https://orcid.org/0000-0002-3105-6117
Key Points
Rare THBD variants of uncertain significance are detected in patients with inherited bleeding and thrombotic disorders.
TM L433P has an impaired effect on the natural anticoagulant pathway by decreased protein C and TAFI activation.
Subjects:
Thrombosis and Hemostasis
Abstract
Thrombomodulin (TM) expressed on endothelial cells regulates coagulation. Specific nonsense variants in the TM gene, THBD, result in high soluble TM levels causing rare bleeding disorders. In contrast, although THBD variants have been associated with venous thromboembolism, this association remains controversial. A multigene panel was used to diagnose 601 patients with inherited bleeding or thrombotic disorders. This resulted in the identification of 8 THBD variants for 6 patients with a thrombotic (C175S, A282P, L433P, P501L, G502R, and P508L) and 2 patients with a bleeding (P260A and T478I) phenotype. These were all classified as variants of uncertain significance, and we here aimed to assess their functional role in coagulation. For this purpose, soluble and cell membrane–bound recombinant TM were produced in Expi293F cells. L433P TM showed a marked decrease in the inhibition of thrombin generation and complete inhibition of protein C and thrombin activatable fibrinolysis inhibitor (TAFI) activation. Soluble C175S TM showed decreased inhibition of thrombin generation and protein C activation, whereas no effect was observed for cell membrane-bound recombinant TM. For the other TM variants, no effect on thrombin generation, protein C, or TAFI activation could be observed. Surface plasmon resonance analysis showed no thrombin-TM binding in the presence of L433P because this residue is located at their interaction site. In conclusion, our study shows the functional effects of L433P TM and potentially C175S TM, which are compatible with an increased thrombosis risk. THBD variants are rare but can be relevant to both bleeding and thrombosis. Functional assays for these variants are critical to understand their roles.
Subjects:
Thrombosis and Hemostasis
References
1.
Heremans
J
, Freson
K
. High-throughput sequencing for diagnosing platelet disorders: lessons learned from exploring the causes of bleeding disorders
. Int J Lab Hematol
. 2018
;40
(suppl 1
):89
-96
.2.
Van Laer
C
, Jacquemin
M
, Baert
S
, et al. Clinical application of multigene panel testing for bleeding, thrombotic, and platelet disorders: a 3-year Belgian experience
. J Thromb Haemost
. 2023
;21
(4
):887
-895
.3.
Ver Donck
F
, Labarque
V
, Freson
K
. Hemostatic phenotypes and genetic disorders
. Res Pract Thromb Haemost
. 2021
;5
(8
):e12637
.4.
Megy
K
, Downes
K
, Simeoni
I
, et al. Curated disease-causing genes for bleeding, thrombotic, and platelet disorders: communication from the SSC of the ISTH
. J Thromb Haemost
. 2019
;17
(8
):1253
-1260
.5.
Ross
JE
, Mohan
S
, Zhang
J
, et al. Evaluating the clinical validity of genes related to hemostasis and thrombosis using the clinical genome resource gene curation framework
. J Thromb Haemost
. 2024
;22
(3
):645
-665
.6.
Dittman
W
, Majerus
P
. Structure and function of thrombomodulin: a natural anticoagulant
. Blood
. 1990
;75
(2
):329
-336
.7.
Suzuki
K
, Kusumoto
H
, Deyashiki
Y
, et al. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation
. EMBO J
. 1987
;6
(7
):1891
-1897
.8.
Weiler
H
, Isermann
BH
. Thrombomodulin
. J Thromb Haemost
. 2003
;1
(7
):1515
-1524
.9.
Neubauer
K
, Zieger
B
. Endothelial cells and coagulation
. Cell Tissue Res
. 2022
;387
(3
):391
-398
.10.
Giri
H
, Biswas
I
, Rezaie
AR
. Thrombomodulin: a multifunctional receptor modulating the endothelial quiescence
. J Thromb Haemost
. 2024
;22
(4
):905
-914
.11.
Myles
T
, Nishimura
T
, Yun
TH
, et al. Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation
. J Biol Chem
. 2003
;278
(51
):51059
-51067
.12.
Nishimura
T
, Myles
T
, Piliponsky
AM
, Kao
PN
, Berry
GJ
, Leung
LLK
. Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo
. Blood
. 2007
;109
(5
):1992
-1997
.13.
Loghmani
H
, Conway
EM
. Exploring traditional and nontraditional roles for thrombomodulin
. Blood
. 2018
;132
(2
):148
-158
.14.
Langdown
J
, Luddington
RJ
, Huntington
JA
, Baglin
TP
. A hereditary bleeding disorder resulting from a premature stop codon in thrombomodulin (p.Cys537Stop)
. Blood
. 2014
;124
(12
):1951
-1956
.15.
Dargaud
Y
, Scoazec
JY
, Wielders
SJH
, et al. Characterization of an autosomal dominant bleeding disorder caused by a thrombomodulin mutation
. Blood
. 2015
;125
(9
):1497
-1501
.16.
Jourdy
Y
, Enjolras
N
, Le Quellec
S
, et al. Why patients with THBD c.1611C>A (p. Cys537X) nonsense mutation have high levels of soluble thrombomodulin?
. PLoS One
. 2017
;12
(11
):e0188213
.17.
Westbury
SK
, Whyte
CS
, Stephens
J
, et al. A new pedigree with thrombomodulin-associated coagulopathy in which delayed fibrinolysis is partially attenuated by co-inherited TAFI deficiency
. J Thromb Haemost
. 2020
;18
(9
):2209
-2214
.18.
Osada
M
, Maruyama
K
, Kokame
K
, et al. A novel homozygous variant of the thrombomodulin gene causes a hereditary bleeding disorder
. Blood Adv
. 2021
;5
(19
):3830
-3838
.19.
