Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin

Qingyu Wu; John P Sheehan; Manuel Tsiang; Steven R Lentz; Jens J Birktoft; J. Evan Sadler

doi:10.1073/pnas.88.15.6775

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Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin

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Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin

Qingyu Wu, John P Sheehan, Manuel Tsiang, Steven R Lentz, Jens J Birktoft and J. Evan Sadler

Proceedings of the National Academy of Sciences - PNAS, Vol.88(15), pp.6775-6779

08/01/1991

DOI: 10.1073/pnas.88.15.6775

PMCID: PMC52171

PMID: 1650482

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Abstract

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.

Details

Title: Subtitle: Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin
Creators: Qingyu Wu - Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110
John P Sheehan - Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110
Manuel Tsiang - Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110
Steven R Lentz - Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110
Jens J Birktoft - Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110
J. Evan Sadler - Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, MO 63110
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.88(15), pp.6775-6779
DOI: 10.1073/pnas.88.15.6775
PMID: 1650482
PMCID: PMC52171
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences
Language: English
Date published: 08/01/1991
Academic Unit: Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
Record Identifier: 9984094583102771

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