Abstract:
: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)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Jhingan A,Zhang L,Christiansen WT,Castellino FJdoi
10.1021/bi00173a033subject
Has Abstractpub_date
1994-02-22 00:00:00pages
1869-75issue
7eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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