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 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)
Wildgoose
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Biopharmaceuticals Division, Novo Nordisk A/S, Gentofte, Denmark.
Foster
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Schiødt
J
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Wiberg
F C
FC
Birktoft
J J
JJ
Petersen
L C
LC
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D005167:Factor VII / Q000737:chemistry / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013725:Terbium; D013925:Thromboplastin / Q000378:metabolism*
D000577:Amides; D011994:Recombinant Proteins; D013725:Terbium; D005167:Factor VII; D013925:Thromboplastin; D010450:Endopeptidases; D015942:Factor VIIa; D002118:Calcium