Role of the N-terminal epidermal growth factor-like domain of factor X/Xa.
The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with approximately 3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with approximately 12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a one-stage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.
Kittur
Farooqahmed S
FS
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Manithody
Chandrashekhara
C
Rezaie
Alireza R
AR
D000682:Amyloid / Q000737:chemistry; D002384:Catalysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005091:Exons; D015943:Factor Va / Q000737:chemistry; D005170:Factor X / Q000737:chemistry*; D015951:Factor Xa / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010314:Partial Thromboplastin Time; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D013925:Thromboplastin / Q000737:chemistry
D000682:Amyloid; D018076:DNA, Complementary; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D015943:Factor Va; D011516:Prothrombin; D005170:Factor X; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa