Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/338411
- author
- Kunz, G ; Öhlin, Ann-Kristin LU ; Adami, A ; Zöller, Bengt LU ; Svensson, P and Lane, DA
- organization
- publishing date
- 2002
- 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
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Division of Clinical Chemistry and Pharmacology (013250300), Emergency medicine/Medicine/Surgery (013240200)
- 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
- 2016-04-01 12:18:22
- date last changed
- 2022-04-21 05:37:16
@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 < .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.}}, 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}}, url = {{http://bloodjournal.hematologylibrary.org/cgi/content/full/99/10/3646}}, volume = {{99}}, year = {{2002}}, }