Functional domains of membrane-bound human thrombomodulin : EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity

Manuel Tsiang; Steven R Lentz; J Evan Sadler

doi:10.1016/S0021-9258(18)42676-2

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Functional domains of membrane-bound human thrombomodulin : EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity

Journal article

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Functional domains of membrane-bound human thrombomodulin : EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity

Manuel Tsiang, Steven R Lentz and J Evan Sadler

The Journal of biological chemistry, Vol.267(9), pp.6164-6170

1992

DOI: 10.1016/S0021-9258(18)42676-2

PMID: 1313430

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Abstract

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.

Fundamental and applied biological sciences. Psychology

Proteins

Biological and medical sciences

Glycoproteins

Analytical, structural and metabolic biochemistry

Details

Title: Subtitle: Functional domains of membrane-bound human thrombomodulin : EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity
Creators: Manuel Tsiang - Washington univ. school medicine, Howard Hughes medical inst., Saint Louis MO 63110, United States
Steven R Lentz - Washington univ. school medicine, Howard Hughes medical inst., Saint Louis MO 63110, United States
J Evan Sadler - Washington univ. school medicine, Howard Hughes medical inst., Saint Louis MO 63110, United States
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.267(9), pp.6164-6170
DOI: 10.1016/S0021-9258(18)42676-2
PMID: 1313430
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology
Language: English
Date published: 1992
Academic Unit: Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
Record Identifier: 9984094631402771

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