Platelet Shape Changes during Thrombus Formation: Role of Actin-Based Protrusions

Bender, Markus; Palankar, Raghavendra

doi:10.1055/a-1325-0993

Cited by 32 publications

(33 citation statements)

References 43 publications

(59 reference statements)

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“…Interestingly, the activation of PAR1 and PAR4 induced morphological changes in MEG-01 cells that were similar to the shape change observed in activated platelets. The shape change of platelets in response to various platelet activators, such as thrombin and ADP, is essential for their spreading and stable adhesion, and requires remodeling of the actin cytoskeleton, which is regulated by complex signaling pathways [ 36 ]. To confirm whether the morphological changes observed in MEG-01 cells involve actin cytoskeleton remodeling, activated cells were labeled with phalloidin for F-actin staining.…”

Section: Resultsmentioning

confidence: 99%

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

Heo

Jeon

Namkung

2022

IJMS

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Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

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Section: Resultsmentioning

confidence: 99%

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

Heo

Jeon

Namkung

2022

IJMS

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“…The next level of shape change is formation of lamellipodia and filopodia leading to increased surface of contact with other platelets and vascular wall [70]. These stages are caused by actin cytoskeleton rearrangements [70,71], potently induced by thrombin, followed by ADP, but less by collagen [71]. Platelets ability to change their shape significantly affects packing of platelets within thrombus, and results in increased platelet density and decreased porosity [72].…”

Section: Other Responsesmentioning

confidence: 99%

Platelet functional responses and signalling: the molecular relationship. Part 1: responses.

Sveshnikova

Stepanyan

Panteleev

2021

SBPR

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Blood platelets are small anucleated cells whose main function is to form a plug upon vascular damage to stop bleeding. This role involves a number of functional responses induced by different agonists and coordinated by an intricate network of signal transduction pathways. Understanding this network is vital from both basic research point of view and for the purposes of drug target identification in thrombosis and hemostasis. This review series will focus on the regulation of platelet signalling, on tracking the molecular relationship between receptor activation and functional responses, and on the networking aspects of these pathways. The present paper, first one out of two, focuses on the description of platelet functional responses and of the conditions for their triggering.

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“…Platelet activation induces a shape change from a resting, discoid to a spherical form. 23 RhoA-deficient platelets show selective defects in platelet activation and shape change after stimulation of agonists that induce in G-protein-coupled (G13, Gq) signaling pathways, which result in impaired intravital thrombosis and a mild bleeding phenotype. 15 To investigate whether absence of RhoB had a similar effect, we analyzed platelet shape change and compared to RhoA-deficient platelets by light transmission aggregometry upon stimulation with a low dose of the stable thromboxane A2 analog U46619 (Figure 2A).…”

Section: Moderately Impaired Platelet Function In Rhob -/Micementioning

confidence: 99%

Microthrombocytopenia caused by impaired microtubule stability in RhoB-deficient mice

Englert

Aurbach

Gerber

et al. 2021

Preprint

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Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are shed into the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with pronouncedly reduced levels of acetylated α-tubulin. Our findings imply that absence of this tubulin posttranslational modification results in decreased microtubule stability leading to microthrombocytopenia in RhoB-deficient mice. Our data thus points to specifically impaired microtubule - but not actin - dynamics as a general mechanism underlying the manifestation of microthrombocytopenia in vivo. We furthermore demonstrate that RhoA and RhoB have specific, non-redundant functions in the megakaryocyte lineage.KEY POINTSRhoB-deficient mice display microthrombocytopeniaRhoB has different functions in the megakaryocyte lineage than RhoA and regulates microtubule dynamics

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Platelet Shape Changes during Thrombus Formation: Role of Actin-Based Protrusions

Cited by 32 publications

References 43 publications

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

Platelet functional responses and signalling: the molecular relationship. Part 1: responses.

Microthrombocytopenia caused by impaired microtubule stability in RhoB-deficient mice

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