Impaired prothrombinase activity of factor X Gly381Asp results in severe familial CRM+ FX deficiency

Summary

We investigated three members of a large Omani family affected by severe factor X (FX) deficiency (coagulant activity <1%) and showing marked differences in the onset of severe hemorrhagic symptoms. All patients were homozygous for a novel FX mutation (Gly381Asp) in the structurally conserved region of the serine protease active site. Expression levels of recombinant 381D-FX were similar to those of wt-FX, indicating the presence of a severe CRM+ FX deficiency, a poorly investigated condition. The 381D-FX was normally activated and did not show a detectable amidolytic activity. Instead, we observed a residual activity in a prothrombin-time based assay (1%) and in prothrombinase assays both in plasma (1%) and in purified systems (3%). Comparison with FX variants characterized by reduced activation suggests that mutations affecting FX activity might result in a more pronounced impairment of coagulation and thus in severe hemorrhagic phenotype. In addition, this study indicates that the hemorrhagic heterogeneity observed in FX deficiencies is only partially explained by molecular analysis of FX gene.

Keywords

CRM+ FX deficiency - hemorrhagic phenotype - recombinant FX - FX activity

• References

• 1 James HL. Physiology and biochemistry of factor X. In Haemostasis and Thrombosis. Bloom AL, Forbes CD, Thomas DP, Tudden-ham EGD. eds Edinburgh: Churchill Living-stone; 1994: 439-64.
• 2 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 29: 10363-70.
• 3 Hedner U, Davie EW. Introduction to hemostasis and the vitamin K-dependent coagulation factors. In The Metabolic Basis of Inherited Disease. Scriver CR, Beaudet AL, Sly WS, Valle D. eds New York: McGraw-Hill; 1989: 2107-27.
• 4 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.
• 5 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 genotype-phenotype relationship in factor X deficiency. Hum Genet 2000; 106: 249-57.
• 6 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.
• 7 Cooper DN, Millar DS, Wacey A, Pemberton S, Tuddenham EGD. Inherited Factor X deficiency: molecular genetics and pathophysiology. Thromb Haemost 1997; 78: 161-72.
• 8 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; 3: 1175-7.
• 9 Bernardi F, Castaman G, Pinotti M, Ferraresi P, DiIasio MG, Lunghi B, Rodeghiero F, Marchetti G. Mutation pattern in clinically asymptomatic coagulation factor VII deficiency. Hum Mutat 1996; 8: 108-15.
• 10 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.
• 11 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.
• 12 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.
• 13 Bezeaud A, Miyata T, Helley D, Zeng YZ, Kato H, Aillaud MF, Juhan-Vague I, Guillin MC. Functional consequence of the Ser334Pro mutation in a human factor X variant. Eur J Biochem 1995; 234: 140-7.
• 14 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.
• 15 Kim DJ, Girolami A, James HL. Characterization of recombinant human coagulation factor X Friuli. Thromb Haemost 1996; 75: 313-7.
• 16 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; 9: 143-52.
• 17 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.
• 18 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 2001; 101: 311-6.
• 19 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.
• 20 Pinotti M, Marchetti G, Baroni M, Cinotti F, Morfini M, Bernardi F. Reduced activation of the Gla19Ala FX variant via the extrinsic coagulation pathway results in symptomatic CRMred FX deficiency. Thromb Haemost 2002; 88: 236-41.
• 21 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.
• 22 Castoldi E, Simioni P, Kalafatis M, Lunghi B, Tormene D, Girelli D, Girolami A, Bernardi F. Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A) affecting the prothrombinase complex in a thrombophilic family. Blood 2000; 96: 1443-8.
• 23 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
• 24 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.
• 25 Camire RM, Larson PJ, Stafford DW, High KA.. Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry 2000; 39: 14322-9.
• 26 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.
• 27 Nichols WC, Amano K, Cacheris PM, Figuei-redo MS, Michaelides K, Schwabb R, Hoyer L, Kaufman RJ, Ginsburg D. Moderation of hemophilia A phenotype by the factor V R506Q mutation. Blood 1996; 88: 1183-87.
• 28 vant'Veer C, Glolden NJ, Kalafatis M, Simioni P, Bertina RM, Mann KG. An in vitro analysis of the combination of hemophilia A and factor V Leiden. Blood 1997; 90: 3067-72.
• 29 Arbini AA, Mannucci PM, Bauer KA. Low prevalence of the factor V Leiden mutation among “severe” hemophiliacs with a “milder” bleeding diathesis. Thromb Haemost 1995; 74: 1255-8.
• 30 Rand MD, Lock JB, van'tVeer C, Gaffney DP, Mann KG. Blood clotting in minimally altered whole blood. Blood 1996; 88: 3432-45..
• 31 Furie B, Bing DH, Feldmann RJ, Robinson DJ, Burnier J, Furie BC. Computer-generated models of blood coagulation factor Xa, factor IXa and thrombin upon structural homology with other serine proteases. J Biol Chem 1982; 257: 3875-82.
• 32 Bode W, Brandstetter H, Mather T, Stubbs MT. Comparative analysis of haemostatic pro-teinases: structural aspects of thrombin, factor Xa, factor IXa and protein C. Thromb Haemost 1997; 78: 501-11.
• 33 Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Blankenship DT, Cardin AD, Kisiel W. Structure of human des (1-45) factor Xa at 2.2 A resolution. J Mol Biol 1993; 232: 947-66.
• 34 Bode W, Turk D, Karshikov A. The refined 1.9-Å X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human -thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Science 1992; 1: 426-71.
• 35 Giannelli F, Green PM, Sommer SS, Poon M, Ludwig M, Schwaab R, Reitsma PH, Goossens M, Yoshioka A, Figueiredo MS, Brownlee GG. Haemophilia B: database of point mutations and short additions and deletions – eighth edition. Nucleic Acids Res 1998; 26: 265-8.