The interaction of human factor VIIa with tissue factor.
The interaction of factor VIIa with tissue factor (TF) results in an increase in the catalytic efficiency for the hydrolysis of several synthetic peptidyl p-nitroanilide substrates by factor VIIa. The binding of human recombinant factor VIIa to recombinant human TF incorporated into vesicles containing phosphatidylcholine (TF/PC) or phosphatidylcholine/phosphatidylserine (TF/PCPS) was studied using the increased rate of H-D-phenylalanyl L-pipecoyl L-arginine p-nitroanilide (S2238) hydrolysis as a signal for the interaction. The saturable dependence of rate on increasing concentrations of factor VIIa or TF/PCPS yielded no obvious evidence for cooperativity and could be analyzed according to the interaction of factor VIIa with independent noninteracting sites (Kd = 259 +/- 60 pM, n = 1.05 +/- 0.12 mol of factor VIIa/mol of TF at saturation). Identical titration curves and equilibrium parameters were derived from titrations using TF/PC or TF in the absence of phospholipids, indicating that possible protein-membrane interactions do not further stabilize the extrinsic Xase complex. The dissociation constant for the interaction of factor VIIa with TF/PCPS inferred from measurements of factor X activation (Kd = 197 +/- 38 pM) was comparable with the values obtained from measurements of S2238 hydrolysis. In contrast to the membrane-independent nature of the enzyme-cofactor interaction, the rate of factor X activation was reduced by approximately 50-fold when the enzyme complex was assembled using solution-phase TF. Collectively, the result indicate that the membrane dependence of extrinsic Xase function primarily results from an influence of the membrane surface on factor X utilization.
Krishnaswamy
S
S
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
D000595:Amino Acid Sequence; D015942:Factor VIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013925:Thromboplastin / Q000378:metabolism*
D008081:Liposomes; D009842:Oligopeptides; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa