The process of blood clotting involves a series of enzymatic reactions in the blood. The protein factor VIII (FVIII) is indispensable in this process in which it markedly enhances the activity of the enzyme factor IX (FIX).
The importance of FVIII is signified by the notion that absence of this protein in individuals is associated with the bleeding disorder hemophilia A.
Treatment of hemophilia A involves intravenous infusion with purified FVIII. An important issue to overcome with the current treatment strategies is the particularly short half-life of FVIII in the circulation. Therefore, hemophilia A patients have to intravenously infuse themselves up to three-times a week. Next to the high cost of treatment, this represents a major burden for the patients. One solution for this issue is a FVIII protein that only requires once weekly intravenous infusions. This project aimed to provide the basis for the development of this novel FVIII protein. FVIII circulates in blood in a tight complex with Von Willebrand Factor (VWF). This protein has a dual role in the biology of FVIII. It protects FVIII from premature clearance from the circulation. At the same time, FVIII is removed from the circulation via VWF. We therefore aimed to design a novel FVIII variant that does not bind VWF but is not internalized and degraded by cells that contribute to the removal of FVIII from the blood.

In the present project, we established that a particular protein domain in FVIII, the socalled C1 domain, comprises binding sites for VWF, FIX and cellular receptors that contribute to FVIII clearance. Replacing this domain with a similar domain from a related protein, Factor V, resulted into impaired binding to VWF and a markedly reduced uptake by cells. A disadvantage of this FVIII variant was that FIX binding was also reduced leaving a residual activity of 20% compared to normal FVIII. Using protein binding studies, site-directed mutagenesis combined with hydrogen/deuterium exchange mass spectrometry analysis, and analysis of the available structures of FVIII, we identified the amino acids in the FVIII C1 domain that contribute to FIX and VWF binding. These results provided novel insight into the biology of FVIII and we could now also explain why certain variants of FVIII and VWF are associated to bleeding disorders.

We next designed novel FVIII/FV-C1 domain variants in which we reintroduced the FIX binding sites. We were, however, unable to successfully produce these proteins by cells. As the contact sites of the FV C1 domain with other domains in FVIII could be impaired as well, we also prepared a FVIII variant in which these contact sites were repaired. This led to a two-fold increase in FVIII activity. Using mice models, we will now test whether or not the in vivo half-life is indeed increased. As cellular uptake of the FVIII/FV-C1 variant is completely impaired, we anticipate that this may indeed be the case. We expect that the FVIII/FV-C1 variant will represents a promising target for further development of a novel FVIII variant with an improved in vivo half-life.