• Our findings identify a previously unrecognized role of the Gab2–MALT1 axis in IL-1β-induced thromboinflammation.

  • Pharmacological inhibition of MALT1 attenuates venous thrombosis induced by flow restriction.

Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1β (IL-1β) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1β and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]–BCL-10 [B-cell lymphoma 10]–MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1β–induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1β–induced NF-κB–dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2–MALT1 axis in thromboinflammation. Targeting the Gab2–MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.

1.
Colling
ME
,
Tourdot
BE
,
Kanthi
Y
.
Inflammation, infection and venous thromboembolism
.
Circ Res.
2021
;
128
(
12
):
2017
-
2036
.
2.
Klemen
ND
,
Feingold
PL
,
Hashimoto
B
, et al
.
Mortality risk associated with venous thromboembolism: a systematic review and Bayesian meta-analysis
.
Lancet Haematol.
2020
;
7
(
8
):
e583
-
e593
.
3.
Søgaard
KK
,
Schmidt
M
,
Pedersen
L
,
Horváth-Puhó
E
,
Sørensen
HT
.
30-year mortality after venous thromboembolism: a population-based cohort study
.
Circulation.
2014
;
130
(
10
):
829
-
836
.
4.
von Brühl
ML
,
Stark
K
,
Steinhart
A
, et al
.
Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo
.
J Exp Med.
2012
;
209
(
4
):
819
-
835
.
5.
Esmon
CT
.
Basic mechanisms and pathogenesis of venous thrombosis
.
Blood Rev.
2009
;
23
(
5
):
225
-
229
.
6.
Payne
H
,
Ponomaryov
T
,
Watson
SP
,
Brill
A
.
Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis
.
Blood.
2017
;
129
(
14
):
2013
-
2020
.
7.
Nopp
S
,
Ay
C
.
Bleeding risk assessment in patients with venous thromboembolism
.
Hamostaseologie.
2021
;
41
(
4
):
267
-
274
.
8.
Klok
FA
,
Kooiman
J
,
Huisman
MV
,
Konstantinides
S
,
Lankeit
M
.
Predicting anticoagulant-related bleeding in patients with venous thromboembolism: a clinically oriented review
.
Eur Respir J.
2015
;
45
(
1
):
201
-
210
.
9.
Khan
F
,
Tritschler
T
,
Kimpton
M
, et al;
MAJESTIC Collaborators
.
Long-term risk for major bleeding during extended oral anticoagulant therapy for first unprovoked venous thromboembolism: a systematic review and meta-analysis
.
Ann Intern Med.
2021
;
174
(
10
):
1420
-
1429
.
10.
Stone
J
,
Hangge
P
,
Albadawi
H
, et al
.
Deep vein thrombosis: pathogenesis, diagnosis, and medical management
.
Cardiovasc Diagn Ther.
2017
;
7
(
suppl 3
):
S276
-
S284
.
11.
Mackman
N
.
New insights into the mechanisms of venous thrombosis
.
J Clin Invest.
2012
;
122
(
7
):
2331
-
2336
.
12.
Stark
K
,
Massberg
S
.
Interplay between inflammation and thrombosis in cardiovascular pathology
.
Nat Rev Cardiol.
2021
;
18
(
9
):
666
-
682
.
13.
Branchford
BR
,
Carpenter
SL
.
The role of inflammation in venous thromboembolism
.
Front Pediatr.
2018
;
6
:
142
.
14.
Pai
RZ
,
Fang
Q
,
Tian
G
,
Zhu
B
,
Ge
X
.
Expression and role of interleukin-1β and associated biomarkers in deep vein thrombosis
.
Exp Ther Med.
2021
;
22
(
6
):
1366
.
15.
Gupta
N
,
Sahu
A
,
Prabhakar
A
, et al
.
Activation of NLRP3 inflammasome complex potentiates venous thrombosis in response to hypoxia
.
Proc Natl Acad Sci USA.
2017
;
114
(
18
):
4763
-
4768
.
16.
Yadav
V
,
Chi
L
,
Zhao
R
, et al
.
Ectonucleotidase tri(di)phosphohydrolase-1 (ENTPD-1) disrupts inflammasome/interleukin 1β-driven venous thrombosis
.
J Clin Invest.
2019
;
129
(
7
):
2872
-
2877
.
17.
Puhlmann
M
,
Weinreich
DM
,
Farma
JM
,
Carroll
NM
,
Turner
EM
,
Alexander
HR
Jr
.
Interleukin-1β induced vascular permeability is dependent on induction of endothelial tissue factor (TF) activity
.
J Transl Med.
2005
;
3
(
1
):
37
.
18.
Bresnihan
B
,
Cunnane
G
.
Infection complications associated with the use of biologic agents
.
