von Willebrand factor protects the Ca2+-dependent structure of the factor VIII light chain.
We have recently reported that cation-exchange iminodiacetate resin completely inactivated factor VIII (FVIII) by direct deprivation of metal ions, predominantly Ca2+, from its molecules, and that von Willebrand factor (VWF) protected FVIII antigen from resin-induced degradation. The present study was further developed to investigate this mechanism. Western blotting analysis and enzyme-linked immunosorbent assay showed that the antigenic structure of the FVIII light chain, especially the C2 domain, was completely impaired by the resin, whilst that of the heavy chain was little affected. However, the complex formation with VWF protected the C2 domain from the resin-induced degradation. The resin-treated C2 domain failed to interact with VWF and phospholipid. Furthermore, the addition of Ca2+ competitively blocked the resin-induced impairment of the C2 domain structure. These results demonstrate that VWF protects the Ca2+-dependent conformational structure of the FVIII light chain, especially the C2 domain, and may indicate that the C2 domain contains the Ca2+-binding site(s).
Takeyama
Masahiro
M
Department of Paediatrics, Nara Medical University, Shijo-cho, Kashihara, Nara, Japan.
Nogami
Keiji
K
Okuda
Masahiro
M
Shima
Midori
M
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000737:chemistry*; D002411:Cation Exchange Resins / Q000494:pharmacology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D007098:Imino Acids / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D014841:von Willebrand Factor / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D002411:Cation Exchange Resins; D007098:Imino Acids; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005169:Factor VIII; D002118:Calcium; C008109:iminodiacetic acid