, Pinotti M, Lunghi B, Ruggieri M, Rodeghiero F, Bernardi F. Molecular bases of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glu102Lys) in the II EGF-like domain. Br J Haematol 1995; 90: 910-5.
  CrossrefPubMedGoogle Scholar
• 33 Leytus SP, Foster DC, Kurachi K, Davie EW. Gene for human factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and Protein C. Biochemistry 1986; 25: 5098-102.
  CrossrefPubMedGoogle Scholar
• 34 Pinotti M, Toso R, Redaelli R, Berrettini M, Marchetti G, Bernardi F. Molecular mechanisms of FVII deficiency: expression of mutations clustered in the IVS7 donor splice site of factor VII gene. Blood 1998; 92: 1646-51.
  PubMedGoogle Scholar
• 35 Rosing J, Bakker HM, Thomassen MCLGD, Hemker HC, Tans G. Characterization of two forms of human factor Va with different cofactor activities. J Biol Chem 1993; 268: 21130-6.
  PubMedGoogle Scholar
• 36 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-85.
  CrossrefPubMedGoogle Scholar
• 37 Kim DJ, Thompson AR, James HL. Factor X Ketchikan: a variant molecule in which Gly replaces a Gla residue at position 14 in the light chain. Hum Genet 1995; 95: 212-4.
  PubMedGoogle Scholar
• 38 Wu W, Bancroft JD, Suttie JW. Differential effects of warfarin on the intracellular processing of vitamin K-dependent proteins. Thromb Haemost 1996; 76: 46-52.
  Article in Thieme ConnectPubMedGoogle Scholar
• 39 Hedner U. Recombinant activated factor VII as a universal haemostatic agent. Blood Coag Fibrinolysis 1998; 09 (suppl) S147-S152.
  PubMedGoogle Scholar
• 40 Santagostino E, Morfini M, Rocino A, Baudo F, Scaraggi FA, Gringeri A. Relationship between factor VII activity and clinical efficacy of recombinant FVIIa given by continuous infusion to patients with factor VIII inhibitors. Thromb Haemost 2001; 86: 954-8.
  Article in Thieme ConnectPubMedGoogle Scholar
• 41 Mertens K, Bertina RM. Pathway in the activation of human coagulation factor X. Biochem J 1980; 185: 647-58.
  CrossrefPubMedGoogle Scholar
• 42 Mizuno H, Fujimoto Z, Atoda H, Morita T. Crystal structure of an anticoagulant protein in complex with the Gla domain of factor X. Proc Natl Acad Sci USA 2001; 98: 7230-4.
  CrossrefPubMedGoogle Scholar
• 43 Soriano-Garcia M, Padmanabhan K, de Vos AM, Tulinsky A. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry 1992; 31: 2554-66.
  CrossrefPubMedGoogle Scholar
• 44 Banner DW, D’Arcy A, Chene C, Winkler FK, Guha A, Konigsberg WH, Nemerson Y, Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996; 380: 41-6.
  CrossrefPubMedGoogle Scholar
• 45 Zhong D, Bajaj MS, Schmidt AE, Bajaj SP. The N-terminal EGF-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor. J Biol Chem 2002; 277: 3622-31.
  CrossrefPubMedGoogle Scholar