Rheum Dis Clin North Am.
2003
;
29
(
1
):
185
-
202
.
19.
Dinarello
CA
,
Simon
A
,
van der Meer
JW
.
Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases
.
Nat Rev Drug Discov.
2012
;
11
(
8
):
633
-
652
.
20.
Wikén
M
,
Hallén
B
,
Kullenberg
T
,
Koskinen
LO
.
Development and effect of antibodies to anakinra during treatment of severe CAPS: sub-analysis of a long-term safety and efficacy study
.
Clin Rheumatol.
2018
;
37
(
12
):
3381
-
3386
.
21.
Wang
M
,
Hao
H
,
Leeper
NJ
,
Zhu
L
;
Early Career Committee
.
Thrombotic regulation from the endothelial cell perspectives
.
Arterioscler Thromb Vasc Biol.
2018
;
38
(
6
):
e90
-
e95
.
22.
Martinod
K
,
Wagner
DD
.
Thrombosis: tangled up in NETs
.
Blood.
2014
;
123
(
18
):
2768
-
2776
.
23.
Kimball
AS
,
Obi
AT
,
Diaz
JA
,
Henke
PK
.
The emerging role of NETs in venous thrombosis and immunothrombosis
.
Front Immunol.
2016
;
7
:
236
-
236
.
24.
Carminita
E
,
Crescence
L
,
Brouilly
N
,
Altié
A
,
Panicot-Dubois
L
,
Dubois
C
.
DNAse-dependent, NET-independent pathway of thrombus formation in vivo
.
Proc Natl Acad Sci USA.
2021
;
118
(
28
):
e2100561118
.
25.
Ruland
J
,
Hartjes
L
.
CARD-BCL-10-MALT1 signalling in protective and pathological immunity
.
Nat Rev Immunol.
2019
;
19
(
2
):
118
-
134
.
26.
Thome
M
.
Multifunctional roles for MALT1 in T-cell activation
.
Nat Rev Immunol.
2008
;
8
(
7
):
495
-
500
.
27.
Blonska
M
,
Lin
X
.
NF-κB signaling pathways regulated by CARMA family of scaffold proteins
.
Cell Res.
2011
;
21
(
1
):
55
-
70
.
28.
Delekta
PC
,
Apel
IJ
,
Gu
S
, et al
.
Thrombin-dependent NF-kappaB activation and monocyte/endothelial adhesion are mediated by the CARMA3·Bcl10·MALT1 signalosome
.
J Biol Chem.
2010
;
285
(
53
):
41432
-
41442
.
29.
McAllister-Lucas
LM
,
Ruland
J
,
Siu
K
, et al
.
CARMA3/Bcl10/MALT1-dependent NF-kappaB activation mediates angiotensin II-responsive inflammatory signaling in nonimmune cells
.
Proc Natl Acad Sci USA.
2007
;
104
(
1
):
139
-
144
.
30.
Martin
D
,
Galisteo
R
,
Gutkind
JS
.
CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex
.
J Biol Chem.
2009
;
284
(
10
):
6038
-
6042
.
31.
Gu
H
,
Neel
BG
.
The “Gab” in signal transduction
.
Trends Cell Biol.
2003
;
13
(
3
):
122
-
130
.
32.
Nishida
K
,
Hirano
T
.
The role of Gab family scaffolding adapter proteins in the signal transduction of cytokine and growth factor receptors
.
Cancer Sci.
2003
;
94
(
12
):
1029
-
1033
.
33.
Wöhrle
FU
,
Daly
RJ
,
Brummer
T
.
Function, regulation and pathological roles of the Gab/DOS docking proteins
.
Cell Commun Signal.
2009
;
7
(
1
):
22
.
34.
Ding
CB
,
Yu
WN
,
Feng
JH
,
Luo
JM
.
Structure and function of Gab2 and its role in cancer (review)
.
Mol Med Rep.
2015
;
12
(
3
):
4007
-
4014
.
35.
Kondreddy
V
,
Magisetty
J
,
Keshava
S
,
Rao
LVM
,
Pendurthi
UR
.
Gab2 (Grb2-associated Binder2) plays a crucial role in inflammatory signaling and endothelial dysfunction
.
Arterioscler Thromb Vasc Biol.
2021
;
41
(
6
):
1987
-
2005
.
36.
Kuhns
DB
,
Priel
DAL
,
Chu
J
,
Zarember
KA
.
Isolation and functional analysis of human neutrophils
Curr Protoc Immunol.
2015
;
111
:
7.23.21
-
27.23.16
.
37.
Hirose
A
,
Tanikawa
T
,
Mori
H
,
Okada
Y
,
Tanaka
Y
.
Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway
.
FEBS Lett.
2010
;
584
(
1
):
61
-
66
.
38.
