Binding of FVIII to Recombinant VWF

Abstract 1408

Von Willebrand factor (VWF) forms a non-covalent complex with FVIII in the circulation, which not only stabilizes the heterodimeric structure of FVIII but also protects this coagulation protein from early proteolytic degradation and cellular uptake. By convention, the physiological ratio in plasma between VWF and FVIII is 1 IU to 1 IU, corresponding to a stoichiometry of approximately 1 FVIII molecule bound per 50 VWF monomers. However, since each VWF monomer contains one FVIII binding site, this ratio does not seem to reflect the genuine binding capacity of VWF for FVIII.

We therefore investigated the interaction between FVIII and a recombinant VWF (rVWF) drug candidate under different conditions. The FVIII binding capacity of VWF (VWF:FVIIIB) was measured by an ELISA system, where the VWF-bound FVIII was quantified by a FVIII chromogenic assay. Under this condition, VWF:FVIIIB of rVWF was similar to that of VWF derived from human normal plasma. The binding affinity of VWF for FVIII was determined using a Biacore system in which VWF is immobilized onto the sensor chip surface. The FVIII binding affinities were similar for rVWF KD=0.50) and pdVWF (KD=0.49 nM) and agreed with data from the literature.

Plasma-derived VWF was reported to show increased FVIII binding when in solution (instead of being immobilized to a surface). To find out whether this also holds true for rVWF, we developed a new assay system based on Biacore technology which measures the binding of FVIII to VWF in solution without any surface binding step of the VWF-FVIII complex. A mixture of rVWF and rFVIII is thereby injected into the flow cell of a Biacore sensor chip with immobilized rVWF. In this indirect assay system, the rFVIII that forms a complex with rVWF in solution is not available for binding to the immobilized rVWF. Under these conditions, rVWF was endowed with a 30-times higher FVIII binding capacity than could be anticipated from the physiological ratio and similar results were obtained for pdVWF.

The combined results showed that the ability to serve as the physiological chaperone protein for FVIII is fully preserved in rVWF, which can therefore be expected to stabilize FVIII in the circulation in a similar manner to VWF in plasma. Results from an ongoing phase 1 study show the good FVIII stabilizing properties of rVWF also when administered in humans with type 3 VWD.