Identification of amino acids in the factor XI apple 3 domain required foractivation of factor IX

Activated coagulation factor XI (factor XIa) proteolytically cleaves its su bstrate, factor IX, in an interaction requiring the factor XI A3 domain (Su n, Y., and Gailani, D, (1996) J, Biol, Chem, 271, 29023-29028). To identify key amino acids involved in factor IX activation, recombinant factor XIa p roteins containing alanine substitutions for wild-type sequence were expres sed in 293 fibroblasts and tested in a plasma clotting assay. Substitutions for Ile(183)-Val(191) and Ser(195)-Ile(197) at the N terminus and for Ser( 258)-Ser(264) at the C terminus of the A3 domain markedly decreased factor XI coagulant activity. The plasma protease prekallikrein is structurally ho mologous to factor XI, but activated factor IX poorly. A chimeric factor XI a molecule with the AS domain replaced with A3 from prekallikrein (FXI/PKA3 ) activated factor M with a K-m 35-fold greater than that of wild-type fact or XI. FXI/PKA3 was used as a template for a series of proteins in which pr ekallikrein A3 sequence was replaced with factor XI sequence to restore fac tor M activation. Clotting and kinetics studies using these chimeras confir med the results obtained with alanine mutants. Amino acids between Ile(183) and Val(191) are necessary for proper factor M activation, but additional sequence between Ser(195) and Ile(197) or between Phe(260) and Ser(265) is required for complete restoration of activation.