PROPERTIES OF RECOMBINANT CHIMERIC HUMAN PROTEIN-C AND ACTIVATED PROTEIN-C CONTAINING THE GAMMA-CARBOXYGLUTAMIC ACID AND TRAILING HELICAL STACK DOMAINS OF PROTEIN-C REPLACED BY THOSE OF HUMAN COAGULATION-FACTOR-IX

The properties of a recombinant (r) chimeric human protein C (PC) containing replacement of its gamma-carboxyglutamic acid (Gla) and helical stack (HS) domains by those of human coagulation factor IX (fIX) have been examined. Titration with Ca2+ of the divalent cation-induced intrinsic fluorescence quenching of this chimera (r-GD(IX)/PC) allowed determination of the [Ca2+], of 1.8 mM, required to produce this alteration in 50% of the protein molecules. These values were 0.41 and 0.61 mM for wtr-PC and fIX, respectively. The chimera did not react with a Ca2+-dependent, Gla domain-directed conformational monoclonal antibody (MAb) to r-PC but did interact with a similar MAb (H5B7)to fIX. The [Ca2+] required to induced H5B7 binding to 50% of the r-GD(IX)/PC molecules was 6.6 mM, while this same value for fIX was a nearly identical 7.2 mM. The [Ca2+] needed for binding of 50% of r-GD(IX)/PC to acidic phospholipid (PL) vesicles was 0.58 mM, while that for wtr-PC and fIX were 1.2 and 0.55 mM, respectively. The [protein] required for 50% binding of r-GD(IX)/PC to PL at 20 mM Ca2+ was 0.29 mu M. These same values for r-PC and fIX were 0.38 and 1.8 mu M, respectively. The Ca2+-mediated inhibition of the thrombin-catalyzed activation of r-GD(IX)/PC was characterized by a K-i of 118 mu M, a value similar to that of 125 mu M obtained for this same inhibition of wtr-PC activation. The thrombin-catalyzed activation of both r-GD(IX)/PC and wtr-PC was stimulated by soluble r-thrombomodulin. Similar to the case of wtr-PC, Ca2+ initially enhanced and, at higher concentrations, inhibited the activation of r-GD(IX)/PC. The K-m and k(cat) values for this latter activation at optimal [Ca2+] (100 mu M) were 4.1 mu M and 2.5 s(-1), respectively. These same kinetic constants for activation of wtr-PC were 4.3 mu M and 2.9 s(-1) respectively. These results show that many of the features needed for functional integrity of the Ca2+-bound Gla/HS domains of PC are also present in those same modules of fIX, a finding that points to a generalized functional role for the Ca2+-induced conformation of the Structural unit consisting of the Gla and HS domains. The data also suggest that the Ca2+-bound form of the Gla/HS region is an independently folded unit in PC and perhaps in fIX. Finally, it also appears that the functional Ca2+ site(s) outside of the Gla domain of PC and APC is independent of the specific nature of the native Gla/HS unit of PC.