A .gamma.-carboxyglutamic acid (.gamma.) variant (.gamma.6D, .gamma.7D) of human activated protein C displays greatly reduced activity as an anticoagulant

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Abstract
Site-specific mutagenesis has been employed to alter the cDNA of human protein C (PC), such that the .gamma.-carboxyglutamic acid (.gamma.) pair at positions 6 and 7 of the recombinant (r) protein would be changed to aspartic acid residues. This variant, [.gamma.6D, .gamma.7D]r-PC, and its wild-type (wt) counterpart have been expressed in human kidney 293 cells. After purification, forms of wtr-PC that were fully .gamma.-carboxylated and .beta.-hydroxylated and of [.gamma.6D, .gamma.7D]r-PC that lacked only the two altered .gamma.-residues at amino acid sequence positions 6 and 7 were obtained. Subsequent to its conversion to activated PC (APC), [.gamma.6D, .gamma.7D]r-APC displayed a greatly reduced activity in the activated partial thromboplastin time of PC-deficient plasma, as compared to wtr-APC and human plasma APC. In addition, the activity of [.gamma.6D, .gamma.7D]r-APC toward inactivation of purified human factor VIII was reduced to less than 5% of that of wtr-APC and human plasma APC. These results, with the first reported mutations at .gamma.-residues of PC produced by recombinant DNA technology, indicate that the paired .gamma.-residues at positions 6 and 7, which are highly conserved in all vitamin K dependent coagulation proteins, are very important to generation of fully functional APC. Additional results demonstrate further that lack of .gamma.-carboxylation at positions 6 and 7 of PC does not substantially affect this same processing reaction at other relevant glutamic acid residues.