Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C

Abstract

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)

Identification of the endothelial cell binding site for factor IX.

“…Moreover, a mutation of lysine to arginine at residue 5, factor IX K5R, results in a factor IX molecule that has a 3-fold increased affinity for the endothelial cell binding site. Our observation that factor IX residues 3-11 are critical for the factor IX endothelial cell interaction have recently been confirmed by Castellino's laboratory (7). They demonstrated that converting the first 11 amino acids of protein C to those of factor IX resulted in a protein C chimera that bound to endothelial cells.…”

The Interaction between the Endothelial Cell Protein C Receptor and Protein C Is Dictated by the γ-Carboxyglutamic Acid Domain of Protein C

“…The major exception to this function of Gla domains is the binding of factor IXa to the factor IXa receptor on endothelial cells. This interaction was shown to be mediated by the Gla domain (21,22).…”

The entire γ‐carboxyglutamic acid‐ and helical stack‐domains of human coagulation factor IX are required for optimal binding to its endothelial cell receptor

“…While these experiments clearly identify the BAEC binding locus of fIX, not fully appreciated is the extent of participation of other regions of the Gla domain, or indeed of other domains of fIX, in contributing to the proper folding of this binding epitope (8). It was the aim of the current investigation to resolve this aspect of the problem.…”

“…Specifically, exchange of the Gla domain/HS regions of fIX with those of protein C (8) and fVII (9,11), two proteins that do not compete for fIX on BAEC, demonstrated that the chimeric proteins developed the ability to compete with fIX for BAEC binding, with IC50 values of the inhibitors being nearly equal to Kd for fIX binding to these cells. In addition, a synthetic polypeptide containing residues 1-47 of fI X interacted with BAEC with the same effectiveness as that of intact fIX (8). Interestingly, replacement of residues 1-13 of protein C with the equivalent 1-14 residues of fIX also resulted in a chimeric protein that was bound at equal strength as fIX to the binding site for this latter protein on BAEC (8), a result similar to that found for a fvII/fIX chimera (9).…”

“…250000 cpm/Ccg. BAEC binding assays.Third passage BAEC were employed for these studies, performed in microtiter plates as described in detail earlier (8). Briefly, a volume of 0.4 mL of a BAEC suspension (3 x lo4 cells/mL) was added to each well of two 48-well microtiter plates.…”