Blood coagulation factor VIII is an essential cofactor in the mammalian blood-clotting cascade. FVIII must bind the phospholipid membrane of activated platelets to function as a cofactor for FIXa. The blood coagulation cascade culminates in the formation of a stable blood clot. In humans, the C1 and C2 domains are implicated in binding phospholipid membranes, however the relative contribution of different residues in the lipid-binding mechanism is unclear. Using site-directed mutagenesis, expression of the isolated C1 and C2 domains in Escherichia coli cells, protein purification with metal affinity chromatography, electrospray ionization mass spectrometry, enzyme-linked immunosorbent assays, liposome sedimentation assays, pull down assays, circular dichroism and intrinsic tryptophan fluorescence, we compare relative binding affinities and stability of five mutations on the C1 and C2 domains. Three of the mutations are on the C2 domain: R2320S, R2320T, and R2215A. The other two mutations are on the C1 domain: R2163H and R2159H. Our results combined with the crystal structure of C1 and C2 soaked with O-phosphatidylserine moieties will help elucidate the roles of these residues in the function and stability of FVIII with regards to Hemophilia A
