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 (flX), was constructed and the gene product was expressed in human 293 cells. A molecular subpopulation of the recombinant chimeric protein (r-[PC/DELTAEGF-1,2/del 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/DELTAEGF-1,2/del 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 Ca2+-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/DELTAEGF-1,2/del 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/DELTAEGF-1,2/del fIXEGF-1,2] with aK(i) of 148 muM, as compared to a K(i) of 125 muM for wtr-PC. At a saturating level of Ca2+, activation of r-[PC/DELTAEGF-1,2/del fIXEGF-1,2] by the thrombin/thrombomodulin (thrombin/TM) complex occurred at approximately 70% of the rate of that of wtr-PC. The results suggest that (1) despite the substitution of substantial domain regions of the light chain of PC with those of a functionally unrelated protein, the chimeric protein retains essential features of PC zymogen; (2) the ability of PC to adopt its Ca2+-dependent conformation is not specifically dependent on its EGF-like regions; (3) the high-affinity Ca2+ sites responsible for inhibition of the thrombin-catalyzed activation of PC, and stimulation of this same activation by thrombin/TM, are not specifically dependent on the EGF-like domains of PC; and (4) determinants present in the EGF-like domains of APC play a role in its anticoagulant properties, perhaps by directing specific alignments with its physiological substrates on the phospholipid surface and/or through general subtle conformational properties of the enzyme that are dependent on the integrity of the EGF-like regions of PC. Additionally, the differences in activity of the mutant APC toward fVa and fVIII may be due to effects resulting from a specific interaction between the fIX EGF regions of [PC/DELTAEGF-1,2/del fIXEGF-1,2] and fVIII, a natural cofactor for fIXa.
