title: Copper-atom identification in the active and inactive forms of plasma-derived FVIII and recombinant FVIII-delta II.
abstract: The plasma-derived factor VIII (pd-FVIII) circulates as different heterodimers of heavy and light chains associated by a metallic ion still present in the functional activated factor VIII trimer of molecular mass 50,000-45,000-70,000 Da. The chelation of the metal leads to the dissociation of these complexes with a concomitant loss of the procoagulant activity. Until now, this ion has not been directly identified and its role in the structure/function relationships remains unclear. We report the first determination of the nature of this metal using atomic-absorption spectroscopy with Zeeman effect. A comparative identification was also performed with the new recombinant factor VIII, FVIII-delta II. In the different active pd-FVIII heterodimers (of molecular mass ranging over 210,000-80,000-90,000-80,000 Da) and in FVIII-delta II, copper was detected. This result is consistent with sequence similarities described between FVIII and copper-binding proteins. The quantification of the copper content in FVIII-delta II and in the corresponding pd-FVIII dimer of 90,000-80,000 Da indicated, for both proteins, the presence of one copper ion/mol FVIII. Copper was also identified in the activated FVIII complex and remained in the dimer of 50,000-70,000 Da generated during FVIII inactivation. Further dissociation into isolated fragments of molecular masses 70,000 Da and 50,000 Da was concomitant with the loss of the copper ion. No copper was detected in the isolated fragment of molecular mass 45,000 Da. These results suggest that the presence of the cation is not directly related to FVIII activity but is an essential structural prerequisite for FVIII heavy-light-chain association.
authors.affiliation: T. M. Innovation (Centre National de Transfusion Sanguine-Institut Merieux) Les Ulis, France.
authors.forename: N
authors.initials: N
authors.lastname: Bihoreau
authors.forename: S
authors.initials: S
authors.lastname: Pin
authors.forename: A M
authors.initials: AM
authors.lastname: de Kersabiec
authors.forename: F
authors.initials: F
authors.lastname: Vidot
authors.forename: M P
authors.initials: MP
authors.lastname: Fontaine-Aupart
mesh_terms: D000818:Animals; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000032:analysis*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D010949:Plasma; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013054:Spectrophotometry, Atomic
chemical_list: D011994:Recombinant Proteins; D003300:Copper; D005169:Factor VIII