THE ACTIVITIES OF RECOMBINANT GAMMA-CARBOXYGLUTAMIC-ACID-DEFICIENT MUTANTS OF ACTIVATED HUMAN PROTEIN-C TOWARD HUMAN COAGULATION-FACTOR VA AND FACTOR-VIII IN PURIFIED SYSTEMS AND IN PLASMA

The dependence of the activity of recombinant activated human protein C (r-APC) on each of its nine gamma-carboxyglutamic (Gla) residues (sequence positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) has been assessed in purified systems and in plasma using r-mutants in which each Gla residue of r-APC was individually altered to an Asp (D) residue. The assays employed included a factor Va inactivation assay in the prothrombinase system with purified components and in plasma. In addition, a factor VIII inactivation assay in the tenase system, also with purified components, was utilized. Compared to wild-type protein (wtr-APC), the r-mutants that possessed nearly full activity in all assays were the Gla(6) --> D variant ([Gla(6)D]r-APC]) as well as [Gla(14)D]r-APC and [Gla(19)D]r-APC. In addition, another mutant (Q(32) --> Gla) in which a Gla was substituted for Gln (Q) at position 32, a situation that exists with other vitamin-K-dependent clotting proteins (e.g., factor IX and prothrombin), displayed full activity in all assays. Those mutants that possessed very-low-to-no activity in all assays included [Gla(16)D]r-APC and [Gla(26)D]r-APC. The other mutants showed partial and, in some cases, differential activity in these assay systems, with [Gla(25)D]r-APC being the most remarkable example. In this case, the factor V/Va plasma assay and the plasma-based activated partial thromboplastin time assay yielded <25% activity, whereas nearly full activity was observed for this variant in the prothrombinase and tenase assays with purified components. Although not as pronounced as with [Gla(25)D]r-APC, similar differences existed for several other mutants in the plasma-based and purified system assays. This suggests that a factor(s) in plasma may diminish the activity of these undercarboxylated forms of r-APC. Finally, none of the r-APC mutants showed significant differential activity toward fVa and fVIII, demonstrating that determinants on APC for each of these substrates are likely the same.