Key Points

  • Aging-associated inflammation by TNF-α plays an important role in the development of platelet hyperreactivity during aging.

  • Aging-associated platelet hyperreactivity is associated with megakaryocytic inflammatory, metabolic, and mitochondrial reprogramming.

Abstract

Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

REFERENCES

REFERENCES
1.
Engbers
MJ
,
van Hylckama Vlieg
A
,
Rosendaal
FR
.
Venous thrombosis in the elderly: incidence, risk factors and risk groups
.
J Thromb Haemost
.
2010
;
8
(
10
):
2105
-
2112
.
2.
Niccoli
T
,
Partridge
L
.
Ageing as a risk factor for disease
.
Curr Biol
.
2012
;
22
(
17
):
R741
-
R752
.
3.
Bruunsgaard
H
,
Andersen-Ranberg
K
,
Hjelmborg
J
,
Pedersen
BK
,
Jeune
B
.
Elevated levels of tumor necrosis factor alpha and mortality in centenarians
.
Am J Med
.
2003
;
115
(
4
):
278
-
283
.
4.
Henry
CJ
,
Casás-Selves
M
,
Kim
J
, et al
.
Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors
.
J Clin Invest
.
2015
;
125
(
12
):
4666
-
4680
.
5.
Yoshida
H
,
Yilmaz
CE
,
Granger
DN
.
Role of tumor necrosis factor-α in the extraintestinal thrombosis associated with colonic inflammation
.
Inflamm Bowel Dis
.
2011
;
17
(
11
):
2217
-
2223
.
6.
Manfredi
AA
,
Baldini
M
,
Camera
M
, et al
.
Anti-TNFα agents curb platelet activation in patients with rheumatoid arthritis
.
Ann Rheum Dis
.
2016
;
75
(
8
):
1511
-
1520
.
7.
Di Minno
MND
,
Iervolino
S
,
Peluso
R
,
Scarpa
R
,
Di Minno
G
.
Platelet reactivity and disease activity in subjects with psoriatic arthritis
.
J Rheumatol
.
2012
;
39
(
2
):
334
-
336
.
8.
Roselli
M
,
Ferroni
P
,
Rolfo
C
, et al
.
TNF-α gene promoter polymorphisms and risk of venous thromboembolism in gastrointestinal cancer patients undergoing chemotherapy
.
Ann Oncol
.
2013
;
24
(
10
):
2571
-
2575
.
9.
Cui
G
,
Wang
H
,
Li
R
, et al
.
Polymorphism of tumor necrosis factor alpha (TNF-alpha) gene promoter, circulating TNF-alpha level, and cardiovascular risk factor for ischemic stroke
.
J Neuroinflammation
.
2012
;
9
:
235
.
10.
Levine
A
,
Shamir
R
,
Wine
E
, et al
.
TNF promoter polymorphisms and modulation of growth retardation and disease severity in pediatric Crohn’s disease
.
Am J Gastroenterol
.
2005
;
100
(
7
):
1598
-
1604
.
11.
Hu
N
,
Cui
Y
,
Yang
Q
,
Wang
L
,
Yang
X
,
Xu
H
.
Association of polymorphisms in TNF and GRN genes with ankylosing spondylitis in a Chinese Han population
.
Rheumatol Int
.
2018
;
38
(
3
):
481
-
487
.
12.
Macedo
LC
,
de Cesare Quintero
F
,
Pagliari-E-Silva
S
, et al
.
Association of TNF polymorphisms with JAK2 (V617F) myeloproliferative neoplasms in Brazilian patients
.
Blood Cells Mol Dis
.
2016
;
57
:
54
-
57
.
13.
Fleischman
AG
,
Aichberger
KJ
,
Luty
SB
, et al
.
TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms
.
Blood
.
2011
;
118
(
24
):
6392
-
6398
.
14.
Lai
HY
,
Brooks
SA
,
Craver
BM
, et al
.
Defective negative regulation of Toll-like receptor signaling leads to excessive TNF-α in myeloproliferative neoplasm
.
Blood Adv
.
2019
;
3
(
2
):
122
-
131
.
15.
Hubbard
RE
,
O’Mahony
MS
,
Savva
GM
,
Calver
BL
,
Woodhouse
KW
.
Inflammation and frailty measures in older people
.
J Cell Mol Med
.
2009
;
13
(
9B
):
3103
-
3109
.
16.
Michaud
M
,
Balardy
L
,
Moulis
G
, et al
.
Proinflammatory cytokines, aging, and age-related diseases
.
J Am Med Dir Assoc
.
2013
;
14
(
12
):
877
-
882
.
17.
Pignatelli
P
,
De Biase
L
,
Lenti
L
, et al
.
Tumor necrosis factor-alpha as trigger of platelet activation in patients with heart failure
.
Blood
.
2005
;
106
(
6
):
1992
-
1994
.
18.
Lee
JL
,
Sinnathurai
P
,
Buchbinder
R
,
Hill
C
,
Lassere
M
,
March
L
.
Biologics and cardiovascular events in inflammatory arthritis: a prospective national cohort study
.
Arthritis Res Ther
.
2018
;
20
(
1
):
171
.
19.
Vaiyapuri
S
,
Jones
CI
,
Sasikumar
P
, et al
.
Gap junctions and connexin hemichannels underpin hemostasis and thrombosis
.
Circulation
.
2012
;
125
(
20
):
2479
-
2491
.
20.
Zhou
Z
,
Gushiken
FC
,
Bolgiano
D
, et al
.
Signal transducer and activator of transcription 3 (STAT3) regulates collagen-induced platelet aggregation independently of its transcription factor activity
.
Circulation
.
2013
;
127
(
4
):
476
-
485
.
21.
Smith
CW
,
Thomas
SG
,
Raslan
Z
, et al
.
Mice lacking the inhibitory collagen receptor LAIR-1 exhibit a mild thrombocytosis and hyperactive platelets
.
Arterioscler Thromb Vasc Biol
.
2017
;
37
(
5
):
823
-
835
.
22.
Fidler
TP
,
Campbell
RA
,
Funari
T
, et al
.
Deletion of GLUT1 and GLUT3 reveals multiple roles for glucose metabolism in platelet and megakaryocyte function [published correction appears in Cell Rep. 2017;20(9):2277 and Cell Rep. 2017;21(6):1705]
.
Cell Rep
.
2017
;
20
(
4
):
881
-
894
.
23.
Fink
BD
,
Herlein
JA
,
O’Malley
Y
,
Sivitz
WI
.
Endothelial cell and platelet bioenergetics: effect of glucose and nutrient composition
.
PLoS One
.
2012
;
7
(
6
):
e39430
.
24.
Branchford
BR
,
Stalker
TJ
,
Law
L
, et al
.
The small-molecule MERTK inhibitor UNC2025 decreases platelet activation and prevents thrombosis
.
J Thromb Haemost
.
2018
;
16
(
2
):
352
-
363
.
25.
Heazlewood
SY
,
Williams
B
,
Storan
MJ
,
Nilsson
SK
.
The prospective isolation of viable, high ploidy megakaryocytes from adult murine bone marrow by fluorescence activated cell sorting
.
Methods Mol Biol
.
2013
;
1035
:
121
-
133
.
26.
Schulze
H
.
Culture, expansion, and differentiation of murine megakaryocytes from fetal liver, bone marrow, and spleen
.
Curr Protoc Immunol
.
2016
;
112
:
22F.6.1
-
22F.6.15
.
27.
Nemkov
T
,
Hansen
KC
,
D’Alessandro
A
.
A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways
.
Rapid Commun Mass Spectrom
.
2017
;
31
(
8
):
663
-
673
.
28.
Yeom
E
,
Park
JH
,
Kang
YJ
,
Lee
SJ
.
Microfluidics for simultaneous quantification of platelet adhesion and blood viscosity
.
Sci Rep
.
2016
;
6
:
24994
.
29.
Branchford
BR
,
Ng
CJ
,
Neeves
KB
,
Di Paola
J
.
Microfluidic technology as an emerging clinical tool to evaluate thrombosis and hemostasis
.
