Factor VIII (fVIII) is an important plasma component required for haemostasis, since genetic defects in this molecule cause a life-threatening coagulation disorder known as Hemophilia A. This genetic disease is treated by repeated infusions of expensive fVIII products. A more effective therapy can be provided if the molecular basis of fVIII clearance is understood and a novel recombinant fVIII protein with a prolonged lifetime in circulation is developed. We have previously found that the low density lipoprotein receptor-related protein (LRP), the main endocytic liver receptor, and cell surface heparan sulfate proteoglycans (HSPGs) cooperate in the clearance of fVIII, since simultaneous blocking of these two receptor systems dramatically prolonged the lifetime of fVIII in mice. While in purified system both LRP and HSPGs were shown to interact with fVIII via the sites located within the A2 domain, the precise molecular events responsible for fVIII catabolism are currently not well characterized. We propose to identify the amino acid residues critical for fVIII interaction with LRP and HSPGs by mutational analysis of the regions previously identified as LRP and HSPGs binding sites of fVIII. The mutations will be introduced into B-domain depleted recombinant fVIII, which is functionally identical to plasma-derived fVIII and is presently used for Hemophilia A therapy. We will express these fVIII mutants in mammalian cells and test them for binding to LRP and heparin, used as model of HSPGs, in purified systems. The catabolism of the mutants will be examined in vitro using LRP-expressing cells and in vivo in a murine model of Hemophilia A. These experiments will identify fVIII mutants with reduced binding to LRP and HSPGs and will clarify the role of these two receptor systems in fVIIII clearance. The proposed studies should develop an insight into the mechanism of fVIII regulation in circulation and will provide a basis for generation of a novel type of recombinant fVIII products, having a prolonged lifetime in circulation. Development of such fVIII derivatives, which may be prospective for less expensive Hemophilia A therapy, is the long-term goal of our studies.