Brill
A
,
Fuchs
TA
,
Chauhan
AK
, et al
.
von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models
.
Blood.
2011
;
117
(
4
):
1400
-
1407
.
39.
Diaz
JA
,
Saha
P
,
Cooley
B
, et al
.
Choosing a mouse model of venous thrombosis
.
Arterioscler Thromb Vasc Biol.
2019
;
39
(
3
):
311
-
318
.
40.
Yau
JW
,
Teoh
H
,
Verma
S
.
Endothelial cell control of thrombosis
.
BMC Cardiovasc Disord.
2015
;
15
(
1
):
130
.
41.
Zhang
Y
,
Cui
J
,
Zhang
G
, et al
.
Inflammasome activation promotes venous thrombosis through pyroptosis
.
Blood Adv.
2021
;
5
(
12
):
2619
-
2623
.
42.
Vischer
UM
,
Barth
H
,
Wollheim
CB
.
Regulated von Willebrand factor secretion is associated with agonist-specific patterns of cytoskeletal remodeling in cultured endothelial cells
.
Arterioscler Thromb Vasc Biol.
2000
;
20
(
3
):
883
-
891
.
43.
Klarenbach
SW
,
Chipiuk
A
,
Nelson
RC
,
Hollenberg
MD
,
Murray
AG
.
Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases
.
Circ Res.
2003
;
92
(
3
):
272
-
278
.
44.
Newton
K
,
Dixit
VM
.
Signaling in innate immunity and inflammation
.
Cold Spring Harb Perspect Biol.
2012
;
4
(
3
):
a006049
.
45.
Singh
R
,
Wang
B
,
Shirvaikar
A
, et al
.
The IL-1 receptor and Rho directly associate to drive cell activation in inflammation
.
J Clin Invest.
1999
;
103
(
11
):
1561
-
1570
.
46.
Klei
LR
,
Hu
D
,
Panek
R
, et al
.
MALT1 protease activation triggers acute disruption of endothelial barrier integrity via CYLD cleavage
.
Cell Rep.
2016
;
17
(
1
):
221
-
232
.
47.
Mao
D
,
Epple
H
,
Uthgenannt
B
,
Novack
DV
,
Faccio
R
.
PLCgamma2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2
.
J Clin Invest.
2006
;
116
(
11
):
2869
-
2879
.
48.
Adams
SJ
,
Aydin
IT
,
Celebi
JT
.
GAB2 – a scaffolding protein in cancer
.
Mol Cancer Res.
2012
;
10
(
10
):
1265
-
1270
.
49.
Wegener
E
,
Krappmann
D
.
CARD-Bcl10-Malt1 signalosomes: missing link to NF-kappaB
.
Sci STKE.
2007
;
2007
(
384
):
pe21
-
pe21
.
50.
Juilland
M
,
Thome
M
.
Holding all the CARDs: how MALT1 controls CARMA/CARD-dependent signaling
.
Front Immunol.
2018
;
9
:
1927
.
51.
Cao
Z
,
Xiong
J
,
Takeuchi
M
,
Kurama
T
,
Goeddel
DV
.
TRAF6 is a signal transducer for interleukin-1
.
Nature.
1996
;
383
(
6599
):
443
-
446
.
52.
Walsh
MC
,
Kim
GK
,
Maurizio
PL
,
Molnar
EE
,
Choi
Y
.
TRAF6 autoubiquitination-independent activation of the NFkappaB and MAPK pathways in response to IL-1 and RANKL
.
PLoS One.
2008
;
3
(
12
):
e4064
.
53.
Jaworski
M
,
Thome
M
.
The paracaspase MALT1: biological function and potential for therapeutic inhibition
.
Cell Mol Life Sci.
2016
;
73
(
3
):
459
-
473
.
54.
Migliacci
R
,
Becattini
C
,
Pesavento
R
, et al
.
Endothelial dysfunction in patients with spontaneous venous thromboembolism
.
Haematologica.
2007
;
92
(
6
):
812
-
818
.
55.
Denis
CV
,
André
P
,
Saffaripour
S
,
Wagner
DD
.
Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice
.
Proc Natl Acad Sci USA.
2001
;
98
(
7
):
4072
-
4077
.
56.
Petri
B
,
Broermann
A
,
Li
H
, et al
.
von Willebrand factor promotes leukocyte extravasation
.
Blood.
2010
;
116
(
22
):
4712
-
4719
.
57.
Fan
Y
,
Yu
Y
,
Shi
Y
, et al
.
Lysine 63-linked polyubiquitination of TAK1 at lysine 158 is required for tumor necrosis factor alpha- and interleukin-1beta-induced IKK/NF-kappaB and JNK/AP-1 activation
.
J Biol Chem.
2010
;
285
(
8
):
5347
-
5360
.
