Thermodynamic and hydrodynamic behavior of the tissue factor:factor VII(a) complex.
Item
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Title
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Thermodynamic and hydrodynamic behavior of the tissue factor:factor VII(a) complex.
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Identifier
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AAI9405597
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identifier
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9405597
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Creator
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Waxman, Evan.
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Contributor
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Advisers: Yale Nemerson | J. B. Alexander Ross
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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The initial event in the extrinsic pathway of blood clotting is the formation of a complex between factor VII(a) and its cofactor, tissue factor (TF). This work concerns the thermodynamics and hydrodynamics of TF:VII(a) complex.;Concerning the thermodynamics of the complex we find that the isolated, extracellular domain of tissue factor (TF{dollar}\sb{lcub}1-218{rcub}{dollar}; sTF) exhibits 4% of the activity of wild-type TF (TF{dollar}\sb{lcub}1-263{rcub}{dollar}). Further, this activity is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70, w/w) are present. To determine whether this decrease results from weakened affinity, we studied their interaction using ultracentrifugation, fluorescence anisotropy, and activities. Ultracentrifugation of the sTF:VIIa complex established a 1:1 stoichiometry and an upper limit of 1 nM for the dissociation constant (K{dollar}\sb{lcub}\rm d{rcub}{dollar}). This value agrees with titrations of dansyl-D-Phe-L-Phe-Arg-chloromethyl ketone active-site-labelled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation was used to obtain quantitative values for binding. These experiments indicate that the K{dollar}\sb{lcub}\rm d{rcub}{dollar} for the interaction of sTF with DF-VIIa is 0.59 nM (25{dollar}\sp\circ{dollar}C). This value may be compared to a K{dollar}\sb{lcub}\rm d{rcub}{dollar} of 7.3 pM obtained for the interaction of DF-VIIa with TF{dollar}\sb{lcub}1-263{rcub}{dollar} in phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol{dollar}\sp{lcub}-1{rcub}{dollar} for the interaction of DF-VIIa with sTF and TF{dollar}\sb{lcub}1-263{rcub}{dollar}, respectively. These binding data show that sTF:VIIa is quantitatively and qualitatively different from TF{dollar}\sb{lcub}1-263{rcub}{dollar}:VIIa.;Ultracentrifugation and fluorescence anisotropy decay techniques were used to evaluate the hydrodynamics of VIIa and sTF:VIIa. Sedimentation velocity experiments showed that both VIIa and sTF:VIIa are highly asymmetric. In each case, the friction ratio, f/f{dollar}\sb{lcub}\rm sphere{rcub}{dollar}, is consistent with a family of ellipsoids ranging from prolate to oblate. Anisotropy decay experiments were then used to limit the family of ellipsoids which can describe the hydrodynamic behavior of VIIa and sTF:VIIa. For both VIIa and sTF:VIIa, the oblate ellipsoid of revolution was eliminated. The fluorescence anisotropy decay data show that upon binding sTF, VIIa loses a segmental motion involving a domain containing its active site.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
