The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.
Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.
Zhong
Degang
D
Department of Internal Medicine and Department of Pharmacological and Physiological Sciences, Saint Louis University Health Sciences Center, St. Louis, Missouri 63110, USA.
Bajaj
Madhu S
MS
Schmidt
Amy E
AE
Bajaj
S Paul
SP
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005167:Factor VII / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D017931:DNA Primers; D010455:Peptides; D004815:Epidermal Growth Factor; D005167:Factor VII; D005164:Factor IX; D005170:Factor X; D013925:Thromboplastin; D012697:Serine Endopeptidases; D015942:Factor VIIa