Basis for the reduced affinity of beta T- and gamma T-thrombin for hirudin.
Partial proteolysis of human alpha-thrombin by trypsin results in the formation of beta T-thrombin and gamma T-thrombin which have a reduced affinity for the inhibitor hirudin and the cell-surface cofactor thrombomodulin as well as reduced activity with fibrinogen. The basis of the reduction in affinity of these thrombin derivatives for hirudin has been investigated by examining their kinetics of interaction with a number of hirudin mutants differing in their C-terminal charge properties as well as with a truncated form of hirudin. The results indicate that the reduced affinity of beta T-thrombin for hirudin is most likely due to a decrease in the strength of nonionic interactions between thrombin and the C-terminal region of hirudin. No decrease in the strength of ionic interactions was observed with beta T-thrombin. In contrast, the reduced affinity of gamma T-thrombin was due to a decrease in the strength of both ionic and nonionic interactions. The N-terminal core region of hirudin, which interacts predominantly with the active-site cleft of thrombin, exhibited similar affinities for alpha-, beta T-, and gamma T-thrombin, indicating that thrombin-hirudin interactions within the active site are largely preserved in beta T- and gamma T-thrombin.
Stone
S R
SR
Friedrich Miescher-Institut, Basel, Switzerland.
Hofsteenge
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin
D006629:Hirudins; D010446:Peptide Fragments; D014357:Trypsin; D013917:Thrombin