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 Gla6-->D variant ([Gla6D]r-APC]) as well as [Gla14D]r-APC and [Gla19D]r-APC. In addition, another mutant (Q32-->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 [Gla16D]r-APC and [Gla26D]r-APC. The other mutants showed partial and, in some cases, differential activity in these assay systems, with [Gla25D]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.(ABSTRACT TRUNCATED AT 250 WORDS)
Jhingan
A
A
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Zhang
L
L
Christiansen
W T
WT
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D005169:Factor VIII / Q000378:metabolism*; D015943:Factor Va / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000737:chemistry / Q000378:metabolism*; D011517:Prothrombin Time; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013925:Thromboplastin
D011486:Protein C; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D015943:Factor Va; D005169:Factor VIII; D013925:Thromboplastin; D002118:Calcium