Two naturally occurring mutations on FVII gene (S3631-W364C) altering intrinsic catalytic activity

Flora Peyvandi, Raimondo De Cristofaro, Sepideh Akhavan, Josephine A. Carew, Raffaele Landolfi, Kenneth A. Bauer, Pier Mannuccio Mannucci

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Factor VII (FVII) requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active FVIIa conformation. This event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex. We have characterized two naturally occurring mutations (S363I - W364C) on FVII gene. Both homozygous patients for each mutation have a normal FVII: Ag level associated to an undetectable FVII coagulant activity. The patient carrying the allele 364C had a more severe hemorrhagic diathesis than the S363I mutant. To understand the mechanism of these deficiency, in vitro expression analysis with further biochemical characterization of recombinant proteins of both mutants FVII-363I, FVII-364C and wild type (WTFVII) FVII constructs were done. The results recapitulated the patients' plasma data with normal Ag level and no detectable coagulant activity. The D-F-Pip-R-pNA and CH3SO2-D-CHA-A-But-R chromogenic substrates were used to evaluate the amidolytic activity of WT and mutant FVII in presence and absence of recombinant tissue factor (rTF). Binding of FVII to rTF by a solid phase binding assay was done using recombinant human rTF. The results of amidolytic assays showed that rTF enhances 28 fold the value of the specificity of constant (kcat/Km) in WT but no activity was detectable in either mutant constructs under any condition. The equilibrium dissociation constant of rTF-FVIIa interaction showed Kd equal to 4.4 ± 0.2nM, 4.9 ± 0.5nM and 6 ± 0.9 of WT, 363I and 364C FVII forms, respectively. The Kd values of the non activated forms were equal to 24.7 ± 3.3, 24.4 ± 3.1 and 20.6 ± 4nM, respectively. These data demonstrate that, compared to the WT form, FVII-363I and FVII-364C have no significant affinity change for TF and that the detrimental effect of these two mutations is attributable to the loss of an efficient catalytic machinery in the FVII molecule causing a severe deficiency of coagulant activities.

Original languageEnglish
Pages (from-to)750-755
Number of pages6
JournalThrombosis and Haemostasis
Volume88
Issue number5
Publication statusPublished - Nov 1 2002

Fingerprint

Factor VII
Thromboplastin
Mutation
Genes
Coagulants
Hemorrhagic Disorders
Chromogenic Compounds
Enzyme Precursors
Recombinant Proteins
Alleles

Keywords

  • Extrinsic coagulation
  • Factor VII
  • Tissue factor

ASJC Scopus subject areas

  • Hematology

Cite this

Two naturally occurring mutations on FVII gene (S3631-W364C) altering intrinsic catalytic activity. / Peyvandi, Flora; De Cristofaro, Raimondo; Akhavan, Sepideh; Carew, Josephine A.; Landolfi, Raffaele; Bauer, Kenneth A.; Mannucci, Pier Mannuccio.

In: Thrombosis and Haemostasis, Vol. 88, No. 5, 01.11.2002, p. 750-755.

Research output: Contribution to journalArticle

Peyvandi, F, De Cristofaro, R, Akhavan, S, Carew, JA, Landolfi, R, Bauer, KA & Mannucci, PM 2002, 'Two naturally occurring mutations on FVII gene (S3631-W364C) altering intrinsic catalytic activity', Thrombosis and Haemostasis, vol. 88, no. 5, pp. 750-755.
Peyvandi F, De Cristofaro R, Akhavan S, Carew JA, Landolfi R, Bauer KA et al. Two naturally occurring mutations on FVII gene (S3631-W364C) altering intrinsic catalytic activity. Thrombosis and Haemostasis. 2002 Nov 1;88(5):750-755.
Peyvandi, Flora ; De Cristofaro, Raimondo ; Akhavan, Sepideh ; Carew, Josephine A. ; Landolfi, Raffaele ; Bauer, Kenneth A. ; Mannucci, Pier Mannuccio. / Two naturally occurring mutations on FVII gene (S3631-W364C) altering intrinsic catalytic activity. In: Thrombosis and Haemostasis. 2002 ; Vol. 88, No. 5. pp. 750-755.
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AU - De Cristofaro, Raimondo

AU - Akhavan, Sepideh

AU - Carew, Josephine A.

AU - Landolfi, Raffaele

AU - Bauer, Kenneth A.

AU - Mannucci, Pier Mannuccio

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N2 - Factor VII (FVII) requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active FVIIa conformation. This event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex. We have characterized two naturally occurring mutations (S363I - W364C) on FVII gene. Both homozygous patients for each mutation have a normal FVII: Ag level associated to an undetectable FVII coagulant activity. The patient carrying the allele 364C had a more severe hemorrhagic diathesis than the S363I mutant. To understand the mechanism of these deficiency, in vitro expression analysis with further biochemical characterization of recombinant proteins of both mutants FVII-363I, FVII-364C and wild type (WTFVII) FVII constructs were done. The results recapitulated the patients' plasma data with normal Ag level and no detectable coagulant activity. The D-F-Pip-R-pNA and CH3SO2-D-CHA-A-But-R chromogenic substrates were used to evaluate the amidolytic activity of WT and mutant FVII in presence and absence of recombinant tissue factor (rTF). Binding of FVII to rTF by a solid phase binding assay was done using recombinant human rTF. The results of amidolytic assays showed that rTF enhances 28 fold the value of the specificity of constant (kcat/Km) in WT but no activity was detectable in either mutant constructs under any condition. The equilibrium dissociation constant of rTF-FVIIa interaction showed Kd equal to 4.4 ± 0.2nM, 4.9 ± 0.5nM and 6 ± 0.9 of WT, 363I and 364C FVII forms, respectively. The Kd values of the non activated forms were equal to 24.7 ± 3.3, 24.4 ± 3.1 and 20.6 ± 4nM, respectively. These data demonstrate that, compared to the WT form, FVII-363I and FVII-364C have no significant affinity change for TF and that the detrimental effect of these two mutations is attributable to the loss of an efficient catalytic machinery in the FVII molecule causing a severe deficiency of coagulant activities.

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