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Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

Kunz, G; Öhlin, Ann-Kristin LU ; Adami, A; Zöller, Bengt LU ; Svensson, P and Lane, DA (2002) In Blood 99(10). p.3646-3653
Abstract
Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly6lAla, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced... (More)
Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly6lAla, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% +/- 5%, P < .001), Pro477Ser (76.8% +/-11%, P <.001), Pro483Leu (82.1% +/- 8%, P < .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (K,) for wild-type thrombin-TM complex was 634 +/- 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had Increased (P < .0001) K, 2967 +/- 283 nM, and 2342 +/- 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations In the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Blood
volume
99
issue
10
pages
3646 - 3653
publisher
American Society of Hematology
external identifiers
  • wos:000175496300025
  • pmid:11986219
  • scopus:0037093228
ISSN
1528-0020
language
English
LU publication?
yes
id
3b724ee6-415d-4848-8cd9-cf642693cbe6 (old id 338411)
alternative location
http://bloodjournal.hematologylibrary.org/cgi/content/full/99/10/3646
date added to LUP
2007-10-31 09:53:39
date last changed
2017-12-10 03:51:08
@article{3b724ee6-415d-4848-8cd9-cf642693cbe6,
  abstract     = {Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly6lAla, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% +/- 5%, P &lt; .001), Pro477Ser (76.8% +/-11%, P &lt;.001), Pro483Leu (82.1% +/- 8%, P &lt; .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (K,) for wild-type thrombin-TM complex was 634 +/- 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had Increased (P &lt; .0001) K, 2967 +/- 283 nM, and 2342 +/- 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations In the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.},
  author       = {Kunz, G and Öhlin, Ann-Kristin and Adami, A and Zöller, Bengt and Svensson, P and Lane, DA},
  issn         = {1528-0020},
  language     = {eng},
  number       = {10},
  pages        = {3646--3653},
  publisher    = {American Society of Hematology},
  series       = {Blood},
  title        = {Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function},
  volume       = {99},
  year         = {2002},
}