Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains.
A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.
Hall
S W
SW
Division of Hematology, Stanford University School of Medicine, Stanford, California 94305-5112, USA. shall@leland.stanford.edu
Nagashima
M
M
Zhao
L
L
Morser
J
J
Leung
L L
LL
D000818:Animals; D001665:Binding Sites; D025901:Carboxypeptidase B2; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005342:Fibrinolysis; D006801:Humans; D017354:Point Mutation; D011485:Protein Binding; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*
D011486:Protein C; D018180:Thrombomodulin; D002268:Carboxypeptidases; D025901:Carboxypeptidase B2; D013917:Thrombin