58.
Wang
C
,
Deng
L
,
Hong
M
,
Akkaraju
GR
,
Inoue
J
,
Chen
ZJ
.
TAK1 is a ubiquitin-dependent kinase of MKK and IKK
.
Nature.
2001
;
412
(
6844
):
346
-
351
.
59.
Abreu
MTH
,
Hughes
WE
,
Mele
K
, et al
.
Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation
.
Mol Biol Cell.
2011
;
22
(
1
):
105
-
116
.
60.
Arnaud
M
,
Mzali
R
,
Gesbert
F
, et al
.
Interaction of the tyrosine phosphatase SHP-2 with Gab2 regulates Rho-dependent activation of the c-fos serum response element by interleukin-2
.
Biochem J.
2004
;
382
(
Pt 2
):
545
-
556
.
61.
Rafferty
BJ
,
Unger
BL
,
Perey
AC
,
Tammariello
SP
,
Pavlides
S
,
McGee
DW
.
A novel role for the Rho-associated kinase, ROCK, in IL-1-stimulated intestinal epithelial cell responses
.
Cell Immunol.
2012
;
280
(
2
):
148
-
155
.
62.
Banerjee
S
,
McGee
DW
.
ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells
.
In Vitro Cell Dev Biol Anim.
2016
;
52
(
8
):
878
-
884
.
63.
Sun
J
.
CARMA3: a novel scaffold protein in regulation of NF-κB activation and diseases
.
World J Biol Chem.
2010
;
1
(
12
):
353
-
361
.
64.
McAuley
JR
,
Freeman
TJ
,
Ekambaram
P
,
Lucas
PC
,
McAllister-Lucas
LM
.
CARMA3 Is a critical mediator of G protein-coupled receptor and receptor tyrosine kinase-driven solid tumor pathogenesis
.
Front Immunol.
2018
;
9
:
1887
.
65.
Mahanivong
C
,
Chen
HM
,
Yee
SW
,
Pan
ZK
,
Dong
Z
,
Huang
S
.
Protein kinase C alpha-CARMA3 signaling axis links Ras to NF-kappa B for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells
.
Oncogene.
2008
;
27
(
9
):
1273
-
1280
.
66.
Amin
AR
,
Ichigotani
Y
,
Oo
ML
, et al
.
The PLC-PKC cascade is required for IL-1beta-dependent Erk and Akt activation: their role in proliferation
.
Int J Oncol.
2003
;
23
(
6
):
1727
-
1731
.
67.
Lin
CH
,
Sheu
SY
,
Lee
HM
, et al
.
Involvement of protein kinase C-gamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells
.
Mol Pharmacol.
2000
;
57
(
1
):
36
-
43
.
68.
Brasier
AR
.
The nuclear factor-kappaB-interleukin-6 signalling pathway mediating vascular inflammation
.
Cardiovasc Res.
2010
;
86
(
2
):
211
-
218
.
69.
Takaesu
G
,
Kishida
S
,
Hiyama
A
, et al
.
TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway
.
Mol Cell.
2000
;
5
(
4
):
649
-
658
.
70.
Budnik
I
,
Brill
A
.
Immune factors in deep vein thrombosis initiation
.
Trends Immunol.
2018
;
39
(
8
):
610
-
623
.
71.
Martinod
K
,
Demers
M
,
Fuchs
TA
, et al
.
Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice
.
Proc Natl Acad Sci USA.
2013
;
110
(
21
):
8674
-
8679
.
72.
Brill
A
,
Fuchs
TA
,
Savchenko
AS
, et al
.
Neutrophil extracellular traps promote deep vein thrombosis in mice
.
J Thromb Haemost.
2012
;
10
(
1
):
136
-
144
.
73.
Thålin
C
,
Hisada
Y
,
Lundström
S
,
Mackman
N
,
Wallén
H
.
Neutrophil extracellular traps: villains and targets in arterial, venous, and cancer-associated thrombosis
.
Arterioscler Thromb Vasc Biol.
2019
;
39
(
9
):
1724
-
1738
.
74.
Carminita
E
,
Crescence
L
,
Panicot-Dubois
L
,
Dubois
C
.
Role of neutrophils and NETs in animal models of thrombosis
.
Int J Mol Sci.
2022
;
23
(
3
):
1411
.
75.
El-Sayed
OM
,
Dewyer
NA
,
Luke
CE
, et al
.
Intact Toll-like receptor 9 signaling in neutrophils modulates normal thrombogenesis in mice
.
J Vasc Surg.
2016
;
64
(
5
):
1450
-
1458.e1
.
76.
Hisada
Y
,
Grover
SP
,
Maqsood
A
, et al
.
Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors
.
Haematologica.
2020
;
105
(
1
):
218
-
225
.
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