The Impact of Glu102Lys on the Factor X Function in a Patient with a Doubly Homozygous Factor X Deficiency (Gla14Lys and Glu102Lys)

Eva Forberg; Iris Huhmann; Ester Jimenez-Boj; Herbert H. Watzke

doi:10.1055/s-0037-1613792

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2000; 83(02): 234-238
DOI: 10.1055/s-0037-1613792

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Schattauer GmbH

The Impact of Glu102Lys on the Factor X Function in a Patient with a Doubly Homozygous Factor X Deficiency (Gla14Lys and Glu102Lys)

Authors

Eva Forberg

Division of Hematology and Hemostaseology, University of Vienna, Austria
Iris Huhmann

Division of Hematology and Hemostaseology, University of Vienna, Austria
Ester Jimenez-Boj

Division of Hematology and Hemostaseology, University of Vienna, Austria
Herbert H. Watzke

Division of Hematology and Hemostaseology, University of Vienna, Austria

Abstract

Summary

Two homozygous point mutations were found in a patient with factor X (FX) deficiency; One results in substitution of Lys for Gla+14 and the second causes a Lys substitution for Glu102. The proposita has a severely reduced FX coagulant activity in the extrinsic (<1% of normal) and in the intrinsic (30% of normal) system of coagulation and after activation with Russel’s viper venom (18% of normal). The FX antigen is reduced in this patient to 20% of normal. The substitution of Lys for Glu102 in FX deficiency has been reported previously in a heterozygous state in conjunction with a Lys for Gla+14 substitution and with a Pro for Ser334 substitution. The contribution of the Lys for Glu102 substitution in the observed combined FX defect in these patients was unclear. The mutation causing the Glu102Lys substitution was introduced by site directed mutagenesis into a wild-type FX cDNA, and recombinant protein was expressed in HEK 293 cells. Compared to the wild-type FX cDNA, the mutant construct had a 67% activity upon activation in the extrinsic system, 93% activity upon activation in the intrinsic system and 72% after activation with RVV. The data presented show that the substitution of Lys for Glu102 results in a minor functional defect of the FX molecule.

Keywords

Factor X - hereditary deficiency - factor X expression - second EGF domain

Full Text

References

References
1 Bauer KA, Kass BL, ten Cate H, Bednarek MA, Hawiger JJ, Rosenberg RD. Detection of factor X activation in humans. Blood 1989; 74 (06) 2007-15.
2 McGee MP, Li CL. Functional difference between intrinsic and extrinsic coagulation pathways. J Biol Chem 1991; 266 (13) 8079-85.
3 Chattopadhyay A, James HL, Fair DS. Molecular recognition sites on factor Xa which participate in the prothrombinase complex. J Biol Chem 1992; 267 (17) 12323-9.
4 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.
5 Fung MR, Hay CW, MacGillivray RTA. Characterization of an almost fulllength cDNA coding for human blood coagulation factor X. Proc Natl Acad Sci USA 1985; 82: 3591-5.
6 De Grouchy J, Dautzemberg MD, Turleau C, Beguin S, Chavin-Colin F. Regional mapping of clotting factors VII and X to 13q34. Expression of factor VII through chromosome 8. Hum Genet 1984; 66: 230-4.
7 Watzke HH, High KA. Factor X. In: High KA, Roberts HR. (eds). Molecular Basis of Thrombosis and Haemostasis. New York: Marcel Dekker Inc; 1995: 239-55.
8 Watzke HH. Molekularbiologische Grundlagen des Faktor-X-Mangels. Hämostaseologie 1994; 14: 190-4.
9 Watzke HH, Lechner K, Roberts HR, Reddy SV, Welsch D, Friedman P, Mähr G, Jagadeeswaran P, Monroe DM, High KA. Molecular defect (Gla 14 to Lys) and its functional consequences in a hereditary factor X deficiency (Factor X “Vorarlberg”). J Biol Chem 1990; 265 (20) 11982-9.
10 Marchetti G, Castaman G, Pinotti M, Lunghi B, Di Iasio MG, Ruggieri M, Rodeghiero F, Bernardi F. Molecular basis of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glu102Lys) in the second EGF-like domain. Br J Haematol 1995; 90: 910-5.
11 Watzke HH, Wallmark A, Hamaguchi N, Giardiana P, Stafford DW, High KA. Factor X Santo Domingo: evidence that the severe clinical phenotype arises from a mutation blocking secretion. J Clin Invest 1991; 88: 1685-9.
12 Rudolph AE, Mullane MP, Porche-Sorbet R, Miletich JP. Expression, purification, and characterization of recombinant human factor X. Prot Expression Purif 1997; 10: 373-8.
13 Higuchi R, Recombinant PCR. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. (eds). PCR protocols. San Diego, CA: Academic Press; 1990: 128-39.
14 Nöbauer-Huhmann IM, Höller 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 FX deficiency (Gla+25 to Lys). Blood Coagulation and Fibrinolysis 1997; 09: 143-52.
15 Bode W, Brandstetter H, Mather T, Stubbe MT. Comparative analysis of haemostatic proteins: structural aspects of thrombin, factor Xa, factor IXa and protein C. Thromb Haemost 1997; 78: 501-11.
16 Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Huber R, Blankenship DT, Cardin Ad, Kisiel W. Structure of human de (1-45) factor Xa at 2.2 A resolution. J Mol Biol 1993; 232: 947-66.
17 Hertzberg MS, Ben-Tal O, Furie B, Furie BC. Construction, expression, and characterization of a chimera of factor IX and factor X. J Biol Chem 1992; 267: 14759-66.