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The effect of Arg 306 → Ala and Arg 506 → Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S
Author(s) -
Egan Jack O.,
Kalafatis Michael,
Mann Kenneth G.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060922
Subject(s) - recombinant dna , chemistry , microbiology and biotechnology , protein c , mutant , cofactor , factor v , cleavage (geology) , thrombin , biochemistry , thermolabile , prothrombinase , biology , enzyme , platelet , gene , medicine , paleontology , surgery , thrombosis , fracture (geology) , immunology
Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg 306 , Arg 506 , and Arg 679 . Site‐directed mutagenesis of human factor V cDNA was used to substitute Arg 306 → Ala (rfVa 306A ) and Arg 506 → Gln (rfVa 506Q ). Both the single and double mutants (rfVa 306A/506Q ) were constructed. The activation of these procofactors by α‐thrombin and their inactivation by APC were assessed in coagulation assays using factor V‐deficient plasma. All recombinant and wild‐type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVa PLASMA ) in HBS Ca 2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild‐type factor Va (rfVa WT ) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVa PLASMA . The rfVa 306A and rfVa 506Q mutants were each inactivated at rates slower than rfVa WT and fVa PLASMA . Following a 90‐min incubation with APC, rfVa 306A and rfva 506Q retain approximately 30‐40% of the initial cofactor activity. The double mutant, rfVa 306A/506Q , was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90‐min incubation in the presence of APC. Recombinant ma WT , rfVa 306A , rfVa 506Q , and rfVa 306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVa WT and rfVa 306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg 506 is not affected by protein S. The initial rate of rfVa 506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg30h. Over‐all, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg 306 and Arg 506 are required for efficient factor Va inactivation.