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 approximately 10% that of wild-type rFVIIa. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The Ca2+ ion binding of factor VIIa and the derivative lacking the γ‐carboxyglutamic acid domain, des(1–38) factor VIIa, was investigated using intrinsic protein fluorescence and Tb3+ ion phosphorescence methods. Binding of Ca2+ ions giving rise to a decrease in the intrinsic protein fluorescence (approximately 50% at saturating conditions) is seen with both proteins. Each of the saturation curves is in accordance with the formation of a 1:1 complex of factor VIIa‐Ca2+ (K D∼30 μM) and des(1–38) factor VIIa‐Ca2+ (K D∼40 μM)). Yet another Ca2+ ion binding site reveals itself in each protein in Tb3+ ion phosphorescence experiments. Ca2+ ion competition studies have showed 1:1 complexes (K D′s∼2 mM). The results are interpreted in terms of two different Ca2+ ion binding sites, one in the EGF‐1 domain and one in the Gly‐209‐Gin‐221 loop of the serine proteinase part.

Des(l-38) factor VII, and des(l--44) factor VII, were obtained by limited proteolysis. The binding of tissue factor to these factor VII,-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic oligopeptide substrates. Compared to native factor VII, (Kay = 0.6 + 0.1 riM), Tissue factor binds to des(l-38) factor VII, with a lower, but still signiticant affinity (Kw = 4.8 + 0.3 nM). The activity of des(I-44) factor VII, was only slightly stimulated by TF (KTF --200 nlVO. Binding of TF depends critically on the presence of Ca 2+ ions. Ca 2+ ions stimulated the activity of factor VIIJTF with an apparent K~ = 0.16 + 0.02 raM. Factor VII, in the absence of tissue factor was stimulated by Ca 2+ with an apparent K~ = 0.05 + 0.01 raM, and similar K~ values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca2+-induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca 2+ ion concentration at which this change occurred was higher for des(I-44) factor VII, (apparent Kc~ = 0.14 mM) than for des(I-38)-and native factor VII, (apparent K~ = 0.04 mM). The Tb 3+ ion luminescence technique was used to further investigate the Ca 2+ binding sites. Tb 3+ ions bound with a lower affinity to des(l-44) factor VII, than to des(1-38)-and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the 'hydrophobic stack' residues 39--44, suggest that this region of factor VII~ provides a structural determinant that together with other regions participates in tissue factor binding.

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The serine endopeptidase subtilisin 309 (Savinase® ) has a preference for substrates containing hydrophobic amino acids in the P4 position. Substitution of Leul35, situated in the hydrophobic S4 binding pocket, for the charged amino acid Glu alters the substrate specificity approximately 500-fold when comparing the ratios of kcat/Km for substrates with Phe and Arg as the P4 residue. This factor can be further increased to 1700-fold by additional replacement of Ilel07 with Val.