Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.
We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.
Waxman
E
E
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
Ross
J B
JB
Laue
T M
TM
Guha
A
A
Thiruvikraman
S V
SV
Lin
T C
TC
Konigsberg
W H
WH
Nemerson
Y
Y
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D001665:Binding Sites; D003619:Dansyl Compounds / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism*; D005454:Fluorescence Polarization; D008081:Liposomes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D008081:Liposomes; D010713:Phosphatidylcholines; C027587:phenylalanyl-phenylalanyl-arginine chloromethyl ketone; D013925:Thromboplastin; D015942:Factor VIIa