Published Ahead of Print on October 9, 2015, as doi:10.3324/haematol.2015.130849.
Copyright 2015 Ferrata Storti Foundation.
Cytoskeletal perturbation leads to platelet dysfunction and
thrombocytopenia in Glanzmann variants
by Loredana Bury, Emanuela Falcinelli, Davide Chiasserini, Timothy A. Springer,
Joseph E. Italiano, and Paolo Gresele
Haematologica 2015 [Epub ahead of print]
Citation: Bury L, Falcinelli E, Chiasserini D, Springer TA, Italiano JE, and Gresele P. Cytoskeletal
perturbation leads to platelet dysfunction and thrombocytopenia in Glanzmann variants.
Haematologica. 2015; 100:xxx
doi:10.3324/haematol.2015.130849
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Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in Glanzmann
variants
Loredana Bury1, Emanuela Falcinelli1, Davide Chiasserini2, Timothy A. Springer3, Joseph E.
Italiano4, and Paolo Gresele1
1
Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia,
Perugia, Italy;
2
Department of Medicine, Section of Neurology, University of Perugia, Perugia, Italy;
3
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and
Program in Cellular and Molecular Medicine, Children’s Hospital, Boston, MA, United States;
4
Hematology Division, Department of Medicine, Brigham and Women's Hospital, Vascular
Biology Program, Boston Children’s Hospital and Harvard Medical School, Boston, MA, United
States
Running title: Mechanism of platelet dysfunction in dominant GT
Corresponding Author: Paolo Gresele, MD, PhD; Department of Medicine, Division of Internal
and Cardiovascular Medicine, University of Perugia, Via E. dal Pozzo, 06126 Perugia, Italy
phone: +390755783989; fax: +390755716083; e-mail: [email protected]
Acknowledgments
This work was supported by a Telethon grant (GGP10155) to PG.
The authors thank Ildo Nicoletti and Alessandra Balduini for help on confocal microscopy, Silvia
Giannini, Teresa Corazzi, Luca Cecchetti, Anna Maria Mezzasoma, Giuseppe Guglielmini and
Viviana Appolloni for help with some experiments and discussion of results. W. Vainchenker
(Université Paris-Sud, Villejuif, France) kindly gave the GP+E-86 TPO-secreting cell line, and F.
Grignani (Department of Experimental Medicine, University of Perugia) the Phoenix cell line. We
finally thank Francesca for her kind and continuous collaboration.
1
ABSTRACT
Several patients with variant dominant forms of Glanzmann Thrombasthenia, associated with
macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to
reduced surface expression and constitutive activation of integrin αIIbβ3, have been reported. The
mechanisms leading to a bleeding phenotype of these patients have never been addressed.
Aim of the present study was to unravel the mechanism by which ITGB3 mutations causing
activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia.
Here we show, using platelets from two patients carrying the β3 del647-686 mutation and CHO
cells expressing different αIIbβ3-activating mutations, that reduced surface expression of αIIbβ3 is due
to receptor internalization. Moreover, we demonstrate that permanent triggering of αIIbβ3-mediated
outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at
the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin
that promotes actin nucleation and prevents stress fibers depolymerization, in control platelets
produced an impairment of platelet function similar to that of patients with dominant Glanzmann
Thrombasthenia variants.
del647-686β3-transduced murine megakaryocytes, generated proplatelets with a reduced number of
large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement
is the cause of macrothrombocytopenia.
These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is
the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in
dominant Glanzmann Thrombasthenia variants.
2
INTRODUCTION
Integrin αIIbβ3 (GPIIb/IIIa), the main platelet receptor, is a heterodimeric calcium-dependent cellsurface glycoprotein expressed on platelets and megakaryocytes that plays a central role in platelet
aggregation and thrombus formation1. Moreover, recent observations suggest that αIIbβ3 is also
involved in proplatelet formation2,3. Integrin αIIbβ3 function depends upon two different signal
transduction pathways: inside-out and outside-in signaling. Under resting conditions, αIIbβ3 is
expressed on platelets in a bent, inactive conformation. Upon platelet activation, inside-out
signaling causes αIIbβ3 extension and headpiece opening, leading the receptor to assume an active
conformation, to acquire the ability to bind its ligands, mainly fibrinogen, and to initiate platelet
aggregation4. On the other hand, ligand binding leads αIIbβ3 complexes to cluster thus triggering
outside-in signaling, with phosphorylation of the β3 cytoplasmic tail, activation of signaling proteins
(e.g. Src-family kinases and Focal Adhesion Kinase), and ultimately reorganization of the actin
cytoskeleton5. Cytoskeletal remodeling, with actin polymerization and depolymerization, is a finely
regulated event6, and when altered it may lead to impaired platelet function and formation, as
observed in patients with mutations of Filamin A or with the MYH9-RD7,8.
Mutations of ITGA2B and ITGB3, the genes coding for integrins αIIb and β3, generate Glanzmann
Thrombasthenia (GT), an autosomal recessive bleeding disorder characterized by absent platelet
aggregation and a normal platelet count and volume, due to quantitative or qualitative defects of
αIIbβ3. Heterozygous carriers of GT are usually asymptomatic because 50% of normal αIIbβ3 is
sufficient for platelet aggregation9, but rare autosomal dominant Glanzmann variants, with platelet
dysfunction and macrothrombocytopenia, have been associated with gain-of-function mutations of
ITGA2B or ITGB3 leading to reduced expression and constitutive activation of αIIbβ33,10.
We have previously described a hereditary Glanzmann-like platelet disorder transmitted in an
autosomal dominant way, associated with macrothrombocytopenia and a bleeding diathesis, due to
a heterozygous G>C transversion (c.2134+1G>C) of ITGB3 leading to the deletion of exon13 and
to the loss of 40 amino acids (del647-686) of integrin β311. This mutation was the first-described,
3
naturally-occurring deletion of the β-tail domain (βTD) of integrin β3, the membrane-proximal
portion of the extracellular domain of the protein. βTD contributes to maintain the bent, inactive
conformation of αIIbβ312, in fact the loss of its disulfide bonds causes constitutive activation of
αIIbβ313-15, but no information is available on its role in outside-in signaling. Recently, a Japanese
family carrying a heterozygous ITGB3 c.2134+1G>A transversion leading to the same integrin β3
deletion and a similar phenotype was reported16.
We have previously reported that del647-686 integrin β3 leads to constitutive activation of αIIbβ3 in
patient megakaryocytes3, a finding recently confirmed in transfected 293T cells16.
A few other heterozygous patients with gain-of-function mutations of ITGB3, mucocutaneous
bleeding and macrothrombocytopenia have been reported17-19 suggesting that, independently from
the
mutation,
constitutive
activation
of
αIIbβ3 leads
to
platelet
dysfunction
and
macrothrombocytopenia by a common mechanism that, however, has never been addressed.
Here we show that constitutive activation of integrin αIIbβ3 decreases surface expression of the
complex through receptor internalization and triggers permanently outside-in signaling that leads to
altered cytoskeletal reorganization that is the main effector of platelet dysfunction and
macrothrombocytopenia, and thus of bleeding, in dominant GT variants.
METHODS
Blood samples were taken from healthy volunteers and from two patients carrying the
ITGB3c.2134+1G>C mutation11. All subjects gave written informed consent in accordance with the
Declaration of Helsinki, the study was approved by the ethical committee of University of Perugia.
Construction of the expression vectors, mutagenesis and transfection
Expression vectors were obtained20 and CHO cells were transfected as described under
Supplemental Data. All experiments were performed two days after transfection.
4
Surface biotinylation, β3 immunoprecipitation and Western Blotting
Surface proteins on CHO cells and platelets were biotinylated, integrin β3 was immunoprecipitated
and analyzed by Western blotting as described under Supplemental Data.
αIIbβ3 expression and internalization and αvβ3 expression
αIIbβ3 expression on resting or activated platelets or CHO cells21 was assessed by flow cytometry22
or by Western blotting after biotinylation of membrane proteins and β3 immunoprecipitation. αvβ3
expression was assessed by flow cytometry22. Internalization of αIIbβ3 was assessed by flow
cytometry as described23. Platelet fibrinogen content was quantified by ELISA (GenWay Biotech.
San Diego, CA). For details see Supplemental Data.
Adhesion assay
CHO cells and platelets were layered on human fibrinogen. Platelets were also layered on human
Von Willebrand Factor after treatment with 0.1U/ml human α-thrombin18,24. For details see
Supplemental Data.
Protein phosphorylation
CHO cells and platelets were plated on human fibrinogen and protein phosphorylation was assessed
as described under Supplemental Data.
Clot retraction
Clot retraction was assessed with CHO cells and PRP25 as described under Supplemental Data.
Actin polymerization
Actin polymerization in CHO cells and platelets was assessed by flow cytometry as described under
Supplemental Data.
5
Analysis of cytoskeletal proteins
Cytoskeleton was extracted from platelets either resting or stimulated with thrombin as described26.
Cytoskeletal proteins were separated on an acrylamide-gel and stained with Comassie-blue26. For
details see Supplemental Data.
Mass Spectrometry
Protein digestion and peptides analysis were performed as reported27. Database searching was
performed using MASCOT v.2.2 and MyriMatch v.2.1.8728. Protein assembly for MyriMatch
analysis was made using IDPicker v.3.029. Spectral-counts were used for semiquantitative
comparison between controls and patients. For details see Supplemental Data.
Effect of the perturbation of actin polymerization on platelet function
αIIbβ3 activation, platelet aggregation30, clot retraction and spreading on fibrinogen30 were assessed
in platelets treated with Jasplakinolide to induce actin polymerization31 as described under
Supplemental Data.
Construction of retroviral vectors, retrovirus production and megakaryocyte infection
The bicistronic pMYs-IRES-GFP/αIIb, pRetroX-IRES-DsRedExpress/β3 and pRetroX-IRESDsRedExpress/β3del647-686 expression vectors were obtained, retroviruses produced and
megakaryocytes double-infected32 as described under Supplemental Data.
Megakaryocyte spreading, proplatelet formation and morphology
Spreading and proplatelet formation in murine megakaryocytes32 were evaluated by
immunofluorescence3.
Proplatelet
morphology
in
human
blood
was
analyzed
by
immunofluorescence as described3,33. For details see Supplemental Data.
6
Perturbation of actin polymerization on proplatelet formation
Human megakaryocyte cultures3 were treated with jasplakinolide (1µM) for 10min and then
cultured for 16 hours. Proplatelet formation, spreading on fibrinogen, proplatelet tips number and
diameter were assessed as described above.
Structural consequences of del647-686
To unravel the possible structural consequences of del647-686 the three-dimensional structure of
the β-TD was derived from the αVβ 3 structure (PDBID-code 4G1E) and visualized using PyMOL
(DeLano Scientific, San Carlos, CA)34.
Statistical analysis
Data are expressed as means±standard deviation. Unpaired t-test or the two-way ANOVA with the
Bonferroni post-test were applied, where appropriate, using GraphPad Prism version5.00
(GraphPad Software, San Diego, California, USA). Differences were considered significant when
p<0.05.
RESULTS
β3 mutations and αIIbβ3 activation
CHO cells transfected with normal αIIb and with four different mutant β3 subunits express on their
surface an αIIbβ3 receptor constitutively activated, i.e. able to bind PAC-1 and fibrinogen under
resting conditions (Supplementary Figure 1A and 1B), confirming previous results3,17-19.
Gain-of-function mutant β3 reduces surface expression of αIIbβ3 by enhancing its internalization
Western blotting and flow-cytometry showed decreased surface expression of β3 but a normal
amount of β3 in cell lysates, both in CHO cells expressing del647-686 integrin β3 and in
heterozygous patient platelets (Figures 1A, 1B). Confocal microscopy of integrin β3 -transfected
7
CHO cells showed that mutant β3 is localized predominantly in the cytoplasm, while normal β3 is
localized predominantly on the cell surface (Supplementary figure 2). Mutant αIIbβ3 co-localized
with concavalin A and with WGA as shown by fluorescence microscopy, confirming normal
synthesis and maturation (Supplementary figure 3). Moreover, in patient’s platelets, cytoplasmic
αIIbβ3 co-localized with P-selectin in α-granules but localized also in other cytoplasmic structures,
probably the open canalicular and dense tubular systems 35 (Supplementary figure 4).
The fraction of internalized αIIbβ3 was significantly higher in unstimulated CHO cells expressing
mutant β3 as compared with cells expressing wild type β3 (Figure 1C). Similar findings were
observed with resting patients’ platelets as compared with control platelets (Figure 1D). In
accordance, patient’s platelets contained more fibrinogen than control platelets (Figure 1E) and
mutant αIIbβ3-expressing CHO cells internalizes fibrinogen under resting conditions differently from
CHO cells expressing normal αIIbβ3 that required activation with DTT to internalize it
(Supplementary figure 1C). However, upon platelet stimulation with ADP or TRAP-6, the internal
pool of αIIbβ3 of patient’s platelets externalizes regularly showing that it is correctly recycled after
internalization (Figure 1F).
αvβ3 expression on patient platelets was comparable to control platelets (control 34.9±8.0% vs
patients 37.6±7.1%, p=ns).
Constitutively activated αIIbβ3 triggers outside-in signaling
CHO cells expressing mutant β3 spread faster on fibrinogen as compared with wild type cells
(Figure 2A). Similarly, spreading of patient’s platelets was initially faster (Figure 2B), but after 30
minutes it became defective11. Moreover, patient’s platelets spread spontaneously on VWF,
differently from control platelets that required αIIbβ3 activation to undergo full spreading18,24
(Figure 2C).
Integrin β3 Tyr773 and Tyr785, as well as Focal Adhesion Kinase (FAK), were constitutively
phosphorylated in mutant CHO cells, while they were phosphorylated only after spreading on
8
fibrinogen in control cells. Similarly, constitutive β3 and FAK phosphorylation was observed in
resting patients’ platelets but not in control platelets (Figure 3A-C).
Finally, clot retraction induced by CHO cells expressing the mutant receptor was decreased (Figure
3D) as well as clot retraction of patient’s PRP (Figure 3E).
Constitutive αIIbβ3 activation leads to impaired cytoskeletal reorganization
The content of polymerized actin (F-actin) of CHO cells expressing each of the heterozygous, β3activating mutants described so far11,17-19 together with normal αIIb was higher than that of CHO
cells expressing normal αIIbβ3, and it did not increase after stimulation with DTT (Figure 4A).
Similarly, F-actin content was significantly higher in resting patient’s platelets as compared with
control platelets. Consistently, stimulation with ADP did not significantly increase F-actin content
of patient’s platelets while it doubled it in control platelets (Figure 4B).
Cytoskeletal protein content was higher in resting patient’s platelets than in resting control platelets.
Moreover, electrophoresis showed three overexpressed bands in patient’s platelets when compared
to resting control platelets. One had a molecular weight of about 70 kDa (band A) while the other
two bands migrated at 55 kDa (bands B and C), (Figure 4C). Mass spectrometry showed that band
A was mainly composed of fibrinogen α-chain, band B of fibrinogen β-chain, and band C of a
fragment of fibrinogen γ-chain (Supplementary Table 1, Supplementary Table 2,
Supplementary figure 5).
Perturbed cytoskeletal reorganization causes platelet dysfunction
To induce actin polymerization in control platelets we used Jasplakinolide, a natural cyclic peptide
that induces actin polymerization and stabilizes actin filaments31. We first assessed that
Jasplakinolide is able to induced actin polymerization in control platelets (Supplementary Figure
6).
9
Incubation with Jasplakinolide did not affect αIIbβ3 surface expression (Figure 5A), but significantly
impaired its activation, induced by ADP (Figure 5B).
Pre-incubation with jasplakinolide impaired, dose-dependently, ADP-induced platelet aggregation
(Figure 5C), clot retraction (Figure 5D) and spreading on fibrinogen (Figure 5E). Platelets treated
with jasplakinolide (1 μM) resembled for their spreading morphology patient platelets11 (Figure
5F). These effects were not a consequence of cell death because platelets were still able to expose
P-Selectin on their surface upon stimulation with 10μM ADP (resting platelets 2.92±1.33%; ADPstimulated platelets 55.03±3.1%; JAS-treated resting platelets 4.52±1.8%; JAS-treated ADPstimulated platelets 57.21±7.9%).
Constitutively activated αIIbβ3 impairs proplatelet formation
The number of mutant-β3-transduced megakaryocytes extending proplatelets in suspension was not
different
from
wild-type
megakaryocytes
(wt
megakaryocytes=73.8±19.3%,
mutant
megakaryocytes=61.4±20.7%; n=5, p=ns). However, proplatelet number was reduced and tip
diameter was larger in mutant megakaryocytes (Figure 6A and B). Moreover, barbell-proplatelets
were significantly more asymmetrical (difference between tips: wt megakaryocytes=1±0.9 µm,
mutant megakaryocytes=2.5±1.1 µm; n=5, p<0.01). Similarly, barbell-proplatelets circulating in
peripheral blood of our patient were more asymmetrical than in normal controls (difference between
tips: controls=0.5±0.4 µm; patient=1.2±0.8 µm, n=5, p<0.05) (Figure 6C).
Mutant
β3-transduced
megakaryocytes
adhered
normally
to
fibrinogen
(wt
megakaryocytes=51.2±15.3%, mutant megakaryocytes=44.6±20.1%; n=5, p=ns) but displayed
abnormal spreading, reminiscent of what previously observed with patient megakaryocytes3
(Figure 6D). On the contrary, when megakaryocytes were plated on VWF, spreading was not
different from controls (wt megakaryocytes=47.9±19.3%, mutant megakaryocytes=50.2±22.6%;
n=5, p=ns), as already observed with patient megakaryocytes3.
10
Perturbed actin polymerization impairs proplatelet formation
The number of jasplakinolide-treated human megakaryocytes extending proplatelets in suspension
was not different from control megakaryocytes (vehicle=26.3±2.6%; jasplakinolide=26.9±9.3%,
n=3, p=ns), however proplatelet formation was abnormal, with most megakaryocytes displaying a
reduced number of proplatelet tips (vehicle=13.9±6.1 tips; jasplakinolide= 1.4±1.4 tips, n=3,
p<0.01); tips diameter was reduced (vehicle=2.9±0.9 µm; jasplakinolide= 1.7±0.8 µm, n=3, p<0.01)
(Figure 7). Adhesion to fibrinogen of jasplakinolide-treated megakaryocytes was strongly impaired
(vehicle=41.4±5.7%; jasplakinolide=4.6±0.5%, n=3, p<0.01) and the few adhering megakaryocytes
did not spread (Figure 7).
Structural consequences of del647-686
The β-TD connects the lower β-leg of integrin β3 to the transmembrane portion and consists of an
amphipathic α-helix lying across a single β-sheet with four β-strands and four disulfide bonds, and
it non-covalently associates with the calf-2 domain of αIIb (Figure 8). Residues spanning from
Lys532 through Gly690 in the lower β-leg stabilize the interactions with integrins αv and αIIb in the
bent conformation, and mutation of these residues activate αIIbβ 3 and trigger fibrinogen binding36.
Del647-686 removes the 2, 3, and 4 β-strands, which represent a large interaction interface with the
calf-2 domain in both αIIbβ 312 and αvβ334 structures, thus destroying the β-TD structure (Figure 8)
hindering the adoption of the bent conformation. The observation that, despite a 40-aminoacid
deletion, αIIbβ3 is still synthesized and expressed on the surface, confirms that β-TD is a domain
important for integrin function/activation but not for β3 synthesis or dimerization with αIIb12.
Del647-686 also removes Cys-655 and 663, partners of Cys-608 and 687 in the formation of
disulfide bonds. Whether the remaining cysteines would then disulfide-link to one another,
exchange with the two α1-helix-loop disulfides, or remain as free sulfhydryls is unknown.
However, previous site-directed mutagenesis of Cys-655 and 663 leading to the loss of disulfide
11
bonds, resulted in constitutive activation of αIIbβ313-15, suggesting that the same mechanism is
responsible for the constitutive αIIbβ 3 activation associated with del647-686.
DISCUSSION
Our results show that constitutive activation of αIIbβ3 due to gain-of-function mutations of ITGB3,
and the consequent permanent triggering of αIIbβ3-mediated outside-in signaling, induce a
perturbation of cytoskeletal remodeling that leads to platelet dysfunction and impaired proplatelet
formation. Starting from the study of platelets from two patients carrying the heterozygous ITGB3
del647-686 mutation11 we extended our observations to three additional ITGB3 gain-of-function
mutations17-19 in order to describe a general mechanism that leads to bleeding in patients with
dominant GT variants.
We show that constitutive activation of αIIbβ3 leads to permanent triggering of αIIbβ3-mediated
outside-in signaling, as shown by the phosphorylation of β3 Tyr773, Tyr785 and FAK in resting
patients’ platelets and in CHO cells expressing del647-686 β3, by faster spreading on fibrinogen and
spontaneous spreading on Von Willebrand factor of patients’ platelets, contrarily to control platelets
that require αIIbβ3 activation for spreading on Von Willebrand factor18,24.
Activation of αIIbβ3 is physiologically followed by receptor internalization, a way for limiting
platelet aggregation37,38. We show here that enhanced αIIbβ3 internalization is the common
mechanism leading to reduced surface expression of αIIbβ3 in patients with GT-like syndromes
associated with constitutive activation of αIIbβ310. In fact, we observed enhanced αIIbβ3
internalization in CHO cells expressing several different ITGB3 gain-of-function mutations17-19 as
well as in platelets of del647-686 β3 patients.
We have previously shown, by flow cytometry using a set of clones directed toward different
epitopes of αIIbβ3, that our patients’ platelets express on average 40% residual αIIbβ3 on their surface
if compared with control platelets11. We show here that in patients’ platelets the ratio between
normal and mutant β3 is maintained on the platelet surface (Figure 1A). The same is observed in
12
CHO cells transfected with both wild type and mutant β3 (heterozygous cell model) which express
an amount of αIIbβ3 approximately equal to that of CHO cells transfected with only mutant β3
(homozygous cell model) (Figure 1A and B). Therefore, it can be speculated that normal αIIbβ3 is
passively internalized along with the mutant one.
Confocal microscopy revealed that in patient’s platelets the internal pool of integrin β3 localizes in
α-granules and in other cytoplasmic compartments, probably corresponding to the open canalicular
and dense tubular systems35, similar to control platelets. Moreover, we show that upon platelet
activation mutant αIIbβ3 externalizes as well as normal αIIbβ3, and therefore is presumably correctly
recycled after internalization.
Enhanced αIIbβ3 internalization was associated with an increased platelet content of fibrinogen. The
intra-platelet fibrinogen pool is largely generated by its internalization mediated by activated
αIIbβ339,40, although internalization by inactive αIIbβ3 has also been reported41. Therefore, increased
fibrinogen content in patients’ platelets is in accordance with a constitutively activated αIIbβ3.
Given that integrin β3 is a component of the vitronectin receptor αvβ3 we measured αvβ3 expression
on patient platelets and found it to be comparable to control platelets. Therefore, our mutation
influences differently αvβ3 and αIIbβ3 expression, similar to others previously reported9.
In normal conditions, αIIbβ3-mediated outside-in signaling leads to a reorganization of the
cytoskeleton that is required for platelet aggregation, clot retraction and spreading. Cytoskeletal
actin reorganization is a finely regulated process with consecutive phases including actin
polymerization, that enhances cytoskeletal rigidity that is required for platelet shape change, and
then depolymerization, that restores the cytoskeletal plasticity required for aggregation and clot
retraction42. An alteration of actin dynamics and cytoskeletal remodeling can thus lead to impaired
platelet function7,8.
We show here that CHO cells expressing ITGB3 gain of function mutations, as well as del647-686
β3-bearing platelets, have an increased F-actin content under resting conditions and show impaired
clot retraction, suggesting that actin turnover is arrested at the stage of polymerization due to
13
permanently triggered outside-in signaling. This is in line with the earlier spreading on fibrinogen
of mutant platelets becoming defective at later time points because actin turnover is arrested at the
stage of polymerization and further cytoskeletal remodeling is not longer possible. Moreover,
patient platelets had an enhanced cytoskeletal-associated protein content as compared with control
platelets, and fibrinogen α, β and γ chains were found to be associated with the cytoskeleton.
In order to assess whether impaired cytoskeletal remodeling recapitulates the platelet features
associated with αIIbβ3-activating ITGB3 mutations we used jasplakinolide, a natural cyclic peptide
that induces actin polymerization by promoting actin nucleation and by preventing stress fibers
depolymerization31. Jasplakinolide-treated control platelets showed impaired αIIbβ3 activation,
reduced aggregation, altered spreading on fibrinogen and clot retraction, a phenotype resembling
del647-686 platelets11. Altogether, these data are compatible with a model in which αIIbβ3
constitutive activation causes the arrest of cytoskeletal remodeling at the stage of polymerization,
thus impairing platelet aggregation, clot retraction and spreading. On the other hand, reduced αIIbβ3
surface expression does not seem to contribute significantly to platelet dysfunction because
incubation of normal platelets with jasplakinolide induced the same platelet dysfunction observed in
a variant GT patient without affecting αIIbβ3 surface expression. Moreover, αIIbβ3 internalization is
not triggered by actin polymerization but depends on the constitutive activation of the receptor.
Cytoskeletal remodeling is crucial also for proplatelet formation, in fact actin polymerization leads
to the amplification of proplatelet ends, thus increasing tips number43, and regulates platelet
size35,36. Impaired actin remodeling in megakaryocytes may thus perturb platelet formation, and in
fact defects in actin-related proteins were shown to lead to thrombocytopenia and to the production
of platelets with altered dimensions44-47. We induced actin polymerization by incubating control
megakaryocytes with jasplakinolide and indeed we observed impaired proplatelet formation.
Murine megakaryocytes transduced with del647-686 human β3 and wild-type human αIIb showed
impaired proplatelet formation, with a reduced number of abnormally large proplatelet tips. These
data confirm our previous observations with peripheral blood CD34+-derived patient
14
megakaryocytes3 and prove that the 2134+1 G>C mutation is the cause of macrothrombocytopenia.
We also observed that barbell-proplatelets produced by transduced murine megakaryocytes are
asymmetrical, similar to those found in patient peripheral blood. This is therefore probably
responsible for the platelet anisocytosis observed in patients with the del647-686 mutation11.
Our observations confirm that αIIbβ3 plays a role in proplatelet formation3,18,48 and show that when
outside-in signaling is constitutively triggered, cytoskeletal reorganization is disturbed and altered
proplatelet formation and preplatelet maturation occur.
In conclusion, we show that gain-of-function mutations of ITGB3 generating constitutive activation
of αIIbβ3 lead to receptor internalization and to the arrest of cytoskeletal remodeling in platelets and
megakaryocytes that in turn generate platelet dysfunction and perturb the finely regulated process of
proplatelet formation leading to macrothrombocytopenia. Reduced platelet number and platelet
dysfunction in patients with dominant GT variants are both consequent to the cytoskeletal
perturbation induced by the constitutive αIIbβ3- mediated outside-in signaling.
15
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19
Legends to the figures
Figure 1: αIIbβ3 surface expression
A) Western blotting of biotinylated (surface) and not-biotinylated (total) integrin β3 of CHO cells
expressing wild type β3 (WT), del647-686 β3 (HOM) or both (HET) and of control and
heterozygous patients’ platelets. Biotinylated proteins were identified using HRP-conjugated avidin.
The first band is wild-type β3 and the second mutant β3. Relative quantification (arbitrary units),
CHO cells: WT=1, HOM=0.18±0.02, HET=0.24±0.09 (n=5, p<0.05 vs WT); Platelets: control=1,
Patient=0.60±0.01 (n=5, p<0.05 vs control).
Total integrin β3 expression was measured in not biotinylated CHO cell and platelet lysates, probing
membranes with a mouse anti human-β3 MoAb. The total amount of integrin β3 was comparable.
Relative quantification (arbitrary units) CHO cells: WT=1, HOM=0.89±0.06, HET=0.97±0.1 (n=5,
p=ns vs WT); Platelets: Control=1, Patients=1.2±0.2 (n=5, p=ns vs control).
B) αIIbβ3 mean fluorescence intensity in CHO cells transfected with empty vectors (mock) or
expressing wild type (WT), del647-686 (HOM) and both (HET) integrin β3 (*p<0.01 vs WT,
#p<0.01 vs mock). αIIbβ3 MFI in CHO cells is comparable with αIIbβ3 MFI in platelets (22.1±3.2 in
control platelets and 11.9±4.1 in patients’ platelets).
C) Internalized αIIbβ3 in wild type and mutant CHO cells, activated or not with DTT (*p<0.05 vs
WT resting).
D) Internalized αIIbβ3 in control and patients’ platelets, stimulated or not with ADP (*p<0.05 vs
control resting). The same experiments were conducted using TRAP-6 obtaining comparable results
(data not shown).
E) Fibrinogen content in patients’ platelets compared with controls as measured by ELISA
(*p<0.05 vs control).
F) Western Blotting of biotinylated β3 and flow cytometry of αIIbβ3 in control and patients’ platelets,
resting or stimulated with ADP or TRAP-6. Biotinylated proteins were identified using HRPconjugated avidin.
20
Figure 2: Spreading
A) Spreading of wild type (WT), mutant homozygous (HOM) and heterozygous (HET) integrin β3expressing CHO cells after 30 and 60 minutes of adhesion to fibrinogen (n=3, *p<0.05 vs WT).
Representative images of wild type (WT) and mutant heterozygous (HET) integrin β3-expressing
CHO cells after 60 minutes of adhesion to fibrinogen. The total surface covered was measured in 20
different high magnification microscopic fields. Mock-transfected CHO cells adhered to fibrinogen
but did not spread. CHO cells adhesion on fibrinogen was comparable using the different cell lines:
WT 30min: 53.4±6.8 adherent cells; WT 60min: 43.5±5.4 adherent cells; HOM 30min: 52.5±7.9
adherent cells; HOM 60min: 51.7±5.3 adherent cells; HET 30min: 47.6±5.4 adherent cells; HET
60min: 51.6±8.4 adherent cells.
B) Spreading of control (Ctrl) and patients’ (Pat) platelets after 10, 30 and 60 minutes of adhesion
to fibrinogen (n=5, *p<0.05, #p<0.01 vs control). Representative images of control and one patient
platelets after 10 minutes of deposition on fibrinogen. Control and patients’ platelet adhesion on
fibrinogen was comparable: Ctrl 10min: 9.5±2.0 adherent platelets; Ctrl 30min: 51.3±11.4 adherent
platelets; Ctrl 60min: 69.9±7.2 adherent platelets; Patients 10min: 11.5±3.1 adherent platelets;
Patients 30min: 48.7±5.3 adherent platelets; Patients 60min: 59.6±8.7 adherent platelets.
C) Representative images of control and patient platelets after 30 minutes of deposition on Von
Willebrand Factor (VWF) under resting conditions or stimulated with human α-thrombin (+Thr).
Both adhesion and spreading of patients’ platelets under resting conditions were increased respect
to control: Ctrl: 52.5±6.2 adherent platelets; 5.2±2.6 % of spreading (% of covered surface);
Patients: 116.0±9.8 adherent platelets; 55.2±10.6% of spreading (% of covered surface). After
stimulation with thrombin both adhesion and spreading of patient platelets were comparable with
those of control platelets: Ctrl Thr: 198.1±11.0 adherent platelets; 73.5±12.4 % of spreading (% of
covered surface); Patients Thr: 214.8±12.9 adherent platelets; 69.7±15.6 % of spreading (% of
covered surface). Platelets were stained with FITC-conjugated phalloidin. Every microscopic field
covers 0.14 square mm.
21
Figure 3: αIIbβ3 –triggered outside-in signaling
A) β3 Tyr773 phosphorylation of wild type (WT) and mutant (MUT) integrin β3-expressing CHO
cells and of control and patient platelets in suspension (susp) and after adhesion to fibrinogen (Fbg).
Relative quantification (arbitrary units), CHO cells: WT susp=1, WT Fbg=29.6±4.7, MUT
susp=55.3±6.8, MUT Fbg,=69.6±12.3 (n=5, p<0.05 vs WT); Platelets: control susp=1, control
Fbg=5.1±1.5, patient susp=7.1±1.8, patient Fbg=8.6±1.3 (n=5, p<0.05 vs control).
B) β3 Tyr785 phosphorylation of wild type and mutant integrin β3-expressing CHO cells and of
control and patients’ platelets in suspension and after adhesion to fibrinogen. Relative quantification
(arbitrary units), CHO cells: WT susp=1, WT Fbg=41.3±2, MUT susp=62.3±7.5, MUT
Fbg=65.7±5.5 (n=5, p<0.05 vs WT); Platelets: control susp=1, control Fbg=5.7±1.6, patients
susp=2.4±1.1, patients Fbg= 2.9±0.6 (n=5, p<0.05 vs control).
C) Focal Adhesion Kinase (FAK) phosphorylation of wild type and mutant integrin β3-expressing
CHO cells and of control and patients’ platelets in suspension and after adhesion to fibrinogen.
Relative quantification (arbitrary units), CHO cells: WT susp=1, WT Fbg=7.9±2.5, MUT
susp=5.2±0.7, MUT Fbg=5.1±0.3 (n=5, p<0.05 vs WT); Platelets: control susp=1, control
Fbg=3.9±0.8, patients susp=2.1±0.7, patients Fbg=4.5±0.9 (n=5, p<0.05 vs control).
D) Clot retraction induced by wild type (WT) and the different mutant integrin β3-expressing CHO
cells after 60 and 90 minutes of incubation at 37°C (*p<0.01 vs WT).
E) Clot retraction induced by control platelets (Ctrl), a Glanzmann Thrombasthenia platelets (GT)
and patients’ (Pat) platelets after 60 and 90 minutes of incubation at 37°C (*p<0.01 vs control).
F) Representative images of WT and the different mutant integrin β3-expressing CHO cells after 90
minutes of incubation at 37°C.
G) Representative images of control, Glanzmann Thrombasthenia and one patient platelets after 90
minutes of incubation at 37°C.
22
Figure 4: Cytoskeleton dynamics
A) Polymerized actin (F-actin) content of controls and patients’ platelets measured by flow
cytometry before and after stimulation with ADP (20 μM) (n=4, *p<0.05 vs resting, #p<0.05 vs
control).
B) Polymerized actin (F-actin) content of wild type (WT) and of different mutant β3-expressing
CHO cells measured by flow cytometry before and after stimulation with DTT (10 μM) (n=6,
*p<0.05 vs resting, #p<0.05 vs control).
C) Representative electrophoresis of cytoskeletal proteins from resting control platelets (Ctrl1,
Ctrl2), control platelets stimulated with thrombin (thr) and platelets from the two different del647686 β3 patients (Pat1, Pat2). Relative quantification (arbitrary units), Control=1; thr=2±0.8, n=3,
p<0.01; Patients=1.2±0.1 (n=3, p<0.01 vs ctrl).
Molecular weight marker on the left, identity of the main bands from MS analysis on the right. All
the proteins found in each band are listed in Supplementary Table 1.
Figure 5: Effect of jasplakinolide on platelet function
A) αIIbβ3 expression in platelets treated with jasplakinolide 5 μM (JAS) or its vehicle (CTRL) as
assessed by flow-cytometry (n=4, p=ns). Data show mean fluorescence intensity.
B) PAC-1 binding to platelets induced by ADP (10 μM) after preincubation with jasplakinolide 5
μM (JAS) or its vehicle (CTRL) (n=4, *p<0.05 vs resting, #p<0.05 vs ctrl ADP). Data show mean
fluorescence intensity.
C) ADP-induced aggregation of platelets treated with jasplakinolide 5 μM (JAS) or its vehicle
(CTRL) (n=4, *p<0.01 vs ctrl).
D) Clot retraction mediated by platelets treated with jasplakinolide 5 μM (JAS) or its vehicle
(CTRL) (n=4, *p<0.01 vs ctrl).
E) Platelet spreading on fibrinogen after treatment with 1 μM jasplakinolide. Platelets were stained
with the CD41 clone P2 mAb.
23
F) Spreading on fibrinogen of platelets treated with jasplakinolide (JAS) or its vehicle (CTRL) after
60 minutes of deposition (n=4, *p<0.01 vs control)
Figure 6: Megakaryocyte spreading and proplatelet formation
A) Number and diameter of proplatelet tips generated by murine megakaryocytes transduced with
the wild type (WT) or the mutant (MUT) integrin β3 (*p<0.01 vs WT). Only cells showing both
green and red fluorescence (i.e. expressing αIIb and β3) were analyzed.
B) Representative image of proplatelet formation by wild type- or mutant β3–expressing murine
megakaryocytes. The number of megakaryocytes expressing normal or mutant αIIbβ3 was
comparable (GFP expression in wt 20.2±1.7% vs mutant 19.6±1.3%; Ds-red expression in wt
18.6±1.2% vs mutant 17.7±1.9%; n=5, p=ns).
Megakaryocytes were stained with a rabbit anti-mouse β1-tubulin antibody; nuclei were stained
with Hoecst.
C) An asymmetric barbell-proplatelet circulating in patient peripheral blood is indicated by a white
arrow (left). Symmetric barbell-proplatelet generated by wild type β3-expressing murine
megakaryocytes compared with an asymmetric barbell-proplatelet generated by mutant β3expressing murine megakaryocytes (right). Barbell-proplatelets were stained with a rabbit antihuman β1-tubulin antibody.
D) Representative picture of wild type- or mutant β3–expressing murine megakaryocytes spreading
on fibrinogen. β1-tubulin was stained with a rabbit anti-human β1-tubulin, actin was stained by
rhodamine-phalloidin, nuclei were stained with Hoecst.
Figure 7: Effect of jasplakinolide on megakaryocyte spreading and proplatelet formation
Representative pictures of proplatelet formation (top and center) and spreading on fibrinogen
(bottom) by megakaryocytes treated with vehicle (Ctrl) or 1μM jasplakinolide (JAS).
Megakaryocytes treated with jasplakinolide display a reduced number of proplatelet tips of altered
diameter (arrowed) and impaired spreading on fibrinogen.
24
Figure 8: Structural consequences of del647-686
Cartoon diagram showing the structure of the β-tail domain, with each domain in a different color.
The deleted region in del647-686 is magenta and the remainder of β-tail domain is green. Disulfides
and Val-664 sidechain are shown as sticks, with sulfur atoms in gold. Ectodomain subunit termini
are marked with α and β. The β-tail domain secondary structure elements are variable in length in
different β3 integrin ectodomain structures, and are long in the αVβ 3 structure depicted here (PDB
ID code 4G1E)36.
25
Supplemental methods
Construction of the expression vectors and mutagenesis
Full-length α IIb and β 3 cDNAs were amplified by Pfu DNA Polymerase. α IIb was cloned into the
HindIII/NotI sites of pcDNA3.1/Hygro(+) (Invitrogen, Life Technologies, Monza, Italy) to obtain
the pcDNA3.1/αIIb expression vector and β 3 was cloned into the HindIII/XhoI sites of pcDNA3
(Invitrogen) to obtain the pcDNA3/β3 expression vector. β 3 cDNA was amplified by PCR from
patient platelets, digested with KpnI and EcoRI and exchanged for the wild-type sequence in the
pcDNA3/β3 construct. β3 S527F, L743P and D748H mutations were introduced into the pcDNA3/β3
vector by site-directed mutagenesis21.
CHO cells-culture and transfection
CHO cells (from European Collection of Cell Cultures, Salisbury, UK) were grown in IMDM
supplemented with 10% FCS, 2mM L-Glutamine, 100U/ml penicillin and 100µg/ml streptomycin.
Cells were transiently transfected with equimolar amounts of pcDNA3.1/αIIb and pcDNA3/β3,
pcDNA3/β3del647-686, pcDNA3/β3S527F, pcDNA3/β3L743P or pcDNA3/β3D748H using
Turbofect (Fermentas, Glen Burnier, MD, USA). Selected experiments were carried out using CHO
cells transfected with pcDNA3.1/αIIb, pcDNA3/β3 and pcDNA3/β3del647-686 together, to obtain a
heterozygous model, mimicking the patients’ genotype. Mock cells were obtained by transfecting
equimolar amounts of pcDNA3 and pcDNA3.1/Hygro(+) plasmids.
CHO cells αIIbβ3 activation
Cells were incubated for 30 minutes with the CD41-FITC clone P2 mAb (Immunotech, Beckman
Coulter, Milan, Italy) to assess αIIbβ3 surface expression or the PAC-1 FITC mAb (BD Biosciences,
Milan, Italy) to measure α IIbβ3 activation. To measure fibrinogen binding cells were incubated with
an anti-human-fibrinogen FITC mAb (Immunsystem AB, Uppsala, Sweden) in the presence of 4
µg/ml of human fibrinogen.
Samples were analyzed in a Cytomics FC500 flow cytometer (Coulter Corporation, Miami, Florida,
USA), equipped with an argon laser operating at 488 nm22.
Surface biotinylation, β3 immunoprecipitation and Western Blotting
CHO cells expressing normal, mutant or heterozygous α IIbβ3 and mock-transfected cells were
detached, washed and resuspended in PBS. Patient PRP was centrifuged at 1000 x g for 10 min,
platelets were washed 3 times with PBS containing 10mM EDTA and resuspended in the same
1 buffer. Samples were incubated with 1 mg/ml of Sulfo-NHS-LC-Biotin reagent (Pierce, Rockford,
IL, USA) for 30 minutes at room temperature, washed in PBS containing 100mM glycine and lysed
with lysis buffer (40 mM Tris- HCl, 0.3 M NaCl, 1 mM EDTA, 1mM NaF, 1 mM Na3VO4, 10 µl
NP-40, 10 µg/ml leupeptin/pepstatin).
Lysates were pre-cleared and β 3 was immunoprecipitated with 10 µg of a mouse anti human-β3
MoAb (Calbiochem, Merck, Darmstadt, Germany) and Protein G Sepharose beads (Invitrogen, Life
Technologies).
Immuno-precipitates
were
then
analyzed
by
SDS-polyacrylamide
gel
electrophoresis (PAGE) and transferred onto nitrocellulose membranes. Biotinylated proteins were
identified using HRP-conjugated avidin (Sigma Aldrich, Milan, Italy) and ECL chemiluminescence
(Amersham, GE Healthcare, Fairfield, CT, USA). Not-biotinylated CHO cell and platelet lysates
were used to measure total β3 expression, by probing membranes with a mouse anti human-β3
MoAb (Calbiochem) and an appropriate HRP-conjugated secondary antibody. Densitometric
analysis was performed on three different experiments using the ImageJ software (NIH, USA).
αIIbβ3 and αVβ3 expression
αIIbβ3 and αVβ3 expression were assessed by flow cytometry using the CD41-FITC clone P2 mAb
and the CD51-FITC Clone AMF7 mAb (Beckman Coulter, Milan, Italy).
For analysis of αIIbβ3 expression upon activation platelets were stimulated with 10µM ADP or 20
µM TRAP-6 and then incubated with the CD41-FITC mAb and analyzed by flow cytometry as
described above. Alternatively, membrane proteins were biotinylated, β3 was immunoprecipitated
and analyzed by SDS-PAGE as described above.
αIIbβ3 and fibrinogen internalization
Internalization of αIIbβ3 and fibrinogen was assessed by flow cytometry. Briefly, washed platelets,
wild type and mutant αIIbβ3-expressing CHO cells were incubated for 60 minutes at 37°C with the
CD41-FITC clone P2 mAb and then treated with 20µM ADP (platelets), 10mM DTT (CHO cells)
or PBS for 20 minutes at 37°C, and finally analyzed by flow cytometry. After the acquisition of the
surface mean fluorescence intensity a saturating concentration of an anti-fluorescein rabbit
polyclonal IgG (Molecular Probes) was added to quench the fluorescence of surface-bound
antibodies, and samples were immediately re-analyzed for residual CD41-FITC fluorescence. The
percentage of internalized αIIbβ3 was calculated as described24.
For fibrinogen internalization CHO cells were treated with 10mM DTT or PBS for 20 minutes at
37°C, incubated for 60 minutes at 37°C with the anti-human-fibrinogen FITC mAb in the presence
2 of 4 µg/ml of human fibrinogen and then fibrinogen internalization was analyzed as above
described.
Adhesion assay
CHO cells expressing mutant, wild type or heterozygous α IIbβ3 (3x106/ml) were resuspended in
IMDM and layered onto glass coverslips coated with 100 µg/ml of human fibrinogen (American
Diagnostica, Stamford CT, USA). May-Grunwald-Giemsa staining was performed and samples
were analyzed by optical microscopy.
Washed platelets (20x106/ml) were layered onto glass slides coated with 100 µg/ml of human
fibrinogen or with 10 µg/ml of human Von Willebrand Factor (VWF) (Haemate P, CSL Behring,
Marburg, Germany). To induce spreading on VWF, which requires activation of α IIbβ3, platelets
were stimulated with 0.1 U/ml of human α-thrombin. Adherent platelets were fixed, permeabilized,
stained with FITC-conjugated phalloidin and analyzed by fluorescence microscopy.
Spreading was assessed at 10, 30 and 60 minutes and the mean percentage of the total surface
covered was calculated by using the ImageJ software (NIH, USA).
Protein phosphorylation
CHO cells stably expressing normal or mutant α IIbβ3, mock-transfected CHO cells or washed
platelets were resuspended in IMDM and plated for 1 h at 37° C in 6-well plates pre-coated with
100 µg/ml of purified human fibrinogen or with 1% BSA. Cells were then washed twice with PBS
and lysed with lysis buffer.
Lysates were recovered, clarified and 20 µg of proteins were analyzed by SDS-PAGE and
transferred onto nitrocellulose membranes. Membranes were probed with a rabbit anti-phosphoFAK MoAb (Cell Signalling Technology, Danvers, MA), a rabbit anti-integrin β3 phosphoY773 and
a rabbit anti-integrin β3 phosphoY785 (AbCam, Cambridge, UK) and immunoreactive bands were
detected using a peroxidase-conjugated anti-rabbit IgG antibody by chemiluminescence and
measured by densitometric analysis using the ImageJ software (NIH, USA).
Clot retraction
CHO cells expressing mutant or wild type α IIbβ3 and mock-transfected cells, resuspended in 800 µl
of IMDM plus 200 µl of human platelet-poor plasma, or alternatively 200 µl of PRP plus 800 µl of
IMDM supplemented with 5mM CaCl2, were placed in tubes pretreated with SigmaCote (Sigma
Aldrich, Milan, Italy). Human α-thrombin (2 U/ml) was then added, tubes were briefly stirred and
3 incubated in a water-bath at 37° C for 30 and 60 min and were photographed at various times. The
two-dimensional sizes of retracted clots on photographs were quantified using Image J software and
clot size was measured. Clot retraction was calculated by the formula: [1-(Vc/Vt)] x100, where Vt
is the total cell suspension volume and Vc the volume occupied by the clot.
Actin polymerization
Actin polymerization was assessed by flow cytometry. Briefly, 20 µl of platelet-rich plasma (PRP)
or of a suspension of wild type or mutant α IIbβ3-expressing CHO cells were stimulated with ADP
(20µM for platelets) or DTT (10mM for CHO cells) for 15 min at 37°C, fixed with 4% PFA,
permeabilized with 0.01% Triton-X and stained with FITC-conjugated Phalloidin (Molecular
Probes, Life Technologies, Monza, Italy).
Extraction of platelet cytoskeleton and analysis of cytoskeletal proteins
Cytoskeleton was extracted from washed platelets, either resting or stimulated with thrombin (0.5
U/ml) for 5 min at 37°C under constant stirring, by the addition of an equal volume of cytoskeleton
extraction buffer 2x26. Samples were carefully mixed and placed on ice for 10 min. Triton X-100insoluble material was recovered by centrifugation at 12,000 x g for 5 min at 4°C, washed twice
with cytoskeletal buffer without Triton X-100, and resuspended in 4% SDS. An equal volume of
sample buffer (5% β -mercaptoethanol, 20% glycerol and 0.02% bromophenol blue) was then
added, samples were boiled for 5 min, and cytoskeletal proteins (20 µg) were separated on a 10%
acrylamide gel and stained with Comassie blue27. Quantitative analysis of cytoskeleton-associated
proteins was performed by densitometric scanning of stained gels by using the ImageJ software
(NIH, USA).
Mass Spectrometry
Protein digestion was performed as previously reported27. Briefly, after Coomassie staining,
selected bands were sliced from the gel, destained in 300 µL of a 100 mM NH4HCO3 in 50% ACN
solution at 37°C for 30 min, and with 25 mM NH4HCO3 in 50% ACN (3x30 min). Destained gel
bands were reduced with a solution of dithiothreitol (DTT) 10 mM in 25 mM of NH4HCO3 for 1h at
56°C and alkylated with a solution of 55 mM iodoacetamide (IAA) in 25 mM NH4HCO3 for 45 min
at room temperature in the dark. Gel bands were then dried in a Speedvac, rehydrated with a 50 mM
NH4HCO3 solution containing 400 ng of proteomic grade trypsin (Promega) and covered with 50
mM NH4HCO3 solution (pH 8) and incubated overnight at 37°C. After centrifugation, oligopeptidecontaining supernatants were recovered and concentrated to dryness. Peptides were redissolved in
4 110 µL of an aqueous solution containing 5% ACN and 0.1% formic acid and analyzed with a LCQ
Deca-XP Plus ion-trap mass spectrometer operating in data-dependent acquisition mode, as
previously reported27.
Database searching was performed using the MASCOT software version 2.2 and MyriMatch
software v. 2.1.8728. Search parameters were as follows: protein sequence database UniProt release
2014_11 (66,922 sequences); enzyme trypsin; fixed modification carbamidomethylation; variable
modification oxidation; two missed cleavages allowed; peptide tolerance ±1.2 Da; MS/MS
tolerance 0.6 Da, peptide charge +1, +2 and +3. Protein assembly for MyriMatch analysis was made
using IDPicker v 3.029 using an FDR both at protein and peptide levels of 1%. Spectral counts were
used for semiquantitative comparison between controls subjects and patients.
Effect of the perturbation of actin polymerization on platelet function
To induce actin polymerization in control platelets we used Jasplakinolide (Vinci Biochem, Vinci,
Italy), a natural cyclic peptide that induces actin polymerization and stabilizes actin filaments. To
prove actin polymerization induced by Jasplakinolide, we incubated control platelets with 5µM
Jasplakinolide, the cytoskeleton was extracted as above described, and the cytoskeletal and Tritonsoluble fractions were analyzed by Western Blotting using an anti-human actin antibody (Sigma
Aldrich), as above described.
Jasplakinolide (5µM) or its vehicle were incubated for 10 minutes with PRP and then α IIbβ3 surface
expression and ADP (10 µM)-induced α IIbβ3 activation were measured by flow-cytometry, as above
described.
Moreover, gel filtered platelets, suspended in Hepes-Tyrode buffer at 2x105/µl, were treated for 10
minutes with jasplakinolide 5 µM or its vehicle, supplemented with 0.5 mM CaCl 2 and 200 µg/ml
of human fibrinogen, and stimulated with ADP (10 µM) 31 and aggregation was followed for 10 min
by light transmission aggregometry (APACT4, Helena Bioscences, UK)32.
Finally, clot retraction and spreading on fibrinogen were evaluated as described above after pretreatment for 10 minutes with increasing concentrations of jasplakinolide (1µM, 3µM or 5µM) or its
vehicle. In this case platelets were stained with CD41-FITC clone P2 mAb because jasplakinolide
inhibits the binding of phalloidin to F-actin33.
Construction of retroviral vectors and retrovirus production
Given that the in-frame fusion of α IIb and β 3 subunits with fluorescent proteins interferes with their
function34 or surface expression (unpublished observations), we decided to use bicistronic vectors
5 that allowed the simultaneous expression of α IIb with the GFP protein and of β 3 with the Ds-Red
protein separately.
Full-length α IIb cDNA was amplified by Pfu DNA Polymerase and cloned into the EcoRI/SnaBI
sites of pMYs-IRES-GFP Bicistronic Retroviral Vector (CellBioLabs, San Diego CA, USA) to
obtain a retroviral vector expressing independently αIIb and GFP.
The full-length wild-type and del647-686 β 3 cDNA was amplified by Pfu DNA Polymerase and
cloned into the BglII/ClaI sites of pRetroX-IRES-DsRedExpress Bicistronic Retroviral Vector
(Clontech, Mountain View, CA, USA) to obtain two retroviral vectors expressing independently
wild-type or mutant β 3 and the Ds-Red protein. Retroviruses were produced by transfecting vectors
with Fugene6 (Roche Applied Science, Mannheim, Germany) in the HEK 293 Phoenix cell line
grown
in
DMEM
supplemented
with
10%
FCS,
2mM
L-Glutamine
and
100U/ml
penicillin/streptomycin. After 72h, retroviral supernatants were obtained and supplemented with 6
µg/ml polybrene (Sigma Aldrich, Milan, Italy).
Megakaryocyte infection
Mouse fetal liver cells were harvested from day 13.5 fetuses and cultured in DMEM supplemented
with 10% FCS, 2mM L-Glutamine, 100U/ml penicillin/streptomycin and 0.1 µg/mL mouse
thrombopoietin (obtained from the GP+E-86 TPO-secreting cell line) for 4 days; at day 4 of
differentiation, cultures were enriched by a single-step gradient (1.5-3% BSA), resuspended in fresh
medium and cultured for an additional 24 h33.
Mouse megakaryocyte cultures at day 3 of differentiation were double-infected with the pMYsIRES-GFP-αIIb and the pRetroX-IRES-DsRed-wt or -mutant β
3
retroviruses by spin-infection.
Transduction efficiency was assessed by flow cytometry measuring the fluorescence emitted by the
GFP and Ds-Red proteins.
Megakaryocyte spreading, proplatelet formation and morphology
Megakaryocyte cultures at day 5 were layered on a single-step gradient and allowed to lay down for
30 min to resolve intermediate stages in proplatelet maturation, as previously described35. To
evaluate spreading, megakaryocytes were plated onto glass coverslips coated with 100 µg/ml of
human fibrinogen, incubated for 4 h at 37°C in a 5% CO2 atmosphere and analyzed by
immunofluorescence microscopy, as previously described3. To evaluate proplatelet formation
megakaryocytes were cytospun on poly-L-lysine coated coverslips, all polynucleated cells
extending protrusions with terminal tips were identified as proplatelet-forming megakaryocytes and
cells showing both green and red fluorescence (i.e. expressing both α IIb and β 3) were analyzed for
6 proplatelet formation. At least 20 megakaryocytes from 5 different samples were analyzed. Tips
were counted and measured using the AxioVision4 Software (Carl Zeiss Inc., Oberkochen,
Germany).
For the assessment of proplatelet morphology in circulating human blood, patient or control PRP
was cytospun on poly-L-lysine-coated coverslips and β1-tubulin distribution was analyzed by
immunofluorescence, as described3,36. At least 20 barbell-proplatelets from 5 different samples were
analyzed. Tips were measured using the AxioVision4 Software. Barbell-proplatelet symmetry was
assessed by measuring the difference in the diameter between the two tips.
CHO cells immunofluorescence and confocal microscopy
CHO cells expressing normal or mutant α IIbβ3 were cultured overnight on poly-L-lysine coated
coverslips, fixed with 3.7% PFA, permeabilized with 0.01% Triton-X, and blocked with 1% BSA.
For fluorescence microscopy β3 integrin was stained with the CD41 clone P2 mAb followed by
labeling with the Alexa Fluor® 488 Goat Anti-Mouse IgG (Molecular Probes). Samples were the
incubated with either Tetramethylrhodamine Conjugate Concanavalin A (ER marker) or Texas
Red®-X Conjugate Wheat Germ Agglutinin (WGA, Golgi marker) (Molecular Probes). Nuclei
were stained blue with Hoecst. Specimens were mounted with the ProLong Antifade medium
(Molecular Probes) and analyzed at room temperature by a Carl Zeiss Axio Observer.A1
fluorescence microscope (Carl Zeiss Inc, Oberkochen, Germany) using a 63x/1.4 Plan-Apochromat
oil-immersion objective and acquired using the AxioVision software (Carl Zeiss Inc).
For confocal microscopy β3 integrin was stained with the CD41 clone P2 mAb followed by labeling
with the Alexa Fluor® 488 Goat Anti-Mouse IgG. Specimens were mounted with the ProLong
Antifade medium. Confocal analysis was performed at room temperature with a confocal
microscope (Bio-Rad MRC 1024) using an Ar/Kr laser. Medial focal planes are shown. Images
were elaborated on a SGI Octane work station (SGI, Mountain View, CA) with the Imaris software
(Bitplane, Zurich, CH).
Confocal microscopy for β3 localization in platelets
Washed platelets were cytospun on poly-L-lysine coated coverslips, fixed with 3.7% PFA,
permeabilized with 0.01% Triton-X, and blocked with 1% BSA. For confocal microscopy β 3
integrin was stained with a rabbit anti-human β3 antibody (Cell Signaling) followed by labeling with
the Alexa Fluor® 594 goat anti-rabbit IgG and P-selectin was stained with the mouse anti-human
CD42P mAb (Beckman Coulter) followed by labeling with the Alexa Fluor® 488 goat anti-mouse
IgG. Confocal analysis was performed using a TCS SPII confocal laser scanning microscopy
7 system equipped with a DM IRBE inverted microscope with a 40x OIL NA objective (Leica,
Bensheim, Germany). Co-localization of and P-selectin was assessed by analyzing the graphics
reporting the intensity of the fluorescence signal along the x axis for each fluorochrome on the
optical section using the LAS-X software (Leica).
8 9 10 11 12 13 14 Supplementary Figures Legends
Supplementary Figure 1: CHO cells expressing the different ITGB3 mutations constitutively
bind PAC-1 and fibrinogen
A) PAC-1 binding measured by flow-cytometry in CHO cells expressing different ITGB3
mutations.
B) Fibrinogen binding measured by flow-cytometry in CHO cells expressing different ITGB3
mutations (*p<0.05 vs WT).
C) Internalized fibrinogen measured by flow cytometry in CHO cells expressing different ITGB3
mutations (*p<0.05 vs WT resting).
Supplementary Figure 2: the mutant β3 integrin preferentially localizes in the cell cytoplasm
Immunolocalization of the wild type (WT) or the mutant (MUT) αIIbβ3 receptor in CHO cells. To
analyze surface expression (surf.) cells were not permeabilized, while to analyze the receptor
retained in the cytoplasm (cyt.) they were permeabilized with 0.1% Triton-X. Integrin β3 was
stained with the CD41 clone P2 mAb followed by labeling with the Alexa Fluor® 488 Goat AntiMouse IgG. Specimens were mounted with the ProLong Antifade medium. Confocal analysis was
performed at room temperature with a confocal microscope (Bio-Rad MRC 1024). Images were
elaborated on a SGI Octane work station (SGI, Mountain View, CA) with the Imaris software
(Bitplane, Zurich, CH).
Supplementary Figure 3: mutant β
3
integrin is correctly synthesized and undergoes
maturation.
Mutant β3 integrin (green signal) co-localizes with the Endoplasmic Reticulum (ER, red signal) and
the Golgi Apparatus (Golgi, red signal) as well as wild-type β3. Co-localization is detectable as
yellow. Integrin β3 was stained with the CD41 clone P2 mAb followed by labeling with the Alexa
Fluor® 488 Goat Anti-Mouse IgG. Samples were the incubated with either Tetramethylrhodamine
Conjugate Concanavalin A (ER marker) or Texas Red®-X Conjugate Wheat Germ Agglutinin
(WGA, Golgi marker). Nuclei were stained with Hoecst. Specimens were mounted with the
ProLong Antifade medium, analyzed at room temperature by a Carl Zeiss Axio Observer.A1
fluorescence microscope using a 63x/1.4 Plan-Apochromat oil-immersion objective and acquired
using the AxioVision software.
Supplementary Figure 4: Colocalization of the β3 integrin with α-granules
15 Immunolocalization of the β3 integrin and P-selectin in control and patient platelets. β3 integrin was
stained with a rabbit anti-human β 3 antibody (Cell Signaling) followed by labeling with the Alexa
Fluor® 594 goat anti-rabbit IgG and P-selectin was stained with the mouse anti-human CD42P mAb
(Beckman Coulter) followed by labeling with the Alexa Fluor® 488 goat anti-mouse IgG. The
graphics on the right report the intensity of the fluorescence signal along the x axis for each
fluorochrome on the optical section and were obtained using the LAS-X software (Leica).
Of note, also single green signals (α-granules) and single red signals (β3 integrin) are detectable,
indicating that β3 integrin localizes both inside and outside α-granules.
Supplementary Figure 5: protein bands selected for mass spectrometry analysis.
Selected protein bands associated with the platelet cytoskeleton from resting control platelets (Ctrl1,
Ctrl2), control platelets stimulated with thrombin (Thr) and platelets from two different patients
(Pat1, Pat2). The molecular weight of the bands is reported on the left, the name of the band on the
left. Supplementary Table 1 lists the identity of the bands.
Supplementary Figure 6: treatment of platelets with Jasplakinolide induces actin
polymerization
Western blotting of actin linked to the platelet cytoskeleton (polymerized actin) in platelets treated
with the vehicle (CTRL) or Jasplakinolide (JAS) and actin present in the Triton-soluble fraction
(not polymerized actin). Platelet cytoskeleton was extracted as described under “Material and
Methods”, proteins were separated on a 10% acrylamide gel and blotted onto nitrocellulose
membranes. Membranes were probed with a mouse anti-human actin mAb (Sigma Aldrich), and
immunoreactive bands were detected using a peroxidase-conjugated anti-rabbit IgG antibody by
chemiluminescence.
16 Supplementary Table 1: Mass spectrometry identification of selected protein bands
Gel Experiment
ban
al MW
d
(KDa)
A*
B*
C*
D*
70
55
55
40
E*
105
F*
35
Protein Name
Serum albumin
Fibrinogen alpha chain
Fermitin family
homolog 3
WD repeat-containing
protein 1
Heat shock cognate 71
kDa protein
Serum deprivationresponse protein
Fibrinogen beta chain
Serum albumin
Tubulin alpha-3C/D
chain
n°
Mascot
identifie Theoretic
Score
al MW
Uniprot ID
d
(maximu
peptide
(kDa)
m)
s
ALBU_HUMAN
292
25
71.317
FIBA_HUMAN
249
18
95.656
URP2_HUMAN
129
9
76.475
81
2
66.836
70
2
71.082
SDPR_HUMAN
58
3
47.202
FIBB_HUMAN
ALBU_HUMAN
TBA3C_HUMA
N
TBA4A_HUMA
Tubulin alpha-4A chain
N
Tubulin beta-1 chain
TBB1_HUMAN
Tubulin beta chain
TBB5_HUMAN
TBB4B_HUMA
Tubulin beta-4B chain
N
Tubulin beta-3 chain
TBB3_HUMAN
Fibrinogen beta chain
FIBB_HUMAN
Fibrinogen alpha chain FIBA_HUMAN
Tubulin beta-1 chain
TBB1_HUMAN
Fibrinogen gamma chain FIBG_HUMAN
Protein disulfidePDIA6_HUMA
isomerase A6
N
ATP synthase subunit
ATPB_HUMAN
beta
Serum albumin
ALBU_HUMAN
Actin, cytoplasmic 1
ACTB_HUMAN
POTE ankyrin domain
POTEE_HUMA
family member E
N
Fibrinogen gamma chain FIBG_HUMAN
ACTN1_HUMA
Alpha-actinin-1
N
Tropomyosin alpha-4
TPM4_HUMAN
chain
Tropomyosin alpha-3
TPM4_HUMAN
chain
270
51
11
2
56.577
71.317
46
4
50.612
83
5
50.634
337
289
19
15
50.865
50.095
277
14
50.255
194
160
140
126
116
8
7
7
4
6
50.856
56.577
95.656
50.865
52.106
113
2
48.49
96
6
56.525
89
441
2
28
71.317
42.052
343
10
122.882
111
8
52.106
45
1
103.563
89
3
28.619
79
2
32.987
WDR1_HUMA
N
HSP7C_HUMA
N
17 *The letters correspond to the different gel bands shown in Supplementary Figure 4.
Supplementary Table 2: Semiquantitative comparison of fibrinogen subunits in patients and
controls by spectral counts
Uniprot ID
Protein name
Fibrinogen alpha
chain
FIBB_HUMAN Fibrinogen beta chain
Fibrinogen gamma
FIBG_HUMAN
chain
FIBA_HUMAN
FC
Patients
CTRL1 CTRL2
vs
controls
4.5
12.3
2.4
PAT1
PAT2
1
0
2
1
3
1
13
36
0
2
1
8
Fibrinogen chains (alpha, beta, gamma) were enriched in patients carrying the β3 del647-686
mutation of ITGB3. Spectral counts relative to the whole lane of the different samples were used in
the comparison.
18