Binding of Factor VIII to Lipid Nanodiscs Increases its Clotting Function in a Mouse Model of Hemophilia A

BACKGROUND Hemophilia A is a congenital bleeding disorder caused by defective or deficient factor VIII (FVIII). The active form of FVIII is the co-factor for the serine protease factor IXa (FIXa) in the membrane-bound intrinsic tenase (FVIIIa-FIXa) complex. The assembly of the FVIIIa-FIXa complex on the activated platelet surface is critical for successful blood clotting. OBJECTIVES To characterize the role of lipid nanodiscs (ND) for on FVIII function in vivo and test the lipid ND as a delivery system for FVIII. To evaluate the potential of binding recombinant FVIII to ND as improved treatment for Hemophilia A. METHODS Recombinant porcine FVIII (rpFVIII) was expressed and characterized in solution, and when bound to ND. The rpFVIII, ND and rpFVIII-ND complexes were characterized via transmission electron microscopy. Functional studies were carried out using aPTT tests and time resolved tail snip studies of hemophilic mice. RESULTS Functional rpFVIII was successfully assembled on lipid ND. When injected in hemophilic mice, the rpFVIII-ND complexes showed a pronounced pro-coagulant effect, which was stronger than that of rpFVIII alone. While injection of the ND alone showed a pro-coagulant effect this effect was not additive, implying that the rpFVIII-ND complexes have a synergistic effect on the clotting process in hemophilic mice. CONCLUSIONS Binding of rpFVIII to ND prior to its injection in hemophilic mice significantly improves the therapeutic function of the protein. This represents a meaningful step towards a new approach to modulate blood coagulation at the membrane-bound FVIII level and the assembly of the intrinsic tenase complex.