Thromb Res
.
2015
;
136
(
1
):
13
-
19
.
30.
Grover
A
,
Sanjuan-Pla
A
,
Thongjuea
S
, et al
.
Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells
.
Nat Commun
.
2016
;
7
:
11075
.
31.
Rossi
DJ
,
Bryder
D
,
Zahn
JM
, et al
.
Cell intrinsic alterations underlie hematopoietic stem cell aging
.
Proc Natl Acad Sci USA
.
2005
;
102
(
26
):
9194
-
9199
.
32.
Pietras
EM
,
Reynaud
D
,
Kang
Y-A
, et al
.
Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions
.
Cell Stem Cell
.
2015
;
17
(
1
):
35
-
46
.
33.
Kovtonyuk
LV
,
Fritsch
K
,
Feng
X
,
Manz
MG
,
Takizawa
H
.
Inflamm-aging of hematopoiesis, hematopoietic stem cells, and the bone marrow microenvironment
.
Front Immunol
.
2016
;
7
:
502
.
34.
Huybers
S
,
Apostolaki
M
,
van der Eerden
BC
, et al
.
Murine TNF(DeltaARE) Crohn’s disease model displays diminished expression of intestinal Ca2+ transporters
.
Inflamm Bowel Dis
.
2008
;
14
(
6
):
803
-
811
.
35.
de Zoeten
EF
,
Pasternak
BA
,
Mattei
P
,
Kramer
RE
,
Kader
HA
.
Diagnosis and treatment of perianal Crohn disease: NASPGHAN clinical report and consensus statement
.
J Pediatr Gastroenterol Nutr
.
2013
;
57
(
3
):
401
-
412
.
36.
Collins
CB
,
Strassheim
D
,
Aherne
CM
,
Yeckes
AR
,
Jedlicka
P
,
de Zoeten
EF
.
Targeted inhibition of heat shock protein 90 suppresses tumor necrosis factor-α and ameliorates murine intestinal inflammation
.
Inflamm Bowel Dis
.
2014
;
20
(
4
):
685
-
694
.
37.
Kontoyiannis
D
,
Pasparakis
M
,
Pizarro
TT
,
Cominelli
F
,
Kollias
G
.
Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies
.
Immunity
.
1999
;
10
(
3
):
387
-
398
.
38.
Liao
Z
,
Tu
JH
,
Small
CB
,
Schnipper
SM
,
Rosenstreich
DL
.
Increased urine interleukin-1 levels in aging
.
Gerontology
.
1993
;
39
(
1
):
19
-
27
.
39.
Barrientos
RM
,
Frank
MG
,
Hein
AM
, et al
.
Time course of hippocampal IL-1 beta and memory consolidation impairments in aging rats following peripheral infection
.
Brain Behav Immun
.
2009
;
23
(
1
):
46
-
54
.
40.
Dinarello
CA
.
Interleukin 1 and interleukin 18 as mediators of inflammation and the aging process
.
Am J Clin Nutr
.
2006
;
83
(
2
):
447S
-
455S
.
41.
Bruunsgaard
H
,
Skinhøj
P
,
Pedersen
AN
,
Schroll
M
,
Pedersen
BK
.
Ageing, tumour necrosis factor-alpha (TNF-alpha) and atherosclerosis
.
Clin Exp Immunol
.
2000
;
121
(
2
):
255
-
260
.
42.
Bruunsgaard
H
,
Pedersen
M
,
Pedersen
BK
.
Aging and proinflammatory cytokines
.
Curr Opin Hematol
.
2001
;
8
(
3
):
131
-
136
.
43.
Peschon
JJ
,
Torrance
DS
,
Stocking
KL
, et al
.
TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation
.
J Immunol
.
1998
;
160
(
2
):
943
-
952
.
44.
Ralser
M
,
Wamelink
MM
,
Kowald
A
, et al
.
Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress
.
J Biol
.
2007
;
6
(
4
):
10
.
45.
Grant
CM
.
Metabolic reconfiguration is a regulated response to oxidative stress
.
J Biol
.
2008
;
7
(
1
):
1
.
46.
Dong
JF
.
