Key Points

  • Cyfip1 plays a crucial role for branching of actin filaments and for lamellipodium formation.

  • Lamellipodium formation is not required for the formation of a hemostatic plug or thrombus.

Abstract

During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.

REFERENCES

REFERENCES
1.
Vögtle
T
,
Cherpokova
D
,
Bender
M
,
Nieswandt
B
.
Targeting platelet receptors in thrombotic and thrombo-inflammatory disorders
.
Hamostaseologie
.
2015
;
35
(
3
):
235
-
243
.
2.
Nieswandt
B
,
Pleines
I
,
Bender
M
.
Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke
.
J Thromb Haemost
.
2011
;
9
(
suppl 1
):
92
-
104
.
3.
Ono
A
,
Westein
E
,
Hsiao
S
, et al
.
Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth
.
Blood
.
2008
;
112
(
1
):
90
-
99
.
4.
Hartwig
JH
.
Mechanisms of actin rearrangements mediating platelet activation
.
J Cell Biol
.
1992
;
118
(
6
):
1421
-
1442
.
5.
McCarty
OJ
,
Larson
MK
,
Auger
JM
, et al
.
Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow
.
J Biol Chem
.
2005
;
280
(
47
):
39474
-
39484
.
6.
Pleines
I
,
Elvers
M
,
Strehl
A
, et al
.
Rac1 is essential for phospholipase C-gamma2 activation in platelets
.
Pflugers Arch
.
2009
;
457
(
5
):
1173
-
1185
.
7.
Aslan
JE
,
McCarty
OJ
.
Rho GTPases in platelet function
.
J Thromb Haemost
.
2013
;
11
(
1
):
35
-
46
.
8.
Yusuf
MZ
,
Raslan
Z
,
Atkinson
L
, et al
.
Prostacyclin reverses platelet stress fibre formation causing platelet aggregate instability
.
Sci Rep
.
2017
;
7
(
1
):
5582
.
9.
Paul
DS
,
Casari
C
,
Wu
C
, et al
.
Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice
.
Blood Adv
.
2017
;
1
(
18
):
1398
-
1408
.
10.
Alekhina
O
,
Burstein
E
,
Billadeau
DD
.
Cellular functions of WASP family proteins at a glance
.
J Cell Sci
.
2017
;
130
(
14
):
2235
-
2241
.
11.
Derivery
E
,
Lombard
B
,
Loew
D
,
Gautreau
A
.
The Wave complex is intrinsically inactive
.
Cell Motil Cytoskeleton
.
2009
;
66
(
10
):
777
-
790
.
12.
Oda
A
,
Miki
H
,
Wada
I
, et al
.
WAVE/Scars in platelets
.
Blood
.
2005
;
105
(
8
):
3141
-
3148
.
13.
Dahl
JP
,
Wang-Dunlop
J
,
Gonzales
C
,
Goad
ME
,
Mark
RJ
,
Kwak
SP
.
Characterization of the WAVE1 knock-out mouse: implications for CNS development
.
J Neurosci
.
2003
;
23
(
8
):
3343
-
3352
.
14.
Calaminus
SD
,
McCarty
OJ
,
Auger
JM
, et al
.
A major role for Scar/WAVE-1 downstream of GPVI in platelets
.
J Thromb Haemost
.
2007
;
5
(
3
):
535
-
541
.
15.
Eto
K
,
Nishikii
H
,
Ogaeri
T
, et al
.
The WAVE2/Abi1 complex differentially regulates megakaryocyte development and spreading: implications for platelet biogenesis and spreading machinery
.
Blood
.
2007
;
110
(
10
):
3637
-
3647
.
16.
Gautreau
A
,
Ho
HY
,
Li
J
,
Steen
H
,
Gygi
SP
,
Kirschner
MW
.
Purification and architecture of the ubiquitous Wave complex
.
Proc Natl Acad Sci USA
.
2004
;
101
(
13
):
4379
-
4383
.
17.
Innocenti
M
,
Zucconi
A
,
Disanza
A
, et al
.
Abi1 is essential for the formation and activation of a WAVE2 signalling complex
.
Nat Cell Biol
.
2004
;
6
(
4
):
319
-
327
.
18.
Steffen
A
,
Rottner
K
,
Ehinger
J
, et al
.
Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation
.
EMBO J
.
2004
;
23
(
4
):
749
-
759
.
19.
Rodríguez
CI
,
Buchholz
F
,
Galloway
J
, et al
.
High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP
.
Nat Genet
.
2000
;
25
(
2
):
139
-
140
.
20.
Tiedt
R
,
Schomber
T
,
Hao-Shen
H
,
Skoda
RC
.
Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo
.
Blood
.
2007
;
