Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin.
Thrombin is a serine protease that acts as a procoagulant by clotting fibrinogen and activating platelets and as an anticoagulant by activating protein C in a thrombomodulin-dependent reaction. Fibrinogen and thrombomodulin bind competitively to an anion-binding exosite on thrombin. We prepared recombinant normal human thrombin and mutant thrombins with single amino acid substitutions in order to localize and distinguish the fibrinogen- and thrombomodulin-binding sites. Normal and mutant thrombins had similar amidolytic activity. Thrombin K52E had approximately 2.5-fold increased protein C-activating activity but only approximately 17% of normal fibrinogen-clotting activity. Thrombin R70E had normal fibrinogen-clotting activity but only approximately 7% of normal protein C-activating activity. Thrombin R68E had markedly reduced activity in both assays. Decreased activation of protein C correlated with decreased binding affinity for thrombomodulin, and ability to activate platelets correlated directly with fibrinogen-clotting activity. These results demonstrate that thrombins with predominantly anticoagulant or procoagulant activity can be created by mutagenesis and that thrombomodulin- and fibrinogen-binding sites on thrombin may overlap but are not identical.
Wu
Q Y
QY
Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110.
Sheehan
J P
JP
Tsiang
M
M
Lentz
S R
SR
Birktoft
J J
JJ
Sadler
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003198:Computer Simulation; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010974:Platelet Aggregation / Q000187:drug effects; D011487:Protein Conformation; D011516:Prothrombin / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013917:Thrombin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D011516:Prothrombin; D005340:Fibrinogen; D013917:Thrombin