The contributions to functional phospholipid (PL) binding of the cluster of amino acid side chains of human protein C (PC) comprising F4, L5, and L8 have been assessed by construction of mutants of PC and activated protein C (APC) designed wherein a hydrophilic side chain replaced the wild-type hydrophobic groups at these positions. The PL-dependent plasma-based anticoagulant activities of [F4Q]-r-APC and [L8Q]r-APC were severely reduced to 5% and < 2%, respectively, of wild-type r-APC. Activity losses of the mutants toward inactivation of coagulation factor VIII, measured in the complete in vitro tenase system, have also been observed. As evidenced through Ca(2+)-induced intrinsic fluorescence changes, both [F4Q]r-PC and [L8Q]r-PC were able to adopt Ca(2+)-dependent conformations that appeared similar to that of wtr-PC, ruling out shortcomings associated with such Ca(2+)-induced transitions as the basis for their anticoagulant activity losses. However, despite this, [L8Q]r-PC showed greatly defective macroscopic binding properties to PL vesicles, as did to a lesser extent [F4Q]r-PC. These findings were similar to those reported previously for [L5Q]r-PC/APC [Zhang, L., & Castellino, F. J. (1994) J. Biol. Chem. 269, 3590-3595]. We thus propose that the PL-dependent activity losses of these mutants are related to their suboptimal binding to PL or to their misorientation on the PL surface leading to poor alignment of the active sites of the r-APC mutants with the complementary cleavage sites on fVIII/fVIIIa and fV/fVa.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

The cDNA encoding a chimeric human protein C (PC), in which its epidermal growth factor-(EGF) like regions have been replaced with equivalent structures from human factor IX (fIX), was constructed and the gene product was expressed in human 293 cells. A molecular subpopulation of the recombinant chimeric protein (r-[PC/delta EGF-1,2/delta fIXEGF-1,2]) was purified that contained the full complement (9 residues/mol) of gamma-carboxyglutamic acid (Gla). After conversion by thrombin to its activated form (r-[APC/delta EGF-1,2/delta fIXEGF-1,2]), this latter enzyme was found to possess approximately 10% of the activity of wild-type recombinant APC (wtr-APC) in an APTT assay. In assay systems employing purified components, the activity of the mutant enzyme toward prothrombinase cofactor Va (fVa) and tenase cofactor VIII (fVIII) was approximately 30% and < 10%, respectively, of that of wtr-APC. The chimeric protein displayed full reactivity with a Ca(2+)-dependent monoclonal antibody to the Gla domain of PC, yielding a C50 for Ca2+ that was very similar to that obtained with wtr-PC (ca. 3.7 mM). Titrations of the dependency on Ca2+ of the intrinsic fluorescence of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] allowed calculation of a C50 value of 0.34 mM, again very similar to that of wtr-PC. As with wtr-PC, Ca2+ inhibited the thrombin-catalyzed activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] with aKi of 148 microM, as compared to a Ki of 125 microM for wtr-PC. At a saturating level of Ca2+, activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2/] by the thrombin/thrombomodulin (thrombin/TM) complex occurred at approximately 70% of the rate of that of wtr-PC.(ABSTRACT TRUNCATED AT 250 WORDS)

No 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)