Functional Consequences of the Ser334→Pro Mutation in a Human Factor X Variant (Factor XMarseille)

Authors


A. Bezeaud, Faculté Xavier Bichat, Laboratoire de Recherche sur l'Hémostase et la Thrombose, 16, rue Henri Huchard, level. B. P. 416, F-75870 Pans Cédex 18, France
Fax:+33 1 40 87 56 83.

Abstract

A factor X molecular variant was identified in a 55-year-old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV-X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV-X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S-2765 (D-Arg-Gly-Arg-NH-Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV-X was the consequence of a threefold decrease in the Kcat for the activation of factor XMarseille while the Km of RVV-X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single-strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T→C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three-dimensional models of various serine proteases, it appears that Ser334 is located within a surface-exposed variable region of factor X. This observation suggests that the Ser334→Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser→Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.

Abbreviations
CBS-31.39

d-Leu-Gly-Arg-NH-Np

S-2238

d-Phe-Pip-Arg-NH-Np

S-2765

d-Arg-Gly-Arg-NH-Np

PEG 8000

poly(ethylene glycol) 8000

SSCP

single-strand coformational polymorphism

RVV

Russell viper venom

NH-Np

4-nitroanilide

Pip

pipecolyl

Enzymes
 

Factor Xa (EC 3.4.21.6)

 

thrombin (EC 3.4.21.5)

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