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Regular Article
PLATELETS AND THROMBOPOIESIS
Defective tubulin organization and proplatelet formation in
murine megakaryocytes lacking Rac1 and Cdc42
Irina Pleines,1 Sebastian Dütting,1 Deya Cherpokova,1 Anita Eckly,2 Imke Meyer,3,4 Martina Morowski,1 Georg Krohne,5
Harald Schulze,4,6 Christian Gachet,2 Najet Debili,7 Cord Brakebusch,8 and Bernhard Nieswandt1
1
University of Würzburg, Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, Deutsche Forschungsgemeinschaft
Research Center for Experimental Biomedicine, Würzburg, Germany; 2Unité Mixte de Recherche-S949 Institut National de la Santé et de la Recherche
Médicale-Université de Strasbourg, Etablissement Francais du Sang-Alsace, Strasbourg, France; 3Freie Universität Berlin, Department of Biochemistry,
Berlin, Germany; 4Laboratory of Pediatric Molekularbiologie, Charité-Medical School, Berlin, Germany; 5Biocenter, University of Würzburg, Würzburg,
Germany; 6Institute for Transfusion Medicine, Human Leukocyte Antigen Tissue Typing Laboratory, Charité, Berlin, Germany; 7Institut National de la Santé
et de la Recherche Médicale, Unité Mixte de Recherche 1009, Institut Gustave Roussy, Villejuif, France; and 8Biotech Research and Innovation Centre,
Biomedical Institute, University of Copenhagen, Copenhagen, Denmark
Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets
are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or socalled proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound
• Rac1 and Cdc42 have
redundant functions in platelet reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA,
Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets;
biogenesis.
however, the specific roles of these proteins during platelet production have not been
• Deficiency of Rac1 and
established. Using conditional knockout mice, we show here that Rac1 and Cdc42
Cdc42 results in highly
possess redundant functions in platelet production and function. In contrast to a singleabnormal megakaryocyte
deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in
morphology associated with
macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function.
severely defective tubulin
Double-deficient bone marrow MKs matured normally in vivo but displayed highly
organization.
abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/
Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was
associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these
results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating
microtubule dynamics. (Blood. 2013;122(18):3178-3187)
Key Points
Introduction
Platelets are synthesized by precursor cells called megakaryocytes
(MKs). MKs reside primarily in the bone marrow (BM) and develop
by differentiation from pluripotent hematopoietic stem cells.1 MK
maturation is initiated by multiple rounds of endomitosis, the
synthesis of platelet-specific granules, and the formation of a demarcation membrane system, which seems to function as a membrane
reservoir for newly formed (pro)platelets.1,2 Finally, mature MKs
extend proplatelet-like protrusions into the sinusoids of the BM, from
which platelets are shed and are finally sized by the shear forces
present in the blood stream.3,4 Microtubule sliding enables proplatelet elongation and mediates organelle trafficking into future
platelets.5 In contrast, actin-dependent mechanisms are thought to be
involved in the branching of proplatelet shafts, thereby increasing
the number of available proplatelet tips.5
Rho GTPases are small proteins (20-25 kDa) belonging to the
superfamily of Ras-related proteins, which are found in all eukaryotic
cells.6 They are best known for regulating actin cytoskeletal
dynamics in virtually all cell types, as well as for their involvement in
the regulation of microtubules.7 The best-characterized Rho GTPases
are RhoA, Rac1, and Cdc42, the activation of which is associated
with the formation of stress fibers, lamellipodia, and filopodia,
respectively. Much knowledge about the functions of Rho GTPases
has been gained by overexpression or knockdown studies in cell
lines, which, however, has often yielded conflicting results. Therefore,
the recent generation of knockout mice for several GTPases has
provided new tools to more reliably study the effect of GTPase
deficiency with a reduced risk for secondary effects.8,9 Several studies
on platelet function using hematopoietic and/or MK- and plateletspecific Rac1 and Cdc42 knockout mice have been performed. The
results have revealed that Rac1 is indispensable not only for
lamellipodia formation but also for activation of the phospholipase
C isoform g2 (PLCg2) in murine platelets.10,11 In contrast, Cdc42deficient platelets, surprisingly, retained the ability to form filopodia
and displayed increased agonist-induced secretion.12 However, the
Submitted March 1, 2013; accepted July 6, 2013. Prepublished online as
Blood First Edition paper, July 16, 2013; DOI 10.1182/blood-2013-03-487942.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
I.P. and S.D. contributed equally to this study.
The online version of this article contains a data supplement.
There is an Inside Blood commentary on this article in this issue.
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© 2013 by The American Society of Hematology
BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
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BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
RAC1 AND CDC42 IN THROMBOPOIESIS
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Figure 1. Absence of Rac1 and Cdc42 leads to macrothrombocytopenia. (A) Western blot analysis of Rac1 and Cdc42 expression in wild-type (wt) and Rac1/Cdc422/2 platelets
generated by the Mx-Cre (left) or the PF4-Cre (right) system. GPIIIa expression was used as loading control. Analysis of peripheral platelet counts (B) and mean platelet volume (C) of
wt (black), Rac12/2 (dark gray), Cdc422/2 (light gray), and Rac1/Cdc422/2 (patterned) mice (n 5 5 per group; ***P , .001). (D-E) Rac1/Cdc422/2 platelets are increased in size.
