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J. Biol. Chem., Vol. 275, Issue 22, 16428-16434, June 2, 2000
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From the Department of Pathology, Vanderbilt University School of
Medicine, Nashville, Tennessee 37232
Activation of prothrombin by factor Xa is
accompanied by expression of regulatory exosites I and II on the blood
coagulation proteinase, thrombin. Quantitative affinity chromatography
and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand,
hirudin54-65 ([5F]Hir54-65
(SO3
Characterization of Proexosite I on Prothrombin*
,
), were employed to
identify and characterize this site on human and bovine prothrombin and
its expression on thrombin.
[5F]Hir54-65(SO3) showed
distinctive fluorescence excitation spectral differences in complexes
with prothrombin and thrombin and bound to human prothrombin and
thrombin with dissociation constants of 3.2 ± 0.3 µM and 25 ± 2 nM, respectively,
demonstrating a 130-fold increase in affinity for the active
proteinase. The bovine proteins similarly showed a 150-fold higher
affinity of
[5F]Hir54-65(SO3)
for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled,
Tyr63-sulfated and nonsulfated hirudin peptides bound
competitively with
[5F]Hir54-65(SO3)
to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir54-65 bound with 7-17-fold lower affinity than the
sulfated analog. These studies characterize proexosite I for the first
time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low
affinity state and is activated with a ~100-fold increase in affinity
when thrombin is formed.
*
This work was supported by National Institutes of Health
Grant HL38779 and Research Career Development Award HL02832 (to
P. E. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported in part by National Institutes of Health (NIH)
Institutional Training Grant HL07751 and, subsequently, by American Heart Association Southeastern Consortium postdoctoral fellowship SE-9820133V.
§
To whom correspondence should be addressed: Dept. of Pathology,
Vanderbilt University School of Medicine, C3321A Medical Center North,
Nashville, TN 37232-2561. Tel.: 615-343-9863; Fax: 615-343-7023; E-mail: paul.bock@mcmail.vanderbilt.edu.
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