Role of the N-terminal EGF module of coagulation factor IX in activation of factors IX and X.
Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder haemophilia B. FIX contains a Gla module, two epidermal growth factor-like (EGF) modules, and a serine protease region. I characterized a monoclonal antibody and found that it recognizes an epitope around residues 72 and 80 in the C-terminal part of EGF1 in human FIX. The antibody exhibited 10-fold greater affinity for activated FIX (FIXa) than for the zymogen FIX, indicating the existence of intra-molecular communication between the serine protease region and EGF1. Binding of the antibody did not affect the amidolytic activity of FIXa, hence I could use the antibody during activation of FIX to show that the C-terminal part of EGF1 is of importance for the interaction with FXIa but not with FVIIa/TF. Considering activation of FX, it is a matter of debate whether EGF1 or FIXa interacts directly with FVIIIa. I activated FX in the presence and absence of the antibody and/or FVIIIa. The addition of antibody caused only a minor decrease in k(cat,app), and the major increase in k(cat,app) caused by the addition of FVIIIa occurred even in the presence of the antibody. This implies that EGF1 of FIXa is not directly involved in interaction with FVIIIa in the Xase complex. A model of the FIXa-FVIIIa complex, based on my findings and results from the literature, was constructed and indicated that EGF1 of FIXa does not interact directly with FVIIIa.
Persson
K E M
KE
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden. Kristina.Persson@klkemi.mas.lu.se
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000918:Antibody Specificity; D000939:Epitopes / Q000276:immunology; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005164:Factor IX; D005170:Factor X; D015942:Factor VIIa; D015949:Factor IXa