Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Apr 18;133(16):1778-1788.
doi: 10.1182/blood-2018-07-861427. Epub 2019 Jan 2.

Disrupted filamin A/αIIbβ3 interaction induces macrothrombocytopenia by increasing RhoA activity

Alessandro Donada  1   2 , Nathalie Balayn  1 , Dominika Sliwa  1 , Larissa Lordier  1 , Valentina Ceglia  1 , Francesco Baschieri  1 , Cyril Goizet  3   4 , Rémi Favier  1   5 , Lucie Tosca  6 , Gérard Tachdjian  6 , Cecile V Denis  7 , Isabelle Plo  1 , William Vainchenker  1 , Najet Debili  1 , Jean-Philippe Rosa  7 , Marijke Bryckaert  7 , Hana Raslova  1
Affiliations

Disrupted filamin A/αIIbβ3 interaction induces macrothrombocytopenia by increasing RhoA activity

Alessandro Donada et al. Blood. .

Abstract

Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/αIIbβ3 interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αIIbβ3-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/αIIbβ3 interaction.

PubMed Disclaimer

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Patient iPSCs display unstable FLNA mRNA and no FLNa. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) primer design for the identification of the expressed X chromosome. Amplification of exon 2 allows detection of both WT and mutated messenger RNA (mRNA); that of exon 43 allows detection of only the WT mRNA. (B) qRT-PCR for FLNa expression on 2 different exons for several iPSC clones (9 FLNAWT and 20 FLNAmut clones for P1; 9 FLNAWT and 10 FLNAmut clones for P2). Results are presented as mean ± standard error of the mean; unpaired Student t test with Welch’s correction was used; each point represents 1 independent experiment. (C-D) Representative immunoblots for FLNa expression on 4 iPSC clones for each patient using a C-terminus–specific antibody (C) and a N-terminal antibody (D). Lines 1, 3, 5, and 7 represent FLNAWT clones, and lines 2, 4, 6, and 8 represent FLNAmut clones. (E) Immunoblot for FLNa expression on iPSCs cultivated for 60 passages using C-terminal antibody. Line 1 represents FLNAWT clone, and lines 2 to 4 represent 3 different FLNAmut clones. ***P < .001.
Figure 2.
Figure 2.
FLNa deficiency induces a marked defect in proplatelet formation. (A) Immunofluorescence staining for the expression of FLNa (green), F-actin (red), and nucleus (blue) in iPSC-derived MKs from P1; scale bar = 30 μm. (B) Flow cytometry analysis of CD41a, CD42a, and CD42b expression: representative dot plot (i) and relative percentages (ii) (n = 10); histogram representative of mean fluorescence intensity for CD41a and CD42a (n = 5) and for CD42b (n = 3); paired Student t test (iii). (C) Proplatelet formation potential of patient iPSC-derived clones. At least 2 clones were assayed for each genotype, for each patient, at 2 different time points. Results are presented as mean ± standard error of the mean; unpaired Student t test with Welch’s correction was used; each point represents 1 independent experiment. (A-B) Experiments were performed at day 15 or 16 of MK differentiation. (C) Proplatelet formation was measured at days 18 and 19. (D) Representative pictures for WT and mutant proplatelet-forming MKs from P1. Immunofluorescence staining of F-actin (green) and β-tubulin (red) was performed after adhesion on fibrinogen for 24 hours. The nucleus is stained with 4′,6-diamidino-2-phenylindole (DAPI; blue); scale bar = 30 μm. *P < .05, **P < .01, ***P < .001.
Figure 3.
Figure 3.
Deletion of αIIbβ3and Rho GTPase FLNa interaction domains, but not of GPIbα-interacting domain, deeply affects proplatelet formation. (A) Schematic representation of FLNa mutants introduced by zinc finger nuclease–mediated gene editing. (B) Representative immunoblot for FLNa expression in the iPSC-edited clones using an N-terminus (left) and C-terminus (right) antibody. (C) Representative pictures of proximity ligation assay for FLNa/β3 and FLNa/RhoA interactions. Red staining represents the interaction between β3 and FLNa and between RhoA and FLNa. The nucleus is stained with 49,6-diamidino-2-phenylindole (blue); scale bar = 30 μm. Two independent experiments for each interaction were performed, with 50 cells analyzed in each experiment. No specific signal was detected when cells were incubated without primary antibodies (data not shown). (D) Flow cytometry analysis of CD41a and CD42a expression in the edited clones at day 15 of differentiation. Results are presented as mean ± standard error of the mean; unpaired Student t test with Welch’s correction was used; each point represents 1 independent experiment. (E) Proplatelet formation potential evaluated at day 18 of MK differentiation for each edited clone (n = 3). Paired Student t test *P < .05, **P < .01.
Figure 4.
Figure 4.
RhoA is overactivated in FLNa-deficient MKs. (A) Immunofluorescence staining of G-actin (red) and F-actin (green) after adhesion on fibrinogen. The nucleus is stained with 4′,6-diamidino-2-phenylindole (blue); scale bar = 30 μm. (B) Quantification of the percentage of stress fiber–forming MKs for each edited clone. Results are presented as mean ± standard error of the mean; each point represents 1 independent experiment. Data were analyzed by performing 1-way analysis of variance (ANOVA) followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). (C) FRET analysis for RhoA activation on different substrates: representative image, scale bar = 10 μm (i) and adhesion on fibrinogen (n = 4), collagen 1 (n = 3), fibronectin (n = 6), VWF (n = 3), and vitronectin (n = 5) (ii). (D) FRET analysis of RhoA activation on fibrinogen of all edited clones: WT and mutant clones (n = 8), del1 and del4 clones (n = 6), and del2 and del3 clones (n = 5). (C-D) At least 15 cells per condition were analyzed. Data were analyzed by performing ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method); all experiments were performed at day 15 or 16 of MK differentiation. *P < .05, **P < .01, ***P < .001.
Figure 5.
Figure 5.
Proplatelet development and stress fiber formation are rescued after inhibition of RhoA pathway. (A) Stress fiber formation assessed at day 15 or 16 of MK differentiation in presence and absence of ROCK1/2 inhibitor Y-27632 for the edited clones: WT and del3 (n = 5), mutant (n = 6), del1 and del2 (n = 3), and del4 (n = 4); paired Student t test. (B) Proplatelet formation potential in presence and absence of ROCK1/2 inhibitor Y-27632 for both patients’ iPSC clones (n = 3); paired Student t test. (C) Proplatelet formation potential in presence and absence of ROCK1/2 inhibitor Y-27632 in all edited clones: WT (n = 6), mutant (n = 8), mutant + WT, del1, del3, and del4 (n = 4), and del2 (n = 3); paired Student t test. The ROCK1 inhibitor was added at day 15 to culture, and proplatelet formation was measured at day 18. *P < .05, **P < .01, ***P < .001.
Figure 6.
Figure 6.
Pathological mechanism for FLNa-deficient MKs. (A) In the presence of FLNa, the interaction between fibrinogen and its receptor αIIbβ3 does not trigger RhoA pathway activation. No anomalies in proplatelet formation could be observed. (B) In the absence of FLNa, the interaction between fibrinogen and αIIbβ3 leads to an increase in RhoA activity. Consequently, the normal actomyosin contractility is disrupted via ROCK1/2 activity, and this leads to deeply flawed proplatelet formation. This increased RhoA activity in the absence of FLNa is specifically dependent on fibrinogen and absent in the presence of other extracellular matrices like fibronectin, vitronectin, collagen 1, or VWF.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zhou AX, Hartwig JH, Akyürek LM. Filamins in cell signaling, transcription and organ development. Trends Cell Biol. 2010;20(2):113-123. - PubMed
    1. Feng Y, Walsh CA. The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. Nat Cell Biol. 2004;6(11):1034-1038. - PubMed
    1. Parrini E, Ramazzotti A, Dobyns WB, et al. . Periventricular heterotopia: phenotypic heterogeneity and correlation with filamin A mutations. Brain. 2006;129(Pt 7):1892-1906. - PubMed
    1. Falet H, Pollitt AY, Begonja AJ, et al. . A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function. J Exp Med. 2010;207(9):1967-1979. - PMC - PubMed
    1. Jurak Begonja A, Hoffmeister KM, Hartwig JH, Falet H. FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly. Blood. 2011;118(8):2285-2295. - PMC - PubMed

Publication types