Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation

Fumie Nakazawa, Takatoshi Koyama, Takako Saito, Misako Shibakura, Haruhiko Yoshinaga, Dong Hui Chung, Ryuichi Kamiyama, Shinsaku Hirosawa

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Thrombomodulin (TM) is an endothelial cell glycoprotein that acts as an anticoagulant. Mutation in the TM gene is a potential risk factor for thrombosis. The first TM mutation identified was a heterozygous substitution of T for G at nucleotide position 1456, which predicted Asp468 with Tyr in a Ser/Thr-rich domain. To evaluate the reported TM gene mutation as a possible cause of thrombosis, we transiently tranfected a vector for TM gene carrying the mutation to mammalian COS7 cells. TM antigen levels in lysates of cells transfected with variant TM were comparable to those in preparations of normal TM. The TM cofactor activity for protein C (PC) activation on the variant TM-expressing cells was similar to that of the control. The Michaelis constant K(m) and V(max). of variant TM for PC activation were shown to be similar compared to those of normal TM. The affinity of each TM for thrombin in PC activation was also similar. We obtained several stable cell lines expressing normal and variant TM. Lysate of the cell lines with normal and variant TM genes had a similar expression level of TM antigen. Pulse-chase analysis showed that normal and variant TM were glycosylated and resistant to endoglycosidase H, indicating that the variant TM was expressed on the cell surface in a mature form. Variant TM protein is apparently expressed on the cell surface with normal cofactor activity for PC activation. It is unlikely that the TM variant directly causes thrombosis by mechanism of reduced expression or impaired cofactor activity for PC activation, which comprises a major anticoagulant activity of TM.

Original languageEnglish
Pages (from-to)416-420
Number of pages5
JournalBritish Journal of Haematology
Volume106
Issue number2
DOIs
Publication statusPublished - 1999
Externally publishedYes

Keywords

  • Cofactor activity for protein C activation
  • Point mutation
  • Protein C
  • Thrombomodulin
  • Thrombosis

ASJC Scopus subject areas

  • Hematology

Fingerprint Dive into the research topics of 'Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation'. Together they form a unique fingerprint.

Cite this