Copper-atom identification in the active and inactive forms of plasma-derived FVIII and recombinant FVIII-delta II.

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.

Metal identification in human anti-hemophilia A factor (factor VIII).

Anti-hemophilia A factor (FVIII) consists in different heterodimers of heavy and light chains associated by a metallic ion. The integrity of this complex is indispensable for procoagulant activity. Atomic absorption spectrometry with Zeeman effect has been applied to determine the nature of this metal. For this purpose, the different active forms of FVIII were separated by FPLC and characterized by SDS-PAGE. Two peaks were observed, the first corresponding to different FVIII complexes of high molecular mass (ranging from 210-80 kDa to 110-80 kDa) and the second to the heavy-light chain dimer of 90-80 kDa. In all these active fractions, copper atom was identified and a proportionality was measured between the metal concentration and the coagulant activity. Furthermore, the determination of copper and FVIII concentrations indicated that only one copper atom is implicated in the 90-80 kDa association.