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

The minimal region of the gamma-carboxyglutamic acid (Gla) domain of human factor (f) IX that interacted with its putative bovine aortic endothelial cell (BAEC) receptor was examined by chemical synthesis of peptides with sequence counterparts in this region of the protein, and assessment of their relative abilities to compete with fIX for receptor binding. We found that IC50 values (total peptide concentrations needed to achieve 50% inhibition of binding of [125I]-fIX to BAEC) were ca. 18 nM for unlabeled fIX and 23 nM for the peptide consisting of the entire Gla domain/helical stack (HS) region (residues 1-47) of fIX. The peptide containing only the Gla domain of fIX (residues 1-38) displayed an IC50 value of > 500 nM for this same competitive binding, whereas peptides containing sequences present in positions 1-14 and 1-24 of the Gla domain of human fIX did not significantly compete with [125I]-fIX for BAEC binding. We conclude that whereas a specific receptor recognition element is present within residues 1-14 of fIX, as has previously been concluded by others and by us, full expression of this epitope requires its presence within the entire Gla domain and HS for proper folding. All determinants for proper folding of fIX that lead to BAEC receptor binding appear to be present within these two domains.