Identification of amino acids in the factor XI apple 3 domain required for activation of factor IX.
Activated coagulation factor XI (factor XIa) proteolytically cleaves its substrate, factor IX, in an interaction requiring the factor XI A3 domain (Sun, Y., and Gailani, D. (1996) J. Biol. Chem. 271, 29023-29028). To identify key amino acids involved in factor IX activation, recombinant factor XIa proteins containing alanine substitutions for wild-type sequence were expressed 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 homologous to factor XI, but activated factor IX poorly. A chimeric factor XIa molecule with the A3 domain replaced with A3 from prekallikrein (FXI/PKA3) activated factor IX with a K(m) 35-fold greater than that of wild-type factor XI. FXI/PKA3 was used as a template for a series of proteins in which prekallikrein A3 sequence was replaced with factor XI sequence to restore factor IX activation. Clotting and kinetics studies using these chimeras confirmed the results obtained with alanine mutants. Amino acids between Ile(183) and Val(191) are necessary for proper factor IX activation, but additional sequence between Ser(195) and Ile(197) or between Phe(260) and Ser(265) is required for complete restoration of activation.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T2 T30 T31 T32 T33 T34 T3 T4 T5 T6 T7 T8 T9
GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-N GENE-Y CHEMICAL GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y CHEMICAL GENE-Y GENE-N GENE-Y GENE-N GENE-Y CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-Y
factor IX factor IX factor XI A3 domain factor IX factor XIa factor XIa A3 domain factor XI plasma protease prekallikrein alanine factor XI factor IX factor XIa A3 domain prekallikrein FXI PKA3 activated factor IX factor IX factor XI Amino acids FXI PKA3 prekallikrein factor XI apple 3 domain factor IX amino acids alanine N C amino acids Activated coagulation factor XI factor XI