Platelet microparticles are not created equal
.
Blood
.
2014
;
124
(
14
):
2161
-
2162
.
47.
Zhao
Z
,
Wang
M
,
Tian
Y
, et al
.
Cardiolipin-mediated procoagulant activity of mitochondria contributes to traumatic brain injury-associated coagulopathy in mice
.
Blood
.
2016
;
127
(
22
):
2763
-
2772
.
48.
Boudreau
LH
,
Duchez
AC
,
Cloutier
N
, et al
.
Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation
.
Blood
.
2014
;
124
(
14
):
2173
-
2183
.
49.
Zhong
Z
,
Liang
S
,
Sanchez-Lopez
E
, et al
.
New mitochondrial DNA synthesis enables NLRP3 inflammasome activation
.
Nature
.
2018
;
560
(
7717
):
198
-
203
.
50.
Boyapati
RK
,
Dorward
DA
,
Tamborska
A
, et al
.
Mitochondrial DNA is a pro-inflammatory damage-associated molecular pattern released during active IBD
.
Inflamm Bowel Dis
.
2018
;
24
(
10
):
2113
-
2122
.
51.
Dayal
S
,
Wilson
KM
,
Motto
DG
,
Miller
FJ
Jr
,
Chauhan
AK
,
Lentz
SR
.
Hydrogen peroxide promotes aging-related platelet hyperactivation and thrombosis
.
Circulation
.
2013
;
127
(
12
):
1308
-
1316
.
52.
Yang
J
,
Zhou
X
,
Fan
X
, et al
.
mTORC1 promotes aging-related venous thrombosis in mice via elevation of platelet volume and activation
.
Blood
.
2016
;
128
(
5
):
615
-
624
.
53.
Jang
JY
,
Blum
A
,
Liu
J
,
Finkel
T
.
The role of mitochondria in aging
.
J Clin Invest
.
2018
;
128
(
9
):
3662
-
3670
.
54.
Ts’ao
CH
,
Rossi
EC
,
Lestina
FC
.
Abnormalities in platelet function and morphology in a case of thrombocythemia
.
Arch Pathol Lab Med
.
1977
;
101
(
10
):
526
-
533
.
55.
Hattori
A
,
Koike
K
,
Ito
S
,
Matsuoka
M
.
Static and functional morphology of the pathological platelets in primary myelofibrosis and myeloproliferative syndrome
.
Ser Haematol
.
1975
;
8
(
1
):
126
-
150
.
56.
Taylor
DD
,
Senhauser
DA
,
Cavazos
F
.
Thrombocytopathy associated with nonleukemic megakaryocytic myelosis. Functional and fine structure observations of the abnormal platelets
.
Am J Clin Pathol
.
1968
;
49
(
5
):
662
-
670
.
57.
Triant
VA
.
Cardiovascular disease and HIV infection
.
Curr HIV/AIDS Rep
.
2013
;
10
(
3
):
199
-
206
.
58.
Triant
VA
,
Perez
J
,
Regan
S
, et al
.
Cardiovascular risk prediction functions underestimate risk in HIV infection
.
Circulation
.
2018
;
137
(
21
):
2203
-
2214
.
59.
Wang
R
,
Stone
RL
,
Kaelber
JT
, et al
.
Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer
.
Proc Natl Acad Sci USA
.
2015
;
112
(
46
):
14266
-
14271
.
60.
Golan
K
,
Kumari
A
,
Kollet
O
, et al
.
Daily onset of light and darkness differentially controls hematopoietic stem cell differentiation and maintenance
.
Cell Stem Cell
.
2018
;
23
(
4
):
572
-
585.e7
.
61.
Puchta
A
,
Naidoo
A
,
Verschoor
CP
, et al
.
TNF drives monocyte dysfunction with age and results in impaired anti-pneumococcal immunity
.
PLoS Pathog
.
2016
;
12
(
1
):
e1005368
.
62.
Hearps
AC
,
Martin
GE
,
Angelovich
TA
, et al
.
Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function
.
Aging Cell
.
2012
;
11
(
5
):
867
-
875
.
You do not currently have access to this content.