Charge-to-alanine mutations of three amino acid residues, viz, D46, D48, and D/Hya71, which are known to be important in stabilizing Ca2+ binding to epidermal growth factor (EGF) domains of vitamin K-dependent blood coagulation proteins, have been engineered into recombinant human protein C (r-PC). The resulting variants were then employed to assess the importance of this Ca2+ binding site in the activation properties of r-PC and in the activity of activated protein C (APC). Another mutation, of D48 to E, was constructed in order that a more conservative mutation at the Ca2+ binding site could be similarly examined. The mutant proteins were fully processed with regard to proper signal peptide cleavage, γ-carboxylation, and β-hydroxylation, except, of course, for the D71A mutant in this latter case. The D48E variant possessed an additional residue of γ-carboxyglutamic acid (Gla), showing that E48 was γ-carboxylated. All of the mutants were reactive against a monoclonal antibody (MAb) specific for a Ca2+-dependent epitope within the amino-terminus of the Gla domain of r-PC, demonstrating that a proper Ca2+-dependent conformation was adopted in this region of the protein. None of the mutants, except for [D48γ]r-PC, were reactive against another Ca2+-dependent MAb which possessed specificity for Ca2+ binding to the EGF1 region of PC-this being the area of the protein that contained the mutated residues. These data strongly suggest that the alanine mutations present at D46, D48, and D71 diminished Ca2+ binding to the EGF1 domain of r-PC. Steady state kinetic analysis demonstrated that determinants for the Ca2+-dependent inhibition of the thrombin (fIIa)-catalyzed activation of r-PC, and for the kinetic recognition of the fIIa/thrombomodulin complex, were not dependent on the integrity of the Ca2+ sites present in EGF1. The lone exception was [D48γ]r-PC, which did not undergo inhibition by Ca2+, an effect likely due to the potential for altered coordination of Ca2+ due to the Gla insertion, rather than to a dependency on D48. Plasma-based anticoagulant assays, as well as individual factor Va and factor VIIIa inactivation assays, showed that only [D71A]r-APC possessed a significantly reduced activity compared to wild-type r-APC. These observations suggest that D/Hya71 is likely an important determinant for activity of APC toward its physiological substrates, factor Va and factor VIIIa.
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