109
(
4
):
1503
-
1506
.
21.
Chen
Z
,
Borek
D
,
Padrick
SB
, et al
.
Structure and control of the actin regulatory WAVE complex
.
Nature
.
2010
;
468
(
7323
):
533
-
538
.
22.
Davidson
AJ
,
Insall
RH
.
Actin-based motility: WAVE regulatory complex structure reopens old SCARs
.
Curr Biol
.
2011
;
21
(
2
):
R66
-
R68
.
23.
Pollitt
AY
,
Insall
RH
.
WASP and SCAR/WAVE proteins: the drivers of actin assembly
.
J Cell Sci
.
2009
;
122
(
Pt 15
):
2575
-
2578
.
24.
Spindler
M
,
van Eeuwijk
JMM
,
Schurr
Y
, et al
.
ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia
.
Blood
.
2018
;
132
(
6
):
635
-
646
.
25.
Heilemann
M
,
van de Linde
S
,
Schüttpelz
M
, et al
.
Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes
.
Angew Chem Int Ed Engl
.
2008
;
47
(
33
):
6172
-
6176
.
26.
Kuijpers
MJ
,
Munnix
IC
,
Cosemans
JM
, et al
.
Key role of platelet procoagulant activity in tissue factor-and collagen-dependent thrombus formation in arterioles and venules in vivo differential sensitivity to thrombin inhibition
.
Microcirculation
.
2008
;
15
(
4
):
269
-
282
.
27.
Munnix
IC
,
Strehl
A
,
Kuijpers
MJ
, et al
.
The glycoprotein VI-phospholipase Cgamma2 signaling pathway controls thrombus formation induced by collagen and tissue factor in vitro and in vivo
.
Arterioscler Thromb Vasc Biol
.
2005
;
25
(
12
):
2673
-
2678
.
28.
Gros
A
,
Syvannarath
V
,
Lamrani
L
, et al
.
Single platelets seal neutrophil-induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice
.
Blood
.
2015
;
126
(
8
):
1017
-
1026
.
29.
Goerge
T
,
Ho-Tin-Noe
B
,
Carbo
C
, et al
.
Inflammation induces hemorrhage in thrombocytopenia
.
Blood
.
2008
;
111
(
10
):
4958
-
4964
.
30.
Durrant
TN
,
van den Bosch
MT
,
Hers
I
.
Integrin αIIbβ3 outside-in signaling
.
Blood
.
2017
;
130
(
14
):
1607
-
1619
.
31.
Di Buduo
CA
,
Wray
LS
,
Tozzi
L
, et al
.
Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies
.
Blood
.
2015
;
125
(
14
):
2254
-
2264
.
32.
Ito
Y
,
Nakamura
S
,
Sugimoto
N
, et al
.
Turbulence activates platelet biogenesis to enable clinical scale ex vivo production
.
Cell
.
2018
;
174
(
3
):
636
-
648.e618
.
33.
Moreau
T
,
Evans
AL
,
Vasquez
L
, et al
.
Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming [published correction appears in Nat Commun. 2017;8:15076]
.
Nat Commun
.
2016
;
7
:
11208
.
34.
Thon
JN
,
Mazutis
L
,
Wu
S
, et al
.
Platelet bioreactor-on-a-chip
.
Blood
.
2014
;
124
(
12
):
1857
-
1867
.
35.
Kahr
WH
,
Pluthero
FG
,
Elkadri
A
, et al
.
Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease
.
Nat Commun
.
2017
;
8
:
14816
.
36.
Falet
H
,
Hoffmeister
KM
,
Neujahr
R
,
Hartwig
JH
.
Normal Arp2/3 complex activation in platelets lacking WASp
.
Blood
.
2002
;
100
(
6
):
2113
-
2122
.
37.
Agbani
EO
,
van den Bosch
MT
,
Brown
E
, et al
.
Coordinated membrane ballooning and procoagulant spreading in human platelets
.
Circulation
.
2015
;
132
(
15
):
1414
-
1424
.
38.
Krause
M
,
Gautreau
A
.
Steering cell migration: lamellipodium dynamics and the regulation of directional persistence
.
Nat Rev Mol Cell Biol
.
2014
;
15
(
9
):
577
-
590
.
39.
Gaertner
F
,
Ahmad
Z
,
Rosenberger
G
, et al
.
Migrating platelets are mechano-scavengers that collect and bundle bacteria
.
Cell
.
2017
;
171
(
6
):
1368
-
1382.e1323
.
40.
Léon
C
,
Eckly
A
,
Hechler
B
, et al
.
Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion
.
Blood
.
2007
;
110
(
9
):
3183
-
3191
.
You do not currently have access to this content.