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 hete rodimers of heavy and light chains associated by a metallic ion still present in the functional activated factor Vm trimer of molecular mass 50000-45000-70000 Da. The chelation of the metal leads to the dissoci ation of these complexes with a concomitant loss of the procoagulant a ctivity. Until now, this ion has not been directly identified and its role in the structure/function relationships remains unclear. We repor t the first determination of the nature of this metal using atomic-abs orption spectroscopy with Zeeman effect. A comparative identification was also performed with the new recombinant factor VIII, FVIII-Delta I I. In the different active pd-FVIII heterodimers (of molecular mass ra nging over 210000-80000-90000-80000 Da) and in FVIII-Delta II, copper was detected. This result is consistent with sequence similarities des cribed between FVIII and copper-binding proteins. The quantification o f the copper content in FVIII-Delta II and in the corresponding pd-FVI II dimer of 90000-80000 Da indicated, for both proteins, the presence of one copper ion/mol FVIII. Copper was also identified in the activat ed FVIII complex and remained in the dimer of 50000-70000 Da generated during FVIII inactivation. Further dissociation into isolated fragmen ts of molecular masses 70000 Da and 50000 Da was concomitant with the loss of the copper ion. No copper was detected in the isolated fragmen t of molecular mass 45000 Da. These results suggest that the presence of the cation is not directly related to FVIII activity but is an esse ntial structural prerequisite for FVIII heavy-light-chain association.