Activation and Inactivation of rVIII-SingleChain
C. Horn, H. J. Metzner (Marburg, Germany)
Time: 11:00 - 12:00
Objective: Activation and inactivation of FVIII is an essential pathway in the regulation of coagulation. Thrombin activates FVIII leading to the formation of active FVIII (FVIIIa), and is also able to inactivate FVIIIa by further proteolytic degradation. Activated protein C (APC)/protein S also mediates the proteolytic degradation of FVIIIa. The dissociation of the A2 subunit from the FVIIIa trimer also results in inactivation. The present study assessed the activation and inactivation of rVIII-SingleChain, a novel recombinant FVIII product with covalently linked heavy and light chain.
Methods: rVIII-SingleChain and various rFVIII products were investigated. The formation of FVIII fragments was visualized by SDS-PAGE / CBB stain and/or Western Blot using anti FVIII antibodies recognizing the FVIIIa components (light chain, A1 and A2 domains). The activity was measured by a one-stage clotting FVIII activity assay using FVIII deficient plasma (Siemens Healthcare) and Pathromtin SL® (Siemens Healthcare) as activator reagent.
Results: The incubation of rVIII-SingleChain and rFVIII comparator products with thrombin resulted in proteolytic activation and formation of the expected FVIIIa band pattern. In particular, SDS-PAGE analysis demonstrated the cumulative appearance of the light chain and the A1 and A2 domain. Further incubation led to the formation of smaller degradation fragment(s). The incubation of FVIIIa with APC/protein S also resulted in the degradation of the A2 domain followed by the A1 domain into smaller fragments. The FVIII activity under these conditions decreased to values of below 1 % of the initial activities. Without APC/Protein S, A2 dissociation was comparable for rVIII-SingleChain and the rFVIII products.
Conclusion: The data of this study demonstrate functional comparability of rVIII-SingleChain and the FVIII comparator products analyzed with respect to the kinetics of FVIIIa inactivation by A2 dissociation and proteolytic degradation via APC/protein S or thrombin. A similar thrombin activation profile of rVIII-SingleChain and rFVIII products was shown. Slight differences in the activation intermediates are most likely due to the different molecular design of rVIII-SingleChain compared to two-chain FVIII molecules.