The chromogenic assay used to monitor the potency of factor VIII does not consistently give the same result as a plasma substrate-based test. The standard measurement of factor VIII at the United States FDA is based on the plasma assay, while the chromogenic test is mandated by the European Pharmacopoeia. Discrepancies between these two tests affect product fill and patient care. To understand and resolve the disparities between the tests, we are making a comprehensive evaluation of molecular parameters that influence the assays. This includes examining the interaction of factor VIII products with von Willebrand factor, factors IXa and X, and phospholipid. Initial strategy: 1. Prepare recombinant vWF protein. We have successfully subcloned the portion of the vWF gene that encodes the FVIII-binding domain in a prokaryotic expression system. Additional mutant proteins will also be generated. Purified proteins will be studied by standard immunological and biochemical techniques. This study examines the address segment and/or precise contact residues on the domain and how the contact residues play a role in both binding and releasing FVIII from vWF. 2. Generate vWF peptides to determine the kinetics of interaction. We will assess whether specifically designed vWF peptides affect different phases of interaction between vWF and rFVIII. BIAcore will be used to further investigate the kinetics of interaction of rFVIII with vWF and vWF peptides, factors IXa and X, and phospholipids. Preliminary findings : 1. Molecular interaction of rFVIII with vWF Surface plasmon resonance (SPR), measured with a BIAcore instrument, was employed in this study. Purified vWF was immobilized on CM5 sensor chip by amine coupling. Various concentrations of rFVIII were passed over the sensor chip to examine the kinetic of binding. rFVIII bound strongly to immobilized vWF and the binding was specific. The interaction of rFVIII with vWF had both fast on- and off-rates. The association rate constant (ka) was about 5-6e6 (M-1S-1). The dissociation rate constant (kd) was from 1.25 to 2.5e-3 (s-1). The dissociation constant (Kd) was approximately 2.5-4.17 e-10 M, in agreement with other studies. The binding of rFVIII to vWF was inhibited in a concentration-dependent manner by vWF native protein and vWF peptides. 2. Bacterial expression of human vWF proteins. To determine the feasibility of expressing human vWF protein (FVIII-binding domain) in a bacterial expression system, the gene encoding the N-terminal portion of vWF (amino acid residues 1-272 and its variants) was amplified by PCR and subcloned into a bacterial expression system. We are currently investigating whether these proteins can be expressed, and, if so, whether they are functionally active.
