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

The cDNA encoding a chimeric human protein C (PC), in which its epider mal growth factor-(EGF) like regions have been replaced with equivalen t 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 g amma-carboxyglutamic acid (Gla). After conversion by thrombin to its a ctivated form (r-[APC/DELTAEGF-1,2/del fIXEGF-1,2]), this latter enzym e 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 prothro mbinase cofactor Va (fVa) and tenase cofactor VIII (fVIII) was approxi mately 30% and < 10%, respectively, of that of wtr-APC. The chimeric p rotein displayed full reactivity with a Ca2+-dependent monoclonal anti body to the Gla domain of PC, yielding a C50 for Ca2+ that was very si milar to that obtained with wtr-PC (ca. 3.7 mM). Titrations of the dep endency on Ca2+ of the intrinsic fluorescence of r-[PC/DELTAEGF-1,2/de l fIXEGF-1,2] allowed calculation of a C50 value of 0.34 mM, again ver y similar to that of wtr-PC. As with wtr-PC, Ca2+ inhibited the thromb in-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 satur ating level of Ca2+, activation of r-[PC/DELTAEGF-1,2/del fIXEGF-1,2] by the thrombin/thrombomodulin (thrombin/TM) complex occurred at appro ximately 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 chime ric protein retains essential features of PC zymogen; (2) the ability of PC to adopt its Ca2+-dependent conformation is not specifically dep endent on its EGF-like regions; (3) the high-affinity Ca2+ sites respo nsible for inhibition of the thrombin-catalyzed activation of PC, and stimulation of this same activation by thrombin/TM, are not specifical ly dependent on the EGF-like domains of PC; and (4) determinants prese nt in the EGF-like domains of APC play a role in its anticoagulant pro perties, perhaps by directing specific alignments with its physiologic al substrates on the phospholipid surface and/or through general subtl e conformational properties of the enzyme that are dependent on the in tegrity of the EGF-like regions of PC. Additionally, the differences i n activity of the mutant APC toward fVa and fVIII may be due to effect s 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 fI Xa.