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The contribution of amino acid residues 1508–1515 of factor V to light chain generation
Author(s) -
ERDOGAN E.,
BUKYS M. A.,
KALAFATIS M.
Publication year - 2008
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2007.02803.x
Subject(s) - recombinant dna , microbiology and biotechnology , chemistry , thrombin , mutant , amino acid , biochemistry , site directed mutagenesis , wild type , clotting factor , gel electrophoresis , immunoglobulin light chain , biology , antibody , gene , platelet , medicine , immunology
Summary. Background: Factor (F) V is activated by α‐thrombin following cleavages at Arg 709 , Arg 1018 and Arg 1545 . Amino acid region 1490–1520 of FV is essential for procofactor activation. Aim: To ascertain which amino acid residues from this region are important for light chain formation and procofactor activation, site‐directed mutagenesis was used to create recombinant FV molecules missing amino acid 1508–1510 (FV Δ1508−1510 ) and 1508–1515 (FV Δ1508−1515 ). We have also created recombinant FV molecules with mutations 1508 DDY 1510 →AAF (FV AAF ), 1514 DY 1515 →AF (FV AF ) and Y 1510 →F (FV Y1510F ). Methods and results: The recombinant mutant molecules were expressed and purified to homogeneity. The clotting activities of all clotting recombinant mutant FV molecules were tested in a two‐stage assay following activation by α‐thrombin and were found to be impaired compared with the clotting activity observed with wild‐type recombinant FV or plasma‐derived FV, with the exception of FV Y1510F , which had normal clotting activity. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) followed by immunoblotting with monoclonal antibodies to FV demonstrated that incubation of 100 n m recombinant wild‐type or plasma‐derived FV with 1 n m α‐thrombin for 5 min was sufficient to generate heavy and light chains and completely activate the procofactor. In contrast, similar experimental conditions were ineffective in fully activating the two deletion mutant molecules as well as FVa AAF and FVa AF , resulting in accumulation of a M r 220 000 fragment representing amino acids 1019–2195. Conclusion: Our data demonstrate that amino acid residues 1508–1515 of FV are required for efficient cleavage by α‐thrombin and light chain formation.