Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis

We have previously determined that the C2-domain of human factor V (residue s 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the ami no terminus of the C2-domain (residues 2037-2087) and blocked PS binding. W e have now investigated the function of individual amino acids within the C 2-domain using charge to alanine mutagenesis. Charged residues located with in the C2-domain were changed to alanine in clusters of 1-3 mutations per c onstruct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS ce lls using a B-domain deleted factor V construct (rHFV des B). All mutants w ere expressed efficiently based on the polyclonal antibody ELISA. The charg ed residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of facto r V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174) ) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope fo r the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) t hrough Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(21 37). The PS-binding site in the factor V C2-domain includes amino acid resi dues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoc lonal antibody HV-1. These factor V C2-domain mutants should provide valuab le tools for further defining the molecular interactions responsible for fa ctor V binding to phospholipid membranes.