Construction, Expression, and Properties of a Recombinant Chimeric Human Protein C with Replacement of Its Growth Factor-like Domains by Those of Human Coagulation Factor IX

Abstract

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)

The Functions of the First Epidermal Growth Factor Homology Region of Human Protein C as Revealed by a Charge-to-Alanine Scanning Mutagenesis Investigation

“…Two mAbs, viz., C3, which was known to possess a Ca 2 *-independent epitope in the EGF1 domain of PC (Heeb ef a/., 1988;Yu et a/., 1994), and RIT, which was found in this study to also react with sites within this same module, were found to be refractive to the variant r-[R 91 A/E 92 A]PC. Not only did this investigation enable us to characterize further the integrity of the mutants, but it also allowed a more precise definition of the epitopes of this important group of mAbs.…”

“…The intrinsic fluorescence of PC and similar proteins of this class is known to undergo large alterations upon binding of Ca 2+ , which are due in part to conformational rearrangements of the Gla domains of these proteins. Since it has been demonstrated previously that this mAb was reactive against the EGF1 domain of PC (Yu et a/., 1994), we are now able to further define this epitope from the above data as primarily residing at the COOH-terminus of this domain with at least some involved amino acid residues located in the last disulfide loop and extending into the polypeptide linker between EGF1 and EGF2. Examples of the titration data are illustrated in Figure 2 for wt r-PC and r-[D 71 A]PC.…”

“…Steady state kinetic analysis of the inhibition of r-PC by Ca 2+ was accomplished using the two-stage assay described earlier (Yu et a/., 1994). First, a solution containing 1 μΜ wt r-PC was incubated with thrombin (20 NIH units), in the absence and presence of Ca 2+ (0-800 μΜ).…”

Identification of a Novel Family of Cell-surface Proteins Expressed in Human Vascular Endothelium

“…fVII, IX, X, and XII) (50,51) and anticoagulant proteins (e.g. thrombomodulin and protein C) (52,53). As shown in Figs.…”

The N-terminal Epidermal Growth Factor-like Domain in Factor IX and Factor X Represents an Important Recognition Motif for Binding to Tissue Factor

“…This mutant was activated by thrombomodulin-thrombin complex at ϳ70% of the rate obtained with the wild type protein C (52). The activated protein C mutant was also defective in inactivating FVa and FVIII/ FVIIIa in a PL-containing system (52). The decreased activity of activated protein C mutant was attributed to direct proteinprotein interaction and/or to misalignment of domains/recognition sites with its physiological substrates.…”