A novel gene mutation in the 60s loop of human coagulation factor VII - inhibition of interdomain crosstalk

Thromb Haemost. 2004 Jan;91(1):28-37. doi: 10.1160/TH03-05-0258.

Abstract

A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively. The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon reso-nance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177-206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC(50) value of 0.5 micro M in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent K(I) of 7 micro M.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / analogs & derivatives*
  • Acetylcysteine / chemistry
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Brain / metabolism
  • Brefeldin A / chemistry
  • CHO Cells
  • Circular Dichroism
  • Cricetinae
  • Cysteine Proteinase Inhibitors / pharmacology
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Precursors / chemistry
  • Exons
  • Factor VII / chemistry*
  • Factor VII / genetics*
  • Factor X / chemistry
  • Factor Xa / chemistry
  • Heterozygote
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Models, Molecular
  • Mutation*
  • Peptides / chemistry
  • Polymerase Chain Reaction
  • Polymorphism, Genetic
  • Polymorphism, Single-Stranded Conformational
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Synthesis Inhibitors / pharmacology
  • Recombinant Proteins / chemistry
  • Restriction Mapping
  • Sequence Analysis, DNA
  • Serine Endopeptidases / chemistry
  • Surface Plasmon Resonance
  • Time Factors
  • Transfection

Substances

  • Antibodies, Monoclonal
  • Cysteine Proteinase Inhibitors
  • Enzyme Precursors
  • Peptides
  • Protein Synthesis Inhibitors
  • Recombinant Proteins
  • lactacystin
  • Brefeldin A
  • Factor VII
  • Factor X
  • Serine Endopeptidases
  • Factor Xa
  • Acetylcysteine