Manderstedt
E
, Halldén
C
, Lind-Halldén
C
, et al. Thrombomodulin (THBD) gene variants and thrombotic risk in a population-based cohort study
. J Thromb Haemost
. 2022
;20
(4
):929
-935
.20.
Wang
L
, Ran
L
, Tian
Y
, Jin
Y
, Yi
J
, He
X
. Effect of thrombomodulin gene polymorphisms on venous thromboembolism: an analysis of evidence involving 6,629 patients
. Vascular
. 2024
;32
(2
):374
-384
.21.
Ohlin
AK
, Marlar
RA
. The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease
. Blood
. 1995
;85
(2
):330
-336
.22.
Ohlin
K
, Marlar
R
. Thrombomodulin gene defects in families with thromboembolic disease--a report on four families
. Thromb Haemost
. 1999
;81
(3
):338
-344
.23.
Kunz
G
, Ohlin
AK
, Adami
A
, Zöller
B
, Svensson
P
, Lane
DA
. Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function
. Blood
. 2002
;99
(10
):3646
-3653
.24.
D’Apolito
M
, Ariano
A
, D’Andrea
G
, et al. Identification of the novel G250R variant indicates a role for thrombomodulin in modulating the risk for venous thromboembolism
. Thromb Haemost
. 2022
;122
(10
):1827
-1832
.25.
Simeoni
I
, Stephens
JC
, Hu
F
, et al. A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders
. Blood
. 2016
;127
(23
):2791
-2803
.26.
Downes
K
, Megy
K
, Duarte
D
, et al. Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders
. Blood
. 2019
;134
(23
):2082
-2091
.27.
Richards
S
, Aziz
N
, Bale
S
, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology
. Genet Med
. 2015
;17
(5
):405
-424
.28.
Cheng
J
, Novati
G
, Pan
J
, et al. Accurate proteome-wide missense variant effect prediction with AlphaMissense
. Science
. 2023
;381
(6664
):eadg7492
.29.
Yang
Z
, Zeng
X
, Zhao
Y
, Chen
R
. AlphaFold2 and its applications in the fields of biology and medicine
. Signal Transduct Target Ther
. 2023
;8
(1
):115
.30.
Lisman
T
, Adelmeijer
J
. Preanalytical variables affect thrombomodulin-modified thrombin generation in healthy volunteers
. Thromb Res
. 2020
;194
:237
-239
.31.
Tripodi
A
. Detection of procoagulant imbalance: modified endogenous thrombin potential with results expressed as ratio of values with-to-without thrombomodulin
. Thromb Haemost
. 2017
;117
(5
):830
-836
.32.
Favaloro
EJ
. Genetic testing for thrombophilia-related genes: observations of testing patterns for factor V Leiden (G1691A) and prothrombin gene mutation (G20210A)
. Semin Thromb Hemost
. 2019
;45
(7
):730
-742
.33.
Baerga-Ortiz
A
, Bergqvist
S
, Mandell
JG
, Komives
EA
. Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles
. Protein Sci
. 2004
;13
(1
):166
-176
.34.
Abramson
J
, Adler
J
, Dunger
J
, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3
. Nature
. 2024
;630
(8016
):493
-500
.35.
Mehic
D
, Colling
M
, Pabinger
I
, Gebhart
J
. Natural anticoagulants: a missing link in mild to moderate bleeding tendencies
. Haemophilia
. 2021
;27
(5
):701
-709
.36.
Elkattawy
S
, Alyacoub
R
, Singh
KS
, Fichadiya
H
, Kessler
W
. Prothrombin G20210A gene mutation-induced recurrent deep vein thrombosis and pulmonary embolism: case report and literature review
. J Investig Med High Impact Case Rep
. 2022
;10
:23247096211058486
.37.
Von Ahsen
N
, Lewczuk
P
, Schü Tz
E
, Oellerich
M
, Ehrenreich
H
. Prothrombin activity and concentration in healthy subjects with and without the prothrombin G20210A mutation
. Thromb Res
. 2000
;99
(6
):549
-556
.38.
Raife
TJ
, Dwyre
DM
, Stevens
JW
, et al. Human thrombomodulin knockin mice reveal differential effects of human thrombomodulin on thrombosis and atherosclerosis
. Arterioscler Thromb Vasc Biol
. 2011
;31
(11
):2509
-2517
.39.
Hernandez
W
, Gamazon
ER
, Smithberger
E
, et al. Novel genetic predictors of venous thromboembolism risk in African Americans
. Blood
. 2016
;127
(15
):1923
-1929
.40.
Key
NS
, Reiner
AP
. Genetic basis of ethnic disparities in VTE risk
. Blood
. 2016
;127
(15
):1844
-1845
.41.
Jiewen
M
, yanyi
T
, Yuanzheng
F
, et al. A missense mutation in lectin domain of thrombomodulin causing functional deficiency
. Transl Res
. 2023
;251
:74
-83
.42.
Demeulenaere
M
, Devreese
K
, Vanbelleghem
H
, et al. Thrombomodulin and endothelial dysfunction: a disease-modifier shared between malignant hypertension and atypical hemolytic uremic syndrome
. Nephron
. 2018
;140
(1
):63
-73
.43.
Kavanagh
D
, Goodship
T
. Genetics and complement in atypical HUS
. Pediatr Nephrol
. 2010
;25
(12
):2431
-2442
.44.
Delvaeye
M
, Noris
M
, De Vriese
A
, et al. Thrombomodulin mutations in atypical hemolytic-uremic syndrome
. N Engl J Med
. 2009
;361
(4
):345
-357
.© 2025 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
2025
You do not currently have access to this content.
This feature is available to Subscribers Only
Sign In or Create an Account Close Modal