Representative scanning (D) and transmission (E) electron microscopy pictures from wt and Rac1/Cdc422/2 platelets are depicted. Bars, 5 and 2 mm.
role of Rac1 and Cdc42 for MK maturation and platelet production has
not been addressed to date. The fact that Rac1-deficient mice
display normal platelet count suggests normal MK and platelet
production in these animals. MK- and platelet-specific Cdc42
knockout mice display moderate thrombocytopenia, which might,
however, be explained by the significantly reduced life span of the
mutant platelets.12
Studies using different cell types have revealed that Rac1 and
Cdc42 may share different downstream signaling molecules.7
Well-investigated examples among these are the actin-polymerizationpromoting proteins mammalian diaphanous 2 (mDia2) and p21activated kinase (PAK), which in turn induces activation of the
LIM kinase (LIMK), leading to phosphorylation-dependent inactivation of cofilin.
In this study, we investigated the potential redundant roles of Rac1
and Cdc42 for platelet production and function by analyzing conditional Rac1 and/or Cdc42 knockout mice. We demonstrate that in
contrast to a single-deficiency in either GTPase, double-deficiency in
Rac1 and Cdc42 virtually abrogates proplatelet formation in vitro.
Interestingly, this phenotype was particularly associated with defective
microtubule stabilization. These results demonstrate for the first time
a functional redundancy of Rac1 and Cdc42 in the hematopoietic
system and point to a novel role of the GTPases in the regulation of
microtubule dynamics during platelet production.
Materials and methods
Mice, platelet preparation, platelet spreading, flow cytometry, determination
of actin polymerization, platelet life span, tail bleeding, in vivo thrombus
formation, western blotting, electron microscopy, histology, determination
of ploidy of MKs, in vitro MK differentiation, analysis of proplatelet
formation, and statistical data analysis are described in detail in the supplemental Methods, available on the Blood website. A list of antibodies and
reagents is also provided in the supplemental Methods. Animal studies were
approved by the district government of Lower Franconia (Bezirksregierung
Unterfranken).
Results
Constitutive deletion of either the Rac113 or the Cdc4214 gene
results in embryonic lethality in mice. To study the function of
Rac1/Cdc42 double-deficiency, mice carrying both the Rac1 and
Cdc42 genes flanked by loxP sites15,16 were generated. The resulting
Rac1/Cdc42fl/fl mice were crossed with transgenic mice expressing
Cre recombinase under the control of either the MK- and plateletspecific platelet factor (Pf) 417 or the Mx promoter,18 which is
specific for the hematopoietic system.
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BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
PLEINES et al
Figure 2. Rac1/Cdc422/2 platelets display abnormal ultrastructure and are rapidly cleared from the circulation. (A-B) Representative TEM pictures are shown.
(A) Normal ultrastructure of wt, Rac12/2 , and Cdc422/2 platelets. Bar, 0.5 mm. (B) Abnormal ultrastructure of Rac1/Cdc422/2 platelets. Bar sizes are depicted. (C)
Increased number of marginal microtubular rings in Rac1/Cdc422/2 platelets. Bar sizes are depicted. (D) Life span from Rac12/2 (dark gray), Cdc422/2 (light gray), and
Rac1/Cdc422/2 (patterned) platelets compared with wt (black) platelets. (E) Infinite tail bleeding times in Rac1/Cdc422/2 (patterned) compared with wt (black) mice (n 5 7 per
group; ***P , .001).
In the PF4-Cre system, gene deletion occurred intrinsically
during MK maturation in Rac1/Cdc42fl/fl Pf4-cre1 mice. In adult Rac1/
Cdc42fl/fl Mx-cre1 mice, gene deletion was induced as described in the
supplemental Methods. Western blot analysis revealed that both
systems efficiently deleted both proteins in platelets (Figure 1A).
However, Rac1/Cdc422/2 Mx-cre1 animals died between 9 and 14
days after Cre induction, which is in line with the observation
that Mx-Cre-induced Cdc42 deficiency alone results in rapid
death of the animals (I.P. and B.N., unpublished data, and Yang
et al19). In contrast, Rac1/Cdc422/2 Pf4-cre1 mice were outwardly
healthy and fertile (not shown). To avoid potential adverse effects on
megakaryopoiesis evoked by Mx-Cre-induced gene deletion, we
therefore exclusively used Rac1/Cdc422/2 Pf4-cre1 (further referred to as Rac1/Cdc422/2) animals in this study, with the indicated
exception of in vitro platelet adhesion assays.
Rac1/Cdc42 double-deficiency leads to
macrothrombocytopenia and abnormal platelet morphology
As expected from previous studies,11,12 Rac12/2 mice displayed
normal and Cdc422/2 mice only moderately decreased platelet
counts. In marked contrast, double-deficiency of Rac1 and Cdc42
resulted in severe thrombocytopenia, with platelet counts lower than
25% those of control mice (wt, 885 3 103/mL [6182 3 103]; Rac1/
Cdc422/2, 202 3 103/mL [6120 3 103]; Figure 1B). The size of
Rac1/Cdc422/2 platelets was markedly increased compared with
that of the single-deficient platelets, as demonstrated by transmission electron microscopy (TEM) and scanning electron
microscopy of resting platelets and determination of the mean platelet
volume (wt, 5.5 femtoliter [fL] [60.2]; Rac1/Cdc422/2, 6.9 fL [6
0.3]; Figure 1C-E).
Detailed analysis by TEM revealed a highly abnormal ultrastructure in Rac1/Cdc422/2 platelets (Figure 2). Whereas Rac12/2
and Cdc422/2 platelets displayed normal granule distribution compared with wt platelets (Figure 2A and data not shown), 51% of the
Rac1/Cdc422/2 platelets were overloaded with granules and/or
vacuoles, whereas 34% were virtually devoid of granules (Figure 2B),
indicating disordered granule formation or trafficking. Interestingly, in the majority of double-deficient platelets, the microtubule
coils in the marginal band were not visible; however, if they were
present, their numbers were increased compared with the wt (wt,
10.1 [60.9] microtubule coils/platelet; Rac1/Cdc422/2, 14 [60.8]
microtubule coils/platelet; Figure 2C). Flow cytometric measurements indicated significantly reduced expression levels of several
prominent platelet surface glycoproteins (GPs) in Rac1/Cdc422/2
platelets (Table 1). Together, these results demonstrated a severe
defect in platelet production in the absence of both Rac1 and Cdc42.
Rac1/Cdc422/2 platelets display a decreased life span
Nonfunctional or preactivated platelets are constantly cleared from
the circulation by the reticulo-endothelial system in spleen and
Table 1. Platelet GP expression in wt and Rac1/Cdc422/2 mice
wt
Rac1/Cdc422/2
Significance
GPIb
368 6 8
298 6 37
*
GPV
368 6 7
312 6 35
*
GPIX
555 6 20
447 6 58
*
CD9
1451 6 47
1380 6 129
n.s.
GP
52 6 2
43 6 2
†
118 6 4
123 6 10
n.s
b1
193 6 4
205 6 18
n.s.
aIIbb3
856 6 45
736 6 47
*
GPVI
a2
Expression of GPs on the platelet surface was determined by flow cytometry.
Diluted whole blood from the indicated mice was incubated with fluorescein
isothiocyanate–labeled antibodies at saturating concentrations for 15 minutes at
room temperature and platelets were analyzed directly using a FACSCalibur. Results
are expressed as mean fluorescence intensity 6 SD for 4 mice per group.
n.s., no significance.
*P , .05.
†P , .01.
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BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
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Figure 3. Defective spreading of Rac1/Cdc422/2
platelets on fibrinogen upon activation. (A) Washed
platelets from wt and Rac1/Cdc422/2Mx mice were allowed
to adhere on immobilized human fibrinogen (100 mg/mL)
upon activation with thrombin (0.01 U/mL, left) or CRP
(10 mg/mL, right). (Upper) DIC images were taken at the
indicated times (5, 15, and 30 minutes), representative of 4
individual experiments. Bar, 5 mm. (Lower) Statistical
analysis of the percentage of spread Rac1/Cdc422/2Mx
and wt platelets. (Bottom) (1) Roundish, no filopodia, no
lamellipodia. (2) Only filopodia. (3) Partial spreading. (4)
Full spreading. Arrows indicate altered morphology in
Rac1/Cdc422/2Mx platelets. (B) Visualization of defective
spreading and actin reorganization of Rac12/2 (middle)
and Rac1/Cdc422/2Mx (right) platelets by scanning electron microscopy. (Upper) Scanning electron microscopy
image of intact platelets. (Lower) Visualization of the
cytoskeleton after denudation of the plasma membrane.
Bar, 2 mm. (C) Determination of relative F-actin contents
in resting, thrombin-activated (1 and 0.01 U/mL), and CRPactivated (10 mg/mL) wt (black) and Rac1/Cdc42 2/2
Mx
(patterned) platelets (n 5 4 per group; ***P , .001).
liver.20 To investigate whether the macrothrombocytopenia in the
double-deficient mice was caused by an increased platelet turnover,
the life span of the cells was determined in Rac1/Cdc422/2 mice.
In agreement with normal platelet counts, the life span of Rac12/2
platelets was approximately 5 days and was thus comparable with
that of the wt platelets (Figure 2D). In contrast, as shown before,12
the life span of Cdc422/2 platelets was reduced to approximately 3 days. Surprisingly, despite their highly abnormal
morphology, the life span of Rac1/Cdc422/2 platelets was not
significantly shorter than that of Cdc422/2 platelets. A per se
preactivation of the double-deficient and Cdc422/2 platelets was
probably not the major cause of their decreased life span, as flow
cytometric measurements showed only minor basal integrin aIIbb3
activation or P-selectin exposure in Rac1/Cdc422/2 and Cdc422/2
platelets under resting conditions or on stimulation with the weak
agonist epinephrine in vitro (supplemental Figure 1C).
after stimulation with ITAM as well as G-protein-coupled-receptor
(GPCR) agonists, despite a moderate decrease in integrin activation.12
In Rac1/Cdc422/2 platelets, integrin aIIbb3 activation (supplemental Figure 1A) was markedly decreased, whereas a-granule
secretion toward GPCR agonists was preserved (supplemental
Figure 1B). Tail bleeding assays revealed that Rac1/Cdc422/2
mice displayed a severe hemostatic defect in vivo characterized
by an inability to arrest bleeding within 20 minutes (mean bleeding
time in wt, 468 6 248 seconds) and by a large volume of lost
blood (Figure 2E and not shown). Similarly, no occlusive
thrombi formed in Rac1/Cdc422/2 mice upon FeCl3-induced
injury of mesenteric arterioles in vivo (supplemental Figure 1D).
Taken together, the combination of reduced platelet counts and
impaired platelet function translated into defective hemostasis and
a profound protection from arterial thrombosis in Rac1/Cdc42
double-deficient animals in vivo.
Rac1/Cdc42 double-deficiency impairs platelet function in vitro
and results in defective hemostasis and arterial thrombus
formation in vivo
Rac1/Cdc422/2 platelets are unable to spread on fibrinogen but
retain the ability to adhere and form filopodia
To investigate the effect of Rac1/Cdc42 double-deficiency on
platelet function, the response of Rac1/Cdc422/2 platelets toward
agonist stimulation was analyzed (supplemental Figure 1). We
have previously shown that Rac12/2 platelets display a selective
defect when activated with immunoreceptor tyrosine-based
activation motif (ITAM)-coupled agonists.11 In Cdc422/2 platelets, degranulation-dependent P-selectin expression was enhanced
To analyze the consequences of Rac1/Cdc42 double-deficiency on
cytoskeletal rearrangements in platelets, thrombin- or collagen-related
peptide (CRP)-activated Rac1/Cdc422/2Mx platelets were allowed to
spread on fibrinogen (Figure 3A). As shown previously,11,12
Rac12/2 platelets were unable to form lamellipodia on fibrinogen
upon thrombin activation, whereas Cdc422/2 platelets spread normally.
Interestingly, Rac1/Cdc422/2Mx platelets retained the ability to
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PLEINES et al
BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
Figure 4. Increased MK numbers in BM of Cdc422/2 and Rac1/Cdc422/2 mice. (A) Determination of MK numbers in hematoxylin and eosin–stained BM sections from wt
(black), Rac12/2 (dark gray), Cdc422/2 (light gray), and Rac1/Cdc422/2 (patterned) mice. (Left) Representative images. (Right) Statistical analysis. (B) Determination of MK
number and morphology within the BM of anti–CD41-stained (green) cryosections. Endothelial cells are stained with anti-CD105 (red) and nuclei with 4,6 diamidino-2phenylindole (DAPI) (blue). Results are expressed as mean MK number per visual field 6 SD of 3 mice per group. (C) Functional endomitosis in Rac12/2, Cdc422/2, and
Rac1/Cdc422/2 MKs. Determination of the mean ploidy of BM-derived MKs from wt (black), Rac12/2 (dark gray), Cdc422/2 (light gray), and Rac1/Cdc422/2 (patterned) mice.
Results are expressed as mean ploidy 6 SD (n 5 4 per group; ***P , .001).
tightly adhere to the fibrinogen matrix under these conditions
(Figure 3A) and, as expected, failed to develop lamellipodia.
However, formation of long and thin filopodia was still observed in
approximately 70% of Rac1/Cdc422/2Mx platelets (Figure 3A).
Consistently, actin distribution was altered in thrombin-stimulated
spread Rac1/Cdc42 2/2Mx platelets denudated of the plasma
membrane (Figure 3B, bottom). Interestingly, CRP-stimulated
Rac1/Cdc422/2Mx platelets also tightly adhered to fibrinogen;
however, the spreading defect was significantly more pronounced
than after thrombin activation (Figure 3A). As a result, the majority
of adherent double-deficient platelets displayed a resting roundish
shape after 30 minutes (Rac1/Cdc422/2Mx, 64% 6 0.05% vs wildtype, 12.5% 6 0.02%), indicating impaired cellular activation
(Figure 3A).
Despite their effect on platelet-spreading morphology, Rac1/
Cdc422/2Mx platelets were still able to assemble F-actin similar
to wt platelets after stimulation with thrombin (0.01 U/mL)
(Figure 3C). In contrast, no significant F-actin assembly could be
detected in double-deficient platelets after CRP activation (10 mg/
mL). Together, these results suggest that defective F-actin assembly
and spreading of Rac1/Cdc422/2Mx platelets on fibrinogen after
CRP stimulation represented a direct consequence of defective GP
VI/ITAM-induced cellular activation caused by Rac1 deficiency. In
contrast, the distinct spreading defect in thrombin-stimulated Rac1/
Cdc422/2Mx platelets was most likely caused predominantly by
defective Rac1-mediated cytoskeletal rearrangement, rather than by
a general defect in F-actin assembly. This assumption is consistent
with the partially preserved activation of double-deficient platelets
toward GPCR-coupled agonists observed by flow cytometry
(supplemental Figure 1A-B). Interestingly, double-deficient platelets retained the ability to firmly adhere to the fibrinogen substrate,
irrespective of the stimulus, indicating that this integrin-mediated
process was largely preserved in absence of Rac1 and Cdc42 (supplemental Figure 1D).
Increased MK number and fragmentation in Rac1/Cdc42
double-deficient mice
The observation that platelet counts were dramatically reduced in
Rac1/Cdc422/2 compared with Cdc422/2 mice despite a similarly
decreased platelet life span implied additional functional defects in the
double-deficient MKs. We therefore investigated whether impaired
MK maturation and/or platelet production contributed to the
thrombocytopenia in Rac1/Cdc422/2 (and Cdc422/2) mice. In
accordance with normal platelet count and morphology, MK numbers
in hematoxylin and eosin–stained spleen and BM sections of Rac12/2
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BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
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Figure 5. Decreased proplatelet formation and abnormal morphology in Cdc422/2 and Rac1/Cdc422/2 MKs. (A) TEM of MKs in BM of adult (6-week-old) mice. (i-ii)
Normal ultrastructure in MKs derived from wt and Rac12/2 mice. (iii-iv) Altered ultrastructure in MKs derived from Cdc422/2 (iii) and Rac1/Cdc422/2 (iv) mice. (Upper)
Overview. Bar sizes are depicted. (Lower) Detailed view. Bar, 0.6 mm. (B-C) Determination of proplatelet formation from cultured MKs derived from wt (black), Rac12/2
(dark gray), Cdc422/2 (light gray), and Rac1/Cdc422/2 (patterned) mice. Results are expressed as percentage of proplatelet-forming MKs per visual field 6 SD from at
least 5 samples per group. (B) Results from BM-derived MKs (day 6 of culture). (C) Results from fetal liver-derived MKs (embryonic day 14.5, day 4 of culture).
***P , .001; **P , .01.
mice were comparable with the control. In contrast, both Cdc422/2
and Rac1/Cdc422/2 mice displayed increased numbers of MKs in
the spleen (not shown). Although BM MK numbers also were
markedly increased in Cdc422/2 mice (Figure 4A), Rac1/Cdc422/2
MKs were hardly identifiable in these sections because of their
altered morphology and decreased demarcation from the surrounding
cells. We therefore visualized Rac1/Cdc422/2 MKs in BM
cryosections, using an anti-CD41 antibody clearly revealing increased
numbers of MKs per visual field in Rac1/Cdc422/2 mice compared
with wt mice (wt, 20 6 4; Rac1/Cdc422/2, 41 6 6; Figure 4B).
Furthermore, mature MKs prematurely extending pseudopods,
as well as MK fragments, were visible in the BM from doubledeficient mice (Figure 4B). Of note, the MK ploidy was similar in
all analyzed mouse lines, indicating that lack of Rac1 and/or
Cdc42 did not affect endomitosis (Figure 4C and data not shown).
Together, these results exclude a global defect in MK
formation from their progenitors as the reason for the thrombocytopenia in Rac1/Cdc422/2 mice but indicate that the doublemutant MKs are unable to efficiently release proplatelets into the
vascular sinusoids.
Rac1/Cdc42 double-deficiency virtually abrogates
proplatelet formation
To investigate MK morphology in more detail, BM sections of wt,
Rac12/2, Cdc422/2, and Rac1/Cdc422/2 mice were analyzed
by TEM (Figure 5A). Mature wt and Rac12/2 MKs displayed
characteristic membrane invaginations formed by the demarcation
membrane system, whereas few invaginations were found in the
periphery and around the nucleus (Figure 5Ai-ii). In Cdc422/2 MKs,
membrane invaginations and the peripheral zone were partially
reduced compared with the wt (Figure 5Aiii). In marked contrast,
Rac1/Cdc422/2 MKs displayed a highly abnormal morphology
characterized by few demarcation membranes and an overall reduction
in granule numbers (Figure 5Aiv). Furthermore, peripheral zones
were virtually absent, resulting in their partial fragmentation. This
phenotype provides an explanation for the decreased visibility of
Rac1/Cdc422/2 MKs in hematoxylin and eosin–stained BM sections
and confirms our data from the CD41-stained BM cryosections
(Figure 4B).
Together, these data strongly indicate redundant functions of
Rac1 and Cdc42 in the late stages of platelet production in vivo.
We therefore investigated the outcome of Rac1 and/or Cdc42
deficiency on proplatelet formation in vitro, using BM- and fetal
liver cell (FLC)-derived MKs (Figure 5B-C). Rac12/2 MKs
formed proplatelets to a similar extent as wt platelets, whereas in
Cdc422/2 MKs, proplatelet formation was moderately, but
significantly, reduced, demonstrating that Cdc42 is of greater
relevance for platelet production than Rac1. Strikingly, proplatelet
formation was markedly reduced in FLC-derived MKs (Figure 5C)
and nearly abrogated in BM-derived MKs (Figure 5B) from Rac1/
Cdc422/2 mice.
These results were in agreement with our observations from
TEM and immunohistochemistry and demonstrated a critical
functional redundancy of Rac1 and Cdc42 in the terminal stages of
platelet production in vitro and in vivo.
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PLEINES et al
BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
Figure 6. Altered tubulin structure in Rac1/Cdc422/2
MKs. (A-B) Analysis of actin and tubulin structure by
confocal microscopy. Representative stainings for actin
(red), tubulin (green), and DAPI (blue) from at least 3
different samples per group are shown. (A) Visualization of
proplatelets from wt (upper), Rac12/2 (second), Cdc422/2
(third), and Rac1/Cdc422/2 (lower) MKs. Bar, 25 mm. (B)
Start of proplatelet formation in mature wt (upper) and
Rac1/Cdc422/2 (lower) MKs. Bar, 25 mm.
Altered microtubule structure in Rac1/Cdc422/2 MKs
To further analyze the effect of Rac1 and/or Cdc42 deficiency on
cytoskeletal rearrangements during proplatelet formation, actin and
tubulin distribution was investigated in cultured FLC-derived MKs.
In wt MKs, actin and tubulin mostly colocalized in the proplatelet
buds, and a similar organization was found in Rac12/2 and most
Cdc422/2 MKs (Figure 6A). In contrast, the tips of the few proplatelets present in double-deficient MKs were mostly devoid of the
marginal tubulin bundles (Figure 6A, bottom). Very interestingly,
further analyses demonstrated that Rac1/Cdc42 double-deficiency
severely affected tubulin organization (Figure 6B). In wt MKs at the
stage of early pseudopod formation, tubulin was mostly organized in
an accurate network (Figure 6B, upper). Early proplatelets from wt
MKs displayed a characteristic structure of homogenously distributed
actin and of organized tubulin bundles resembling bunches of grapes
(Figure 7A, upper). In contrast, in double-deficient MKs, tubulin was
organized in thick bundles, which only partially colocalized with
actin. The observation that altered tubulin organization was most
evident in very mature Rac1/Cdc422/2 MKs supports our hypothesis
that specifically, the terminal stages of platelet production are affected
by Rac1/Cdc42 double-deficiency, whereas MK maturation is still
functional in the absence of both GTPases.
To examine potential mechanisms underlying the observed
tubulin organization, we investigated the expression of different
potential activators and effectors of Rac1/Cdc42 in FLC-derived
MKs by western blot. Phosphorylation of the Rac1 and Cdc42
activating protein p70 S6 kinase21 was similar in wt and Rac1/
Cdc422/2 MKs (supplemental Figure 3). An intense regulatory
crosstalk between the 3 major Rho GTPases Rac1, Cdc42, and
RhoA has been described8; however, we found that the expression
levels of RhoA and its downstream effector mDia1 were not
significantly altered in Rac1/Cdc422/2 MKs (supplemental Figure 1). Interestingly, double-deficient MKs displayed a pronounced
increase in the amounts of the phosphorylated (inactive) form of the
actin turnover-regulating protein cofilin, which is also seen in
Cdc422/2 platelets (Figure 7B and Pleines et al12). Cofilin phosphorylation is induced downstream of Rac1 and Cdc42 via sequential
phosphorylation of PAK and LIMK. Levels of phosphorylated LIMK
were indeed significantly increased in double-deficient MKs
(P 5 .04); however, we were not able to detect phosphorylated
PAK isoforms in either wt or Rac1/Cdc422/2 MKs (Figure 7B and
data not shown).
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BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
RAC1 AND CDC42 IN THROMBOPOIESIS
3185
Figure 7. Altered tubulin structure in Rac1/Cdc42-/- MKs is associated with changes in cofilin activation. (A) Analysis of actin and tubulin structure in early proplateletforming MKs by confocal microscopy. Representative stainings for actin, tubulin, and DAPI from at least 3 different samples per group are shown. Bar, 25 mm. (B) Increased
phosphorylation of cofilin and LIMK, and reduced expression level of IQGAP1 in Rac1/Cdc422/2 MKs. Western blot analysis of FLC-derived MKs. MKs on day 3 of culture
were purified with a bovine serum albumin gradient, and protein lysates were obtained. Equal amounts of proteins were loaded, and the expression was detected with specific
antibodies and quantified by densitometry with Image J software (National Institutes of Health). Shown blots are representative of at least 3 independent samples. **P , .01;
*P , .05.
Another recently emerging effector protein of Rac1 and Cdc42 is
the scaffolding protein IQGAP1, which is implicated in the coordination of multiple cellular signaling processes leading to actin
polymerization and tubulin multimerization in cell types other than
platelets.22 Intriguingly, IQGAP1 expression was significantly
reduced in Rac1/Cdc422/2 MKs compared with in the wt MKs
(P 5 .007). Together, these results indicate potential roles for cofilin
and IQGAP1 in regulating actin and microtubule dynamics downstream of Rac1/Cdc42 during platelet production.
Discussion
To date, little is known about the detailed roles of Rho GTPases in the
regulation of cytoskeletal rearrangements during MK maturation and
platelet formation. However, the observations that deficiency of
Rac1,11 Cdc42,12 or RhoA23 has differential effects on platelet counts
indicate important roles for Rho GTPases in platelet production. Our
data now show that whereas deletion of either Rac1 or Cdc42 had no
or only a mild effect on platelet counts, respectively, combined
deletion of both Rho GTPases resulted in severe macrothrombocytopenia. The few circulating Rac1/Cdc422/2 platelets displayed a
severely abnormal morphology and multiple functional defects,
resulting in a bleeding phenotype and abrogated thrombus formation
in vivo (Figure 2E; supplemental Figure 2). The thrombocytopenia
in the double-deficient mice was associated with normal MK ploidy
(Figure 4C), suggesting that Rac1 and Cdc42 are not essentially
required to establish a polyploid nucleus during MK maturation.
Importantly, our findings indicate that overlapping Rac1- and
Cdc42-mediated signaling is crucial, specifically for the final stages
of platelet production, as loss of both Rac1 and Cdc42 virtually
abrogated proplatelet formation in vitro (Figure 5B-C). Likewise,
mature BM MKs from Rac1/Cdc422/2 mice exhibited a highly
abnormal morphology in vivo (Figure 5A). Thus, Rac1- and
Cdc42-induced signaling seems to be required not only for efficient
formation of demarcation membranes and synthesis and trafficking
of granules but also for the maintenance of the structural integrity
of mature MKs. The resulting uncontrolled fragmentation of Rac1/
Cdc422/2 MKs within the BM provides a further explanation for
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3186
PLEINES et al
the severe thrombocytopenia seen in the double-deficient mice
(Figure 4B).
Of note, our study also provides novel insights into the individual
roles of Rac1 and Cdc42 in platelet production. Whereas our results
confirm that Rac1 is dispensable for platelet production, we interestingly found that proplatelet formation of Cdc42 single-deficient
MKs was significantly reduced in vitro, and BM MKs showed
a partially altered morphology. In line with these observations,
Cdc42 also may positively regulate proplatelet formation in human
MKs.24 Together with the similarly reduced platelet life span upon
Cdc42 single- and Rac1/Cdc42 double-deficiency, this indicates that
Cdc42 represents the major regulator of platelet production and
function, whereas Rac1 fulfills supporting functions. This is reflected
by the complex phenotype of Cdc42-deficient platelets.12
Our results reveal that defective proplatelet formation in doubledeficient MKs (and to some extent also in Cdc42 single-deficient
MKs) was associated with a severely altered microtubule structure,
whereas actin distribution was not dramatically affected (Figures 6
and 7A).
To our knowledge, our results for the first time demonstrate
redundant roles of Rac1 and Cdc42 in the regulation of microtubule dynamics during platelet production. Microtubules are
crucial for the movements of granules and organelles within
proplatelets,25 as well as for final platelet sizing in the blood,26 and
mice lacking the hematopoietic-specific b tubulin isoform display
defective platelet production, reduced platelet counts, and roundish
platelet morphology.27 Thus, the defect in microtubule stabilization
provides a plausible explanation for the macrothrombocytopenia in
Rac1/Cdc422/2 platelets. Supporting our hypothesis, we observed
that tubulin was virtually absent in most proplatelet tips from doubledeficient MKs and that the marginal tubulin band was abnormally
organized in double-deficient platelets (Figures 2C, 6A, and 7A).
The exact mechanism by which Rac1 and Cdc42 regulate
microtubule dynamics in MKs remains to be investigated. Interestingly, our results reveal altered regulation/expression of 2 Rac1 and
Cdc42 downstream signaling molecules: cofilin and IQGAP1. We
found significantly increased amounts of phosphorylated (inactive)
cofilin and its upstream regulator LIMK in Rac1/Cdc422/2 MKs and
platelets (Figure 7B), a phenotype to some extent also present in
Cdc422/2 platelets.12 As Cdc42 single-deficiency alone already
significantly reduced proplatelet formation (Figure 5B-C), this
supports our assumption of the involvement of cofilin in proplatelet
formation and platelet production, potentially by regulating microtubule stabilization in MKs. It is noteworthy in this context that
cofilin-deficiency in MKs significantly impairs platelet production
while not leading to obvious defects in MK microtubule morphology
and proplatelet formation.28 However, it is difficult to directly
compare the outcome of the complete absence of cofilin with that of
altered protein activity present here.
In line with our findings in MKs, knockout studies demonstrated
that both Rac1 and Cdc42 play important roles in neurite polarity and
outgrowth, processes that are in many respects similar to proplatelet formation and are also highly dependent on microtubule
stabilization.29,30 Intriguingly, increased cofilin phosphorylation
was also observed in Cdc422/2 -deficient neurons, and cofilin
knockdown in wt neurons resulted in a phenotype similar to
Cdc42 deficiency.29
One important Rac1/Cdc42 effector mediating cofilin phosphorylation is PAK, via activation of the downstream effector
LIMK. However, whereas LIMK phosphorylation was significantly increased, we were not able to detect significant amounts of
(phosphorylated) PAK in double-deficient or wt MKs (Figure 7B
BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
and not shown). This might indicate that as observed in neurons,
increased cofilin and LIMK phosphorylation is mediated by decreased phosphatase activity, rather than increased PAK activity.29
Of note, cofilin phosphorylation can also occur by RhoA-mediated
activation of LIMK. However, levels of RhoA and its downstream
effector mDia1 were not significantly altered in double-deficient
compared with wt MKs, which is in line with observations made in
Rac1- or Cdc42-deficient neurons (supplemental Figure 3).29,30 It
therefore seems unlikely that a compensatory upregulation of RhoA
is responsible for the observed phenotype in our system.
A direct role of cofilin in modulating tubulin rearrangements has
not been described to date. Recently, however, a pharmacologic
approach demonstrated that inhibition of the cofilin phosphorylating
enzyme LIMK stabilizes microtubules.31 Moreover, the formation of
aggregates consisting of active cofilin and actin (so-called cofilin
rods) were observed in the brains of patients with Alzheimer
disease.32 Studies using mice later demonstrated that cofilin rods
might influence microtubule organization and intracellular trafficking
by affecting the redistribution and phosphorylation of microtubule
associated protein.33,34
Together, these studies strongly support the hypothesis that
cofilin may be involved in the regulation of tubulin dynamics and
stability, and thereby platelet production in MKs.
In addition to cofilin deregulation, our study interestingly reveals
reduced expression of the scaffolding protein IQGAP122 in Rac1/
Cdc422/2 MKs, for the first time indicating IQGAP1 as a regulator of
actin/microtubule dynamics during platelet production. The exact
function of IQGAP1 in these processes remains to be investigated.
However, its established function as a linker of actin and microtubule
dynamics downstream of Rac1 and Cdc42, but not RhoA,35 makes it
a promising novel candidate for the regulation of these processes
in MKs.
In summary, the results presented here demonstrate for the first
time that Rac1/Cdc42-controlled regulation of microtubule dynamics, potentially via effectors such as cofilin and IQGAP1, is critical
for the terminal stages of platelet production in vivo.
Acknowledgments
The authors thank Sylvia Hengst and Jonas Müller for excellent
technical assistance. The authors also thank the microscopy platform
of the Bioimaging Center (Rudolf Virchow Center).
This work was supported by the Deutsche Forschungsgemeinschft
(NI 556/9-1 and Sonderforschungsbereich 688 [B.N.]) and the
Rudolf Virchow Center.
Authorship
Contribution: I.P. and S.D. performed experiments, analyzed data,
and wrote the paper; D.C., A.E., I.M., and M.M. performed
experiments and analyzed data; G.K., H.S., C.G., N.D., and C.B.
provided vital new reagents and contributed to the writing of the
paper; and B.N. designed research, analyzed data and wrote the
paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Bernhard Nieswandt, Department of Experimental Biomedicine-Vascular Medicine, University Hospital
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BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18
and Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-
RAC1 AND CDC42 IN THROMBOPOIESIS
3187
Str 2, 97080 Würzburg; e-mail: bernhard.nieswandt@virchow.
uni-wuerzburg.de.
References
1. Schulze H, Shivdasani RA. Mechanisms of
thrombopoiesis. J Thromb Haemost. 2005;3(8):
1717-1724.
2. Schulze H, Korpal M, Hurov J, et al.
Characterization of the megakaryocyte
demarcation membrane system and its role in
thrombopoiesis. Blood. 2006;107(10):3868-3875.
3. Italiano JE Jr, Patel-Hett S, Hartwig JH.
Mechanics of proplatelet elaboration. J Thromb
Haemost. 2007;5(Suppl 1):18-23.
4. Junt T, Schulze H, Chen Z, et al. Dynamic
visualization of thrombopoiesis within bone
marrow. Science. 2007;317(5845):1767-1770.
14. Chen F, Ma L, Parrini MC, et al. Cdc42 is required
for PIP(2)-induced actin polymerization and early
development but not for cell viability. Curr Biol.
2000;10(13):758-765.
15. Chrostek A, Wu X, Quondamatteo F, et al.
Rac1 is crucial for hair follicle integrity but is not
essential for maintenance of the epidermis. Mol
Cell Biol. 2006;26(18):6957-6970.
16. Wu X, Quondamatteo F, Lefever T, et al. Cdc42
controls progenitor cell differentiation and betacatenin turnover in skin. Genes Dev. 2006;20(5):
571-585.
5. Patel SR, Hartwig JH, Italiano JE Jr. The
biogenesis of platelets from megakaryocyte
proplatelets. J Clin Invest. 2005;115(12):
3348-3354.
17. 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.
6. Jaffe AB, Hall A. Rho GTPases: biochemistry
and biology. Annu Rev Cell Dev Biol. 2005;
21:247-269.
18. Kühn R, Schwenk F, Aguet M, Rajewsky K.
Inducible gene targeting in mice. Science. 1995;
269(5229):1427-1429.
7. Etienne-Manneville S, Hall A. Rho GTPases in
cell biology. Nature. 2002;420(6916):629-635.
19. Yang L, Wang L, Kalfa TA, et al. Cdc42 critically
regulates the balance between myelopoiesis and
erythropoiesis. Blood. 2007;110(12):3853-3861.
8. Pedersen E, Brakebusch C. Rho GTPase function
in development: how in vivo models change our
view. Exp Cell Res. 2012;318(14):1779-1787.
9. Heasman SJ, Ridley AJ. Mammalian Rho
GTPases: new insights into their functions from
in vivo studies. Nat Rev Mol Cell Biol. 2008;9(9):
690-701.
10. 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.
11. 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.
12. Pleines I, Eckly A, Elvers M, et al. Multiple
alterations of platelet functions dominated by
increased secretion in mice lacking Cdc42 in
platelets. Blood. 2010;115(16):3364-3373.
13. Sugihara K, Nakatsuji N, Nakamura K, et al. Rac1
is required for the formation of three germ layers
during gastrulation. Oncogene. 1998;17(26):
3427-3433.
20. Grozovsky R, Hoffmeister KM, Falet H. Novel
clearance mechanisms of platelets. Curr Opin
Hematol. 2010;17(6):585-589.
21. Ip CK, Cheung AN, Ngan HY, Wong AS.
p70 S6 kinase in the control of actin cytoskeleton
dynamics and directed migration of ovarian
cancer cells. Oncogene. 2011;30(21):2420-2432.
22. Malarkannan S, Awasthi A, Rajasekaran K, et al.
IQGAP1: a regulator of intracellular spacetime
relativity. J Immunol. 2012;188(5):2057-2063.
23. Pleines I, Hagedorn I, Gupta S, et al.
Megakaryocyte-specific RhoA deficiency causes
macrothrombocytopenia and defective platelet
activation in hemostasis and thrombosis. Blood.
2012;119(4):1054-1063.
24. Bluteau D, Lordier L, Di Stefano A, Chang Y,
Raslova H, Debili N, Vainchenker W. Regulation
of megakaryocyte maturation and platelet
formation. J Thromb Haemost. 2009;7(Suppl 1):
227-234.
25. Patel SR, Richardson JL, Schulze H, et al.
Differential roles of microtubule assembly and
sliding in proplatelet formation by
megakaryocytes. Blood. 2005;106(13):
4076-4085.
26. Thon JN, Macleod H, Begonja AJ, et al.
Microtubule and cortical forces determine platelet
size during vascular platelet production. Nat
Commun. 2012;3:852.
27. Schwer HD, Lecine P, Tiwari S, Italiano JE Jr,
Hartwig JH, Shivdasani RA. A lineage-restricted
and divergent beta-tubulin isoform is essential for
the biogenesis, structure and function of blood
platelets. Curr Biol. 2001;11(8):579-586.
28. Bender M, Eckly A, Hartwig JH, et al.
ADF/n-cofilin-dependent actin turnover
determines platelet formation and sizing. Blood.
2010;116(10):1767-1775.
29. Garvalov BK, Flynn KC, Neukirchen D, et al.
Cdc42 regulates cofilin during the establishment
of neuronal polarity. J Neurosci. 2007;27(48):
13117-13129.
30. Tahirovic S, Hellal F, Neukirchen D, et al. Rac1
regulates neuronal polarization through the
WAVE complex. J Neurosci. 2010;30(20):
6930-6943.
31. Prudent R, Vassal-Stermann E, Nguyen CH, et al.
Pharmacological inhibition of LIM kinase
stabilizes microtubules and inhibits neoplastic
growth. Cancer Res. 2012;72(17):4429-4439.
32. Minamide LS, Striegl AM, Boyle JA, Meberg PJ,
Bamburg JR. Neurodegenerative stimuli induce
persistent ADF/cofilin-actin rods that disrupt distal
neurite function. Nat Cell Biol. 2000;2(9):628-636.
33. Whiteman IT, Minamide LS, Goh L, Bamburg JR,
Goldsbury C. Rapid changes in phospho-MAP/tau
epitopes during neuronal stress: cofilin-actin rods
primarily recruit microtubule binding domain
epitopes. PLoS ONE. 2011;6(6):e20878.
34. Cichon J, Sun C, Chen B, et al. Cofilin
aggregation blocks intracellular trafficking and
induces synaptic loss in hippocampal neurons.
J Biol Chem. 2012;287(6):3919-3929.
35. Bashour AM, Fullerton AT, Hart MJ, Bloom GS.
IQGAP1, a Rac- and Cdc42-binding protein,
directly binds and cross-links microfilaments.
J Cell Biol. 1997;137(7):1555-1566.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2013 122: 3178-3187
doi:10.1182/blood-2013-03-487942 originally published
online July 16, 2013
Defective tubulin organization and proplatelet formation in murine
megakaryocytes lacking Rac1 and Cdc42
Irina Pleines, Sebastian Dütting, Deya Cherpokova, Anita Eckly, Imke Meyer, Martina Morowski,
Georg Krohne, Harald Schulze, Christian Gachet, Najet Debili, Cord Brakebusch and Bernhard
Nieswandt
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