Localization of thrombomodulin-binding site within human thrombin.
A binding site for thrombomodulin on human thrombin (alpha-thrombin) was elucidated by identifying an epitope for a monoclonal antibody for thrombin (MT-6) which inhibited the activation of protein C by the thrombin-thrombomodulin complex by directly inhibiting the binding of thrombin to thrombomodulin. An 8.5-kDa fragment isolated by digestion of thrombin with Staphylococcus aureus V8 protease followed by reversed-phase high performance liquid chromatography (HPLC) and a peptide isolated by reversed-phase HPLC after reduction of the 8.5-kDa fragment, which was composed of three peptides linked by disulfide-bonds, bound directly to MT-6 and thrombomodulin. The amino acid sequence of the peptide coincided with the sequence of residues Thr-147 to Asp-175 of the B-chain of thrombin. A synthetic peptide corresponding to Thr-147 to Ser-158 of the B-chain inhibited the binding of thrombin to thrombomodulin. Elastase-digested thrombin, which was cleaved between Ala-150 and Asn-151, lost its binding affinity for both MT-6 and thrombomodulin. These findings indicate that the binding site for thrombomodulin is located within the sequence between Thr-147 and Ser-158 of the B-chain.
Suzuki
K
K
Division of Enzyme Cytology, University of Tokushima, Japan.
Nishioka
J
J
Hayashi
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004220:Disulfides; D010446:Peptide Fragments; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D010196:Pancreatic Elastase; D013917:Thrombin