IDENTIFICATION OF A CALCIUM-BINDING SITE IN THE PROTEASE DOMAIN OF HUMAN BLOOD-COAGULATION FACTOR-VII - EVIDENCE FOR ITS ROLE IN FACTOR-VII TISSUE FACTOR INTERACTION

Previous studies have identified a putative calcium binding site involving two glutamic acid residues located in the protease domain of coagulation factor IX. Amino acid sequence homology considerations suggest that factor VII (FVII) possesses a similar site involving glutamic acid residues 210 and 220. In the present study, we have constructed site-specific mutants of human factor VII in which Glu-220 has been replaced with either a lysine (E220K FVII) or an alanine (E220A FVII). These mutants were indistinguishable from wild-type factor VII by SDS-PAGE but only possessed 0.1% the coagulant activity of factor VII. Incubation of E220K/E220A FVII with factor Xa resulted in a slower than normal activation rate which eventually yielded a two-chain factor VIIa molecule possessing a coagulant activity of almost-equal-to 10% that of wild-typer FVIIa. Amidolytic activity measurements indicated that E220K/E220A FVIIa, unlike wild-type factor VIIa, possessed no measurable amidolytic activity toward the chromogenic substrate S-2288, even at high CaCl2 concentrations. Addition of tissue factor apoprotein, however, induced the amidolytic activity of the mutant molecule to a level 30% of that observed for wild-type factor VIIa. This tissue factor dependent enhancement of E220K/E220A FVIIa amidolytic activity was calcium dependent and required a CaCl2 concentration in excess of 5 mM for maximal rate enhancement. This was in sharp contrast to wild-type factor VIIa which required CaCl2 levels of 0.5 mM for maximal enhancement of tissue factor dependent amidolytic activity. Competition binding experiments suggest that the decrease in amidolytic and coagulant activity observed in the factor VII mutants is a direct result of impaired tissue factor binding. Evidence for the participation of Glu-220 in calcium binding was obtained using the luminescent lanthanide Tb3+. The binding of Tb3+ to wild-type factor VIIa was described by a simple binding curve (K(d) = 9 muM). All binding monitored by this technique was independent of the factor VII-Gla domain and could be displaced by addition of Ca2+ (K(d) = 1.5 mM). Substitution of Glu-220 with a Lys (E220K FVII) resulted in a 25-fold decrease in the affinity of the FVII molecule for Tb3+. Our results are consistent with the existence of a low-affinity calcium binding site located in the protease domain of FVII and necessary for normal factor VII-tissue factor interaction.