The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.
The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.
Ye
J
J
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Liu
L W
LW
Esmon
C T
CT
Johnson
A E
AE
D000595:Amino Acid Sequence; D000838:Anions; D001665:Binding Sites; D001667:Binding, Competitive; D019793:Fluorescein; D005452:Fluoresceins; D006629:Hirudins / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D018179:Receptors, Thrombin; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000838:Anions; D005452:Fluoresceins; D006629:Hirudins; D010446:Peptide Fragments; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C062510:hirugen; D013917:Thrombin; D019793:Fluorescein