Summary
A chimeric cDNA, encoding residues 1-46 (the γ-carboxyglutamic acid module and its
trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419
of human anticoagulant protein C (PC), was constructed and expressed. The resulting
protein, r-[∆GD-HSPC /∇GD-HSfVII ]PC, was properly processed with regard to signal/ propeptide release, cleavage of
the K156 R dipeptide, Gla and Hya contents, and the presence of glycosylation.
The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid
vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for
the Ca2+ -induced conformation of the Gla domain (GD) of PC, but did react with another MAb
directed in part to the Ca2+ -dependent conformation of the GD of fVII. Further, this chimeric protein possessed
similar steady state constants as wild-type r-PC toward activation by thrombin and
thrombin/thrombomodulin. The activated form of the chimera was very similar to that
of its wild- type counterpart in its whole plasma anticoagulant activity, as well
as its activity toward inactivation of coagulation factor VIII. The chimeric protein
did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region
of fVII is insufficient for that particular interaction.
The results demonstrate that the GD/HS of fVII, when present in the PC and APC background,
serves to maintain the Ca2+ /PL-related functions of these latter proteins, and suggest that the Ca2+ and PL- dependent interactions of the GD-HS of PC are sufficiently general in nature
such that the GD-HS regions of other proteins of this type can satisfy most of the
requirements of PC and APC. The data presented also offer support for the independent
nature of the domain unit consisting of the GD/HS module.
