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Blood, 15 May 2002, Vol. 99, No. 10, pp. 3646-3653
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Naturally occurring mutations in the thrombomodulin gene leading
to impaired expression and function
Gabriella Kunz,
Ann-Kristin Öhlin,
Antonella Adami,
Bengt Zöller,
Peter Svensson, and
David A. Lane
From the Department of Haematology, Imperial College
School of Medicine, London, United Kingdom; Department of Coagulation
Disorders, University of Lund, University Hospital Malmö, Sweden;
and Department of Clinical Chemistry, University of Lund, University
Hospital, Lund, Sweden.
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,
Gly61Ala, 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% ± 1%, 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 (Km) for
wild-type thrombin-TM complex was 634 ± 6 nmol/L. Two
mutants, with Arg385Ser and Pro477Ser, had increased
(P < .0001) Km, 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.
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