Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C.
A series of recombinant (r) chimeric mutants of human coagulation protein C (PC) and activated protein C (APC) containing replacements of homologous PC domains by those of human coagulation factor IX (fIX) were generated, with the intention of determining whether the specific bovine aortic endothelial cell (BAEC) receptor-binding characteristics of fIX could be incorporated into the chimeric r-PC while maintaining the essential properties of PC and APC. Using a competitive BAEC displacement assay with [125I]fIX, we found that a chimeric r-PC (r[delta PC1-46/delta fIX1-47]PC), consisting of the entire gamma-carboxyglutamic domain ([GDIX], residues 1-38) and helical stack ([HSIX], residues 38-47) of fIX as replacements for these same domains of PC, provided an IC50 for fIX-related BAEC binding of 13 nM, as compared to 10 nM for that of unlabeled fIX. This showed that all of the BAEC tight binding determinants for fIX existed within the [GDIX/HSIX]. Additionally, this chimera reacted to the same extent as fIX with the Ca(2+)-dependent, [GDIX]-specific monoclonal antibody H5B7 and lost its reactivity to a similar antibody specific for the [GDPC], JTC1. A synthetic peptide containing residues 1-47 of fIX also competed effectively (IC50 = 16 nM) with intact fIX for BAEC binding. Displacement of [125I]fIX from BAEC did not occur with a chimera containing the [HSIX] alone or with another mutant protein possessing a replacement of the two epidermal growth factor (EGF) homology regions of r-PC (residues 47-137) with those same domains of fIX.(ABSTRACT TRUNCATED AT 250 WORDS)
Geng
J P
JP
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Christiansen
W T
WT
Plow
E F
EF
Castellino
F J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D005164:Factor IX / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D000939:Epitopes; D010446:Peptide Fragments; D011486:Protein C; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C052276:factor IX-IXa receptor; D005164:Factor IX