Ca2+ binding to the first epidermal growth factor-like domain of factor VIIa increases amidolytic activity and tissue factor affinity.
Coagulation factor VIIa belongs to a family of homologous enzymes, including factors IXa and Xa and activated protein C, composed of two epidermal growth factor-like domains located between an N-terminal domain rich in gamma-carboxyglutamic acid residues and a C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca2+ binding site, the function of which is largely unknown. Site-directed mutagenesis of two Ca2+-liganding Asp residues in this domain abolished Ca2+ binding and resulted in a 2-3-fold decrease in amidolytic activity at optimal Ca2+ concentrations. The lower amidolytic activity persisted in complex with soluble tissue factor, apparently due to a lower kcat of the mutant factor VIIa. Mutant and wild-type factor VIIa bound to lipidated tissue factor were equally efficient activators of factor X. The dissociation constants, derived from amidolytic activity and surface plasmon resonance measurements, were 2-5 nM and 50-60 nM for the interactions between wild-type and mutant factor VIIa, respectively, and soluble tissue factor. Binding to lipidated tissue factor was characterized by dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional Ca2+ binding site in the first epidermal growth factor-like domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.
Persson
E
E
Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Niels Steensens Vej 1, DK-2820 Gentofte, Denmark. egpe@novo.dk
Olsen
O H
OH
Ostergaard
A
A
Nielsen
L S
LS
D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002846:Chromatography, Affinity; D004815:Epidermal Growth Factor / Q000378:metabolism*; D015942:Factor VIIa / Q000235:genetics / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013925:Thromboplastin / Q000378:metabolism*
D001224:Aspartic Acid; D004815:Epidermal Growth Factor; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium