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Sample records for platelet actin-cytoskeleton structures

  1. Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

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    Irina V. Gorudko

    2013-07-01

    Myeloperoxidase (MPO is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist. In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE. Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

  2. Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton.

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    Casati, Lavinia; Celotti, Fabio; Negri-Cesi, Paola; Sacchi, Maria Cristina; Castano, Paolo; Colciago, Alessandra

    2014-01-01

    Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30-150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.

  3. Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbβ3-actin cytoskeleton complex in platelets.

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    Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

    2016-09-01

    Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

  4. The Role of Actin Cytoskeleton in Memory Formation in Amygdala

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    Raphael eLamprecht

    2016-03-01

    Full Text Available The central, lateral and basolateral amygdala nuclei are essential for the formation of long-term memories including emotional and drug-related memories. The study of cellular and molecular mechanisms underpinning memory in amygdala may shed light on the formation of memory and on fear and addiction-related disorders. A challenge is to identify molecules activated by learning that subserve cellular changes needed for memory formation and maintenance in amygdala. Recent studies show that activation of synaptic receptors during fear and drug-related learning leads to alteration in actin cytoskeleton dynamics and structure in amygdala. Such changes in actin cytoskeleton in amygdala are essential for fear and drug-related memories formation. Moreover, the actin cytoskeleton subserves, after learning, changes in neuronal morphogenesis and glutamate receptors trafficking in amygdala. These cellular events are involved in fear and drug-related memories formation. Actin polymerization is also needed for the maintenance of drug-associated memories in amygdala. Thus, the actin cytoskeleton is a key mediator between receptor activation during learning and cellular changes subserving long-term memory in amygdala. The actin cytoskeleton may serve as a target for pharmacological treatment of fear memory associated with fear and anxiety disorders and drug addiction to prevent the debilitating consequences of these diseases.

  5. Imaging the fine-scale structure of the cellular actin cytoskeleton by Single Particle Tracking and Atomic Force Microscopy

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    Mustata, Gina-Mirela

    It has been proposed that diffusion in the plasma membrane of eukaryotic cells it is compartmentalized due to the interaction with the underlying actin-based membrane skeleton that comes into close proximity to the lipid bilayer. The cytoskeleton is a dynamic structure that maintains cell shape, enables cell motion, and plays important roles in both intra-cellular transport and cellular division. We show here the evidence of plasma membrane compartmentalization using Single Particle Tracking (SPT) and Atomic Force Microscopy (AFM) imaging. SPT of Quantum dot labeled lipid in the plasma membrane of live normal rat kidney cells show compartments ranging from 325 nm to 391 nm depending on the sampling time. Using AFM imaging of live NRK cell in the presence of phalloidin, the membrane compartmentalization it is visible with the average size of the compartments of 325 +/- 10 nm (the main peak is centered at 260 nm). Further, the underlying membrane skeleton in fixed cells was directly imaged after partial removal of the plasma membrane to reveal size of the membrane skeleton meshwork of 339 +/- 10 nm. A new method of measuring the characteristics of the actin meshwork was proposed. Probing the local compliance of the plasma membrane through the deflection of a soft AFM cantilever we can expect that the stiffness of the membrane will be higher at locations directly above a cortical actin. This new method provided information about the structure of the skeletal meshwork of neuronal cell body predicting an average compartment size of about 132 nm. This was confirmed through SPT of QD-lipid incorporated into the neuronal cell membrane.

  6. Multiscale modeling and mechanics of filamentous actin cytoskeleton.

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    Yamaoka, Hidetaka; Matsushita, Shinji; Shimada, Yoshitaka; Adachi, Taiji

    2012-03-01

    The adaptive structure and functional changes of the actin cytoskeleton are induced by its mechanical behavior at various temporal and spatial scales. In particular, the mechanical behaviors at different scales play important roles in the mechanical functions of various cells, and these multiscale phenomena require clarification. To establish a milestone toward achieving multiscale modeling and simulation, this paper reviews mathematical analyses and simulation methods applied to the mechanics of the filamentous actin cytoskeleton. The actin cytoskeleton demonstrates characteristic behaviors at every temporal and spatial scale, and mathematical models and simulation methods can be applied to each level of actin cytoskeletal structure ranging from the molecular to the network level. This paper considers studies on mathematical models and simulation methods based on the molecular dynamics, coarse-graining, and continuum dynamics approaches. Every temporal and spatial scale of actin cytoskeletal structure is considered, and it is expected that discrete and continuum dynamics ranging from functional expression at the molecular level to macroscopic functional expression at the whole cell level will be developed and applied to multiscale modeling and simulation.

  7. Formins: Bringing new insights to the organization of actin cytoskeleton

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    GUO Chunqing; REN Haiyun

    2006-01-01

    The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, functions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.

  8. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

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    Fernando Navarro-Garcia

    2013-01-01

    Full Text Available The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

  9. Interconnection between actin cytoskeleton and plant defense signaling.

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    Janda, Martin; Matoušková, Jindřiška; Burketová, Lenka; Valentová, Olga

    2014-01-01

    Actin cytoskeleton is the fundamental structural component of eukaryotic cells. It has a role in numerous elementary cellular processes such as reproduction, development and also in response to abiotic and biotic stimuli. Remarkably, the role of actin cytoskeleton in plant response to pathogens is getting to be under magnifying glass. Based on microscopic studies, most of the data showed, that actin plays an important role in formation of physiological barrier in the site of infection. Actin dynamics is involved in the transport of antimicrobial compounds and cell wall fortifying components (e.g. callose) to the site of infection. Also the role in PTI (pathogen triggered immunity) and ETI (effector triggered immunity) was recently indicated. On the other hand much less is known about the transcriptome reprogramming upon changes in actin dynamics. Our recently published results showed that drugs inhibiting actin polymerization (latrunculin B, cytochalasin E) cause the induction of genes which are involved in salicylic acid (SA) signaling pathway. In this addendum we would like to highlight in more details current state of knowledge concerning the involvement of actin dynamics in plant defense signaling.

  10. Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

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    Roberts, Brett J.; Pashaj, Anjeza; Johnson, Keith R.; Wahl, James K.

    2011-01-01

    Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration. PMID:21945137

  11. Yeast studies reveal moonlighting functions of the ancient actin cytoskeleton

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    Sattlegger, Evelyn; Chernova, Tatiana A.; Gogoi, Neeku M.; Pillai, Indu V.; Chernoff, Yury O.; Munn, Alan L.

    2014-01-01

    Classic functions of the actin cytoskeleton include control of cell size and shape and the internal organisation of cells. These functions are manifest in cellular processes of fundamental importance throughout biology such as the generation of cell polarity, cell migration, cell adhesion and cell division. However, studies in the unicellular model eukaryote Saccharomyces cerevisiae (Baker's yeast) are giving insights into other functions in which the actin cytoskeleton plays a critical role. These include endocytosis, control of protein translation and determination of protein 3-dimensional shape (especially conversion of normal cellular proteins into prions). Here we present a concise overview of these new "moonlighting" roles for the actin cytoskeleton and how some of these roles might lie at the heart of important molecular switches. This is an exciting time for researchers interested in the actin cytoskeleton. We show here how studies of actin are leading us into many new and exciting realms at the interface of genetics, biochemistry and cell biology. While many of the pioneering studies have been conducted using yeast, the conservation of the actin cytoskeleton and its component proteins throughout eukaryotes suggests that these new roles for the actin cytoskeleton may not be restricted to yeast cells but rather may reflect new roles for the actin cytoskeleton of all eukaryotes. PMID:25138357

  12. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

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    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  13. Visualization of endothelial actin cytoskeleton in the mouse retina.

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    Alessia Fraccaroli

    Full Text Available Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs, orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs, enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.

  14. Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

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    López-Contreras, L; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Montaño, Sarita; Chávez-Munguía, B; Sánchez-Ramírez, B; Talamás-Rohana, P

    2013-01-01

    Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  15. Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

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    L. López-Contreras

    2013-01-01

    Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  16. Profilin as a regulator of the membrane-actin cytoskeleton interface in plant cells

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    Tiantian eSun

    2013-12-01

    Full Text Available Membrane structures and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell. Recently, the molecular mechanisms operating at this interface have been progressively addressed. Many experiments have revealed that the actin cytoskeleton can interact with membranes through various discrete membrane domains. The actin-binding protein, profilin has been proven to inhibit actin polymerization and to promote F-actin elongation. This is dependent on many factors, such as the profilin/G-actin ratio and the ionic environment of the cell. Additionally, profilin has specific domains that interact with phosphoinositides and poly-L-proline rich proteins; theoretically, this gives profilin the opportunity to interact with membranes, and a large number of experiments have confirmed this possibility. In this article, we summarize recent findings in plant cells, and discuss the evidence of the connections among actin cytoskeleton, profilin and biomembranes through direct or indirect relationships.

  17. How cellular membrane properties are affected by the actin cytoskeleton.

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    Lemière, J; Valentino, F; Campillo, C; Sykes, C

    2016-11-01

    Lipid membranes define the boundaries of living cells and intracellular compartments. The dynamic remodelling of these membranes by the cytoskeleton, a very dynamic structure made of active biopolymers, is crucial in many biological processes such as motility or division. In this review, we present some aspects of cellular membranes and how they are affected by the presence of the actin cytoskeleton. We show that, in parallel with the direct study of membranes and cytoskeleton in vivo, biomimetic in vitro systems allow reconstitution of biological processes in a controlled environment. In particular, we show that liposomes, or giant unilamellar vesicles, encapsulating a reconstituted actin network polymerizing at their membrane are suitable models of living cells and can be used to decipher the relative contributions of membrane and actin on the mechanical properties of the cellular interface. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. The actin Cytoskeleton in Root Hairs: a cell elongation device

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    Ketelaar, T.; Emons, A.M.C.

    2009-01-01

    The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the

  19. Dynamics and Regulation of Actin Cytoskeleton in Plant Cells

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    Ren Haiyun

    2007-01-01

    @@ The actin cytoskeleton constituted of globular actin (G-actin) is a ubiquitous component of eukaryotic cells and plays crucial roles in diverse physiological processes in plant cells, such as cytoplasmic streaming, organelle and nucleus positioning, cell morphogenesis, cell division, tip growth, etc.

  20. The actin Cytoskeleton in Root Hairs: a cell elongation device

    NARCIS (Netherlands)

    Ketelaar, T.; Emons, A.M.C.

    2009-01-01

    The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the

  1. Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes.

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    Nicchia, Grazia Paola; Rossi, Andrea; Mola, Maria Grazia; Procino, Giuseppe; Frigeri, Antonio; Svelto, Maria

    2008-12-01

    Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4. Copyright 2008 Wiley-Liss, Inc.

  2. Regulation of actin cytoskeleton architecture by Eps8 and Abi1

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    Miller Jeffrey R

    2005-10-01

    Full Text Available Abstract Background The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. Results Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. Conclusion Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.

  3. Fractal dimension as a measure of altered actin cytoskeleton in MC3T3-E1 cells under simulated microgravity using 3-D/2-D clinostats.

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    Qian, A R; Li, D; Han, J; Gao, X; Di, S M; Zhang, W; Hu, L F; Shang, Peng

    2012-05-01

    Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

  4. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

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    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; Bryce, Nicole S; Whan, Renee M; Hardeman, Edna C; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  5. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

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    Lila, T; Drubin, D G

    1997-02-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.

  6. The actin cytoskeleton may control the polar distribution of an auxin transport protein

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    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  7. Molecular architecture of synaptic actin cytoskeleton in hippocampal neurons reveals a mechanism of dendritic spine morphogenesis.

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    Korobova, Farida; Svitkina, Tatyana

    2010-01-01

    Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.

  8. Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophila tracheal morphogenesis.

    Science.gov (United States)

    Okenve-Ramos, Pilar; Llimargas, Marta

    2014-02-01

    A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.

  9. Actin cytoskeleton-dependent pathways for ADMA-induced NF-κB activation and TGF-β high expression in human renal glomerular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Liyan Wang; Dongliang Zhang; Junfang Zheng; Yiduo Feng; Yu Zhang; Wenhu Liu

    2012-01-01

    Asymmetric dimethylarginine (ADMA),an endogenous nitric oxide synthase inhibitor,is considered to be an independent risk factor in the progression of chronic kidney diseases (CKD).It can induce kidney fibrosis by increasing transforming growth factor (TGF)-β1 expression,but its molecular mechanism is unclear.The aim of the present study was to investigate the role of actin cytoskeleton in ADMA-induced TGF-β1 high expression in human renal glomerular endothelial cells (HRGECs).The structure of stress fibers was visualized by immunofluorescence,nuclear factor-κB (NF-κB) DNA-binding activity was assessed by an electrophoretic mobility shift assay and TGF-β1 expression was assessed by western blot analysis.Results showed that ADMA induced the assembly of stress fibers,DNA binding of NF-κB,and increasing expression of TGF-β1.When the dynamics of actin cytoskeleton was perturbed by the actin-depolymerizing agent cytochalasin D and the actin-stabilizing agent jasplakinolide,or ablation of stress fiber bundles by the nicotineamide adenine dinucleotide phosphate oxidase inhibitor apocynin and p38 mitogen-activated protein kinase inhibitor SB203580,ADMA-induced DNA binding of NF-κB and TGF-β1 expression were inhibited.These results revealed an actin cytoskeleton-dependent mechanism in ADMA-induced NF-κB activation and TGF-β1 high expression in HRGECs.The specific targeting of the actin cytoskeleton may be a useful strategy to prevent ADMA-activated kidney fibrosis in CKD.

  10. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-12-01

    Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

  11. Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

    Institute of Scientific and Technical Information of China (English)

    XU Xia; Zl Huijun; SUN Yina; REN Haiyun

    2004-01-01

    The formation of the polarity of pollen protoplast and the dynamics of actin cytoskeleton were observed by non-fixation, Alexa-Phalloidin probing and confocal laser scanning microscopy. Our results showed that the protoplast obtained from stored pollen contained numerous crystalline fusiform bodies to constitute a storage form of actin. When dormant pollen was hydrated, the actin cytoskeleton forms a fine network spreading uniformly in the protoplast. In the process of polarity formation and germination of pollen protoplast, actin filaments marshaled slowly to the brim, and then formed multilayer continuous actin filament bundles surrounding the cortical of the protoplast. When the protoplast was exposed to actin filament-disrupting drugs, such as Latrunculin A and Cytochalasin D, continuously arranged actin bundles were disturbed and in this condition, the protoplast could not germinate. But when exposed to actin filament stabiling drug-phalliodin, the dynamics of actin filaments in the protoplasts behaved normally and the protoplasts could germinate normally. These results were also confirmed by the pharmacology experiments on pollen grains. And when Latrunculin A or Cytochalasin D was washed off, the ratio of pollen germination was resumed partly. All the results above show that the dynamic organization of the actin cytoskeleton are critical in the cell polarity formation and germination of pollen protoplast, and that the reorganization of actin cytoskeleton is mainly due to the rearrangement of actin filament arrays.

  12. Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans.

    Science.gov (United States)

    Ketelaar, Tijs; Meijer, Harold J G; Spiekerman, Marjolein; Weide, Rob; Govers, Francine

    2012-12-01

    The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin

  13. Downregulation of tumorogenicity and changes in the actin cytoskeleton of murine hepatoma after irradiation with polychromatic visible and IR light.

    Science.gov (United States)

    Knyazev, Nickolay A; Samoilova, Kira A; Abrahamse, Heidi; Filatova, Natalia A

    2015-04-01

    This study evaluated the function and structural consequences of direct exposure of murine hepatoma MH-22a cells to polychromatic polarized light, to determine potential risk of malignancy following irradiation. Visible (VIS) and infrared (IR) light have been actively used for prevention and treatment of complications developed after conventional tumor therapy. However, the safety associated with this irradiation has not been determined. Polychromatic light (480-3400 and 385-750 nm), were used at different doses (4.8-38.4 J/cm(2)) to determine the viability, proliferation, and actin cytoskeleton in vitro by flow cytometry and confocal microscopy. Tumorogenic properties of cells were studied in vivo after transplantation in C3HA mice. Polychromatic light of a wide range of doses did not change the viability and proliferation of cells. After transplantation of cells irradiated with VIS-IR light (4.8 and 9.6 J/cm(2)) and VIS light (38.4 J/cm(2)) the tumor volume was lower in the treated group than in the control group in vivo. Transplantability of the irradiated cells also decreased, whereas survival of tumor-bearing mice increased. Three cell populations with different cytoskeleton structure were identified. After irradiation, the reorganized part of the actin cytoskeleton changed its localization to the submembranous area. A decrease of tumorigenicity in cells irradiated with polychromatic light used in non-damaging doses correlated with an increase in the number of cells with reorganized actin in the submembranous area. The results of the present study argue in favor of the oncological safety of polychromatic VIS-IR light (480-3400 nm).

  14. Change in the actin cytoskeleton during seismonastic movement of Mimosa pudica.

    Science.gov (United States)

    Kanzawa, Nobuyuki; Hoshino, Yoshinori; Chiba, Makiko; Hoshino, Daisuke; Kobayashi, Hidetaka; Kamasawa, Naomi; Kishi, Yoshiro; Osumi, Masako; Sameshima, Masazumi; Tsuchiya, Takahide

    2006-04-01

    The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement.

  15. Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Chalmel Frédéric

    2007-08-01

    Full Text Available Abstract Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our

  16. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    OpenAIRE

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of gi...

  17. Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells.

    Science.gov (United States)

    Efremov, Yu M; Dokrunova, A A; Efremenko, A V; Kirpichnikov, M P; Shaitan, K V; Sokolova, O S

    2015-11-01

    The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding protein abundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanical properties, which are important for many processes, including invasion and metastasis. Here, we studied the effects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632 and detachment from the surface by trypsinization on the mechanical properties of normal Vero and prostate cancer cell line DU145. The Young's modulus of Vero cells was 1300±900 Pa, while the prostate cancer cell line DU145 exhibited significantly lower Young's moduli (600±400 Pa). The Young's moduli exhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverse viscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant to specific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from the substrate. This article is part of a Special Issue entitled: Mechanobiology.

  18. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging.

    Science.gov (United States)

    Tormos, Ana M; Rius-Pérez, Sergio; Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M; Nebreda, Ángel R; Sastre, Juan; Taléns-Visconti, Raquel

    2017-01-01

    Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. p38α MAPK is essential for actin dynamics with age in hepatocytes.

  19. Actin cytoskeleton contributes to the elastic modulus of embryonic tendon during early development.

    Science.gov (United States)

    Schiele, Nathan R; von Flotow, Friedrich; Tochka, Zachary L; Hockaday, Laura A; Marturano, Joseph E; Thibodeau, Jeffrey J; Kuo, Catherine K

    2015-06-01

    Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  20. The Hippo pathway polarizes the actin cytoskeleton during collective migration of Drosophila border cells.

    Science.gov (United States)

    Lucas, Eliana P; Khanal, Ichha; Gaspar, Pedro; Fletcher, Georgina C; Polesello, Cedric; Tapon, Nicolas; Thompson, Barry J

    2013-06-10

    Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.

  1. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging

    Science.gov (United States)

    Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M.; Nebreda, Ángel R.; Sastre, Juan

    2017-01-01

    Background Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Methods Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. Results We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. Conclusions p38α MAPK is essential for actin dynamics with age in hepatocytes. PMID:28166285

  2. F-actin cytoskeleton and the fate of organelles in chromaffin cells.

    Science.gov (United States)

    Villanueva, José; Gimenez-Molina, Yolanda; Viniegra, Salvador; Gutiérrez, Luis M

    2016-06-01

    In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss

  3. Regulation of the actin cytoskeleton by PIP2 in cytokinesis.

    Science.gov (United States)

    Logan, Michael R; Mandato, Craig A

    2006-06-01

    Cytokinesis is a sequential process that occurs in three phases: assembly of the cytokinetic apparatus, furrow progression and fission (abscission) of the newly formed daughter cells. The ingression of the cleavage furrow is dependent on the constriction of an equatorial actomyosin ring in many cell types. Recent studies have demonstrated that this structure is highly dynamic and undergoes active polymerization and depolymerization throughout the furrowing process. Despite much progress in the identification of contractile ring components, little is known regarding the mechanism of its assembly and structural rearrangements. PIP2 (phosphatidylinositol 4,5-bisphosphate) is a critical regulator of actin dynamics and plays an essential role in cell motility and adhesion. Recent studies have indicated that an elevation of PIP2 at the cleavage furrow is a critical event for furrow stability. In this review we discuss the role of PIP2-mediated signalling in the structural maintenance of the contractile ring and furrow progression. In addition, we address the role of other phosphoinositides, PI(4)P (phosphatidylinositol 4-phosphate) and PIP3 (phosphatidylinositol 3,4,5-triphosphate) in these processes.

  4. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Dov, Nadav [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel); Korenstein, Rafi, E-mail: korens@post.tau.ac.il [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel)

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  5. The Actin Cytoskeleton in SMA and ALS: How Does It Contribute to Motoneuron Degeneration?

    Science.gov (United States)

    Hensel, Niko; Claus, Peter

    2017-04-01

    Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are neurodegenerative diseases with overlapping clinical phenotypes based on impaired motoneuron function. However, the pathomechanisms of both diseases are largely unknown, and it is still unclear whether they converge on the molecular level. SMA is a monogenic disease caused by low levels of functional Survival of Motoneuron (SMN) protein, whereas ALS involves multiple genes as well as environmental factors. Recent evidence argues for involvement of actin regulation as a causative and dysregulated process in both diseases. ALS-causing mutations in the actin-binding protein profilin-1 as well as the ability of the SMN protein to directly bind to profilins argue in favor of a common molecular mechanism involving the actin cytoskeleton. Profilins are major regulators of actin-dynamics being involved in multiple neuronal motility and transport processes as well as modulation of synaptic functions that are impaired in models of both motoneuron diseases. In this article, we review the current literature in SMA and ALS research with a focus on the actin cytoskeleton. We propose a common molecular mechanism that explains the degeneration of motoneurons for SMA and some cases of ALS.

  6. Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Extracellular calmodulin(CaM)plays significant roles in many physiological processes,but little is known about its mechanism of regulating stomatal movements.In this paper,whether CaM exists in the guard cell walls of Arabidopsis and whether depolymerization of actin cytoskeleton is involved in extracellular CaM-induced stomatal closing are investigated.It is found that CaM exists in guard cell walls of Arabidopsis,and its molecular weight is about 17 kD.Bioassay using CaM antagonists W7-agarose and anti-CaM serum shows that the endogenous extracellular CaM promotes stomatal closure and delays stomatal opening.The long radial actin filaments in guard cells undergo disruption in a time-dependent manner during exogenous CaM-induced stomatal closing.Pharmacological experiments show that depolymerization of actin cytoskeleton enhances the effect of exogenous CaM-induced stomatal closing and polymerization reduces the effect.We also find that exogenous CaM triggers an increase in [Ca2+]cyt of guard cells.If [Ca2+]cyt increase is blocked with EGTA,exogenous CaM-induced stomatal closure is inhibited.These results indicate that extracellular CaM causes elevation of [Ca2+]cyt in guard cells,subsequently resulting in disruption of actin filaments and finally leading to guard cells closure.

  7. MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

    Science.gov (United States)

    Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M

    2006-10-19

    Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (PMRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (PMRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (PMRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

  8. Cellular chirality arising from the self-organization of the actin cytoskeleton.

    Science.gov (United States)

    Tee, Yee Han; Shemesh, Tom; Thiagarajan, Visalatchi; Hariadi, Rizal Fajar; Anderson, Karen L; Page, Christopher; Volkmann, Niels; Hanein, Dorit; Sivaramakrishnan, Sivaraj; Kozlov, Michael M; Bershadsky, Alexander D

    2015-04-01

    Cellular mechanisms underlying the development of left-right asymmetry in tissues and embryos remain obscure. Here, the development of a chiral pattern of actomyosin was revealed by studying actin cytoskeleton self-organization in cells with isotropic circular shape. A radially symmetrical system of actin bundles consisting of α-actinin-enriched radial fibres (RFs) and myosin-IIA-enriched transverse fibres (TFs) evolved spontaneously into the chiral system as a result of the unidirectional tilting of all RFs, which was accompanied by a tangential shift in the retrograde movement of TFs. We showed that myosin-IIA-dependent contractile stresses within TFs drive their movement along RFs, which grow centripetally in a formin-dependent fashion. The handedness of the chiral pattern was shown to be regulated by α-actinin-1. Computational modelling demonstrated that the dynamics of the RF-TF system can explain the pattern transition from radial to chiral. Thus, actin cytoskeleton self-organization provides built-in machinery that potentially allows cells to develop left-right asymmetry.

  9. Aurora A kinase modulates actin cytoskeleton through phosphorylation of Cofilin: Implication in the mitotic process.

    Science.gov (United States)

    Ritchey, Lisa; Chakrabarti, Ratna

    2014-11-01

    Aurora A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by the modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through the regulation of the actin cytoskeleton. Aurora A phosphorylates Cofilin at multiple sites including S(3) resulting in the inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during the progression of prophase to metaphase. Inhibition of Aur-A activity induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the regulation of actin cytoskeleton reorganization, through Cofilin phosphorylation during early mitotic stages.

  10. HGF Modulates Actin Cytoskeleton Remodeling and Contraction in Testicular Myoid Cells

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    Angela Catizone

    2015-01-01

    Full Text Available The presence of the HGF/Met system in the testicular myoid cells was first discovered by our group. However, the physiological role of this pathway remains poorly understood. We previously reported that HGF increases uPA secretion and TGF-β activation in cultured tubular fragments and that HGF is maximally expressed at Stages VII–VIII of the seminiferous epithelium cycle, when myoid cell contraction occurs. It is well known that the HGF/Met pathway is involved in cytoskeletal remodeling; moreover, the interaction of uPA with its receptor, uPAR, as well as the activation of TGF-β have been reported to be related to the actin cytoskeleton contractility of smooth muscle cells. Herein, we report that HGF induces actin cytoskeleton remodeling in vitro in isolated myoid cells and myoid cell contraction in cultured seminiferous tubules. To better understand these phenomena, we evaluated: (1 the regulation of the uPA machinery in isolated myoid cells after HGF administration; and (2 the effect of uPA or Met inhibition on HGF-treated tubular fragments. Because uPA activates latent TGF-β, the secretion of this factor was also evaluated. We found that both uPA and TGF-β activation increase after HGF administration. In testicular tubular fragments, HGF-induced TGF-β activation and myoid cell contraction are abrogated by uPA or Met inhibitor administration.

  11. Effects of altered gravity on the cell cycle, actin cytoskeleton and proteome in Physarum polycephalum

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    He, Jie; Zhang, Xiaoxian; Gao, Yong; Li, Shuijie; Sun, Yeqing

    Some researchers suggest that the changes of cell cycle under the effect of microgravity may be associated with many serious adverse physiological changes. In the search for underlying mechanisms and possible new countermeasures, we used the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony to study the effects of altered gravity on the cell cycle, actin cytoskeleton and proteome. In parallel, the cell cycle was analyzed in Physarum incubated (1) in altered gravity for 20 h, (2) in altered gravity for 40 h, (3) in altered gravity for 80 h, and (4) in ground controls. The cell cycle, the actin cytoskeleton, and proteome in the altered gravity and ground controls were examined. The results indicated that the duration of the G2 phase was lengthened 20 min in high aspect ratio vessel (HARV) for 20 h, and prolonged 2 h in altered gravity either for 40 h or for 80 h, whereas the duration of other phases in the cell cycle was unchanged with respect to the control. The microfilaments in G2 phase had a reduced number of fibers and a unique abnormal morphology in altered gravity for 40 h, whereas the microfilaments in other phases of cell cycle were unchanged when compared to controls. Employing classical two-dimensional electrophoresis (2-DE), we examined the effect of the altered gravity on P. polycephalum proteins. The increase in the duration of G2 phase in altered gravity for 40 h was accompanied by changes in the 2-DE protein profiles, over controls. Out of a total of 200 protein spots investigated in G2 phase, which were reproducible in repeated experiments, 72 protein spots were visually identified as specially expressed, and 11 proteins were up-regulated by 2-fold and 28 proteins were down-regulated by 2-fold over controls. Out of a total of three low-expressed proteins in G2 phase in altered gravity for 40 h, two proteins were unknown proteins, and one protein was spherulin 3b by MALDI-TOF mass spectrometry (MS

  12. Beta adrenergic overstimulation impaired vascular contractility via actin-cytoskeleton disorganization in rabbit cerebral artery.

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    Hyoung Kyu Kim

    Full Text Available BACKGROUND AND PURPOSE: Beta adrenergic overstimulation may increase the vascular damage and stroke. However, the underlying mechanisms of beta adrenergic overstimulation in cerebrovascular dysfunctions are not well known. We investigated the possible cerebrovascular dysfunction response to isoproterenol induced beta-adrenergic overstimulation (ISO in rabbit cerebral arteries (CAs. METHODS: ISO was induced in six weeks aged male New Zealand white rabbit (0.8-1.0 kg by 7-days isoproterenol injection (300 μg/kg/day. We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Systemic properties of 2DE proteomics result were analyzed using bioinformatics software. ROS generation and following DNA damage were assessed to evaluate deteriorative effect of ISO on CAs. Intracellular Ca(2+ level change and vascular contractile response to vasoactive drug, angiotensin II (Ang II, were assessed to evaluate functional alteration of ISO treated CAs. Ang II-induced ROS generation was assessed to evaluated involvement of ROS generation in CA contractility. RESULTS: Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1 in ISO-CAs. As a cause of dysregulation of actin-cytoskeleton organization, we found decreased level of RhoA and ROCK1, which are major regulators of actin-cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca(2+ efflux and constriction response to angiotensin II and high K(+ in ISO-CAs. ISO also increased basal ROS generation and induced oxidative damage in CA; however, it decreased the Ang II-induced ROS generation rate. These results indicate that ISO disrupted actin cytoskeleton proteome network

  13. Crosstalk between the actin cytoskeleton and Ran-mediated nuclear transport

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    Steward Ruth

    2005-08-01

    Full Text Available Abstract Background Transport of macromolecules into and out of the nucleus is a highly regulated process. The RanGTP/RanGDP gradient controls the trafficking of molecules exceeding the diffusion limit of the nuclear pore across the nuclear envelope. Results We found genetic interaction between genes establishing the Ran gradient, nuclear transport factor 2 (ntf-2, Ran GTPase activating protein (Sd, and the gene encoding Drosophila Profilin, chickadee (chic. The severe eye phenotype caused by reduction of NTF2 is suppressed by loss of function mutations in chic and gain of function mutations in Sd (RanGAP. We show that in chic mutants, as in Sd-RanGAP, nuclear export is impaired. Conclusion Our data suggest that Profilin and the organization of the actin cytoskeleton play an important role in nuclear trafficking.

  14. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

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    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  15. WIP modulates dendritic spine actin cytoskeleton by transcriptional control of lipid metabolic enzymes.

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    Franco-Villanueva, Ana; Fernández-López, Estefanía; Gabandé-Rodríguez, Enrique; Bañón-Rodríguez, Inmaculada; Esteban, Jose Antonio; Antón, Inés M; Ledesma, María Dolores

    2014-08-15

    We identify Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) as a novel component of neuronal synapses whose absence increases dendritic spine size and filamentous actin levels in an N-WASP/Arp2/3-independent, RhoA/ROCK/profilinIIa-dependent manner. These effects depend on the reduction of membrane sphingomyelin (SM) due to transcriptional upregulation of neutral sphingomyelinase (NSM) through active RhoA; this enhances RhoA binding to the membrane, raft partitioning and activation in steady state but prevents RhoA changes in response to stimulus. Inhibition of NSM or SM addition reverses RhoA, filamentous actin and functional anomalies in synapses lacking WIP. Our findings characterize WIP as a link between membrane lipid composition and actin cytoskeleton at dendritic spines. They also contribute to explain cognitive deficits shared by individuals bearing mutations in the region assigned to the gene encoding for WIP.

  16. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

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    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

  17. The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns.

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    Jessica L Henty-Ridilla

    Full Text Available Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs during pattern-triggered immunity (PTI and perturbations by effector proteins during effector-triggered susceptibility (ETS. We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

  18. Modulating the actin cytoskeleton affects mechanically induced signal transduction and differentiation in mesenchymal stem cells.

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    Petra Müller

    Full Text Available Mechanical interactions of mesenchymal stem cells (MSC with the environment play a significant role in controlling the diverse biological functions of these cells. Mechanical forces are transduced by integrins to the actin cytoskeleton that functions as a scaffold to switch mechanical signals into biochemical pathways. To explore the significance of cytoskeletal mechanisms in human MSC we modulated the actin cytoskeleton using the depolymerising drugs cytochalasin D (CytD and latrunculin A (LatA, as well as the stabilizing drug jasplakinolide (Jasp and examined the activation of the signalling molecules ERK and AKT during mechanical loading. All three drugs provoked significant changes in cell morphology and organisation of the cytoskeleton. Application of mechanical forces to β1-integrin receptors using magnetic beads without deformation of the cell shape induced a phosphorylation of ERK and AKT. Of the two drugs that inhibited the cytoskeletal polymerization, LatA completely blocked the activation of ERK and AKT due to mechanical forces, whereas CytD inhibited the activation of AKT but not of ERK. Activation of both signalling molecules by integrin loading was not affected due to cell treatment with the cytoskeleton stabilizing drug Jasp. To correlate the effects of the drugs on mechanically induced activation of AKT and ERK with parameters of MSC differentiation, we studied ALP activity as a marker for osteogenic differentiation and examined the uptake of fat droplets as marker for adipogenic differentiation in the presence of the drugs. All three drugs inhibited ALP activity of MSC in osteogenic differentiation medium. Adipogenic differentiation was enhanced by CytD and Jasp, but not by LatA. The results indicate that modulation of the cytoskeleton using perturbing drugs can differentially modify both mechanically induced signal transduction and MSC differentiation. In addition to activation of the signalling molecules ERK and AKT, other

  19. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

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    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity.

  20. Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa.

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    Bucci, D; Galeati, G; Tamanini, C; Vallorani, C; Rodriguez-Gil, J E; Spinaci, M

    2012-04-01

    Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after "in vitro" capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.

  1. Identification and Characterization of a Candidate Wolbachia pipientis Type IV Effector That Interacts with the Actin Cytoskeleton

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    Kathy B. Sheehan

    2016-07-01

    Full Text Available Many bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that of Wolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte and horizontally (by environmental transmission. Maintenance of the infection within Drosophila melanogaster is sensitive to the regulation of actin, as Wolbachia inefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized that Wolbachia must depend on the host actin cytoskeleton. In this study, we identify and characterize a Wolbachia protein (WD0830 that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeast Saccharomyces cerevisiae induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actin in vitro and cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughout Drosophila development and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increased Wolbachia titers in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitate Wolbachia’s replication or transmission. Therefore, this candidate secreted effector may play a role in Wolbachia’s infection of and persistence within host niches.

  2. Antiamoebic Activity of Adenophyllum aurantium (L.) Strother and Its Effect on the Actin Cytoskeleton of Entamoeba histolytica

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    Herrera-Martínez, Mayra; Hernández-Ramírez, Verónica I.; Hernández-Carlos, Beatriz; Chávez-Munguía, Bibiana; Calderón-Oropeza, Mónica A.; Talamás-Rohana, Patricia

    2016-01-01

    In Mexico, the Adenophyllum aurantium (L.) Strother plant is consumed as an infusion to treat intestinal diseases such as amoebiasis, which is an endemic health problem in Mexico and other countries. However, the effect of A. aurantium on Entamoeba histolytica, the causative agent of amoebiasis, is unknown. An aerial part methanolic extract (AaMeA), a root methanolic extract (AaMeR) and a root ethyl acetate extract (AaEaR) were tested on E. histolytica trophozoites. AaMeA and AaMeR did not show antiproliferative activity; however, AaEaR exhibited an in vitro GI50 of 230 μg/ml, and it was able to inhibit the differentiation of Entamoeba invadens trophozoites into cysts. The intraperitoneal administration of AaEaR (2.5 or 5 mg) to hamsters that were infected with E. histolytica inhibited the development of amoebic liver abscesses in 48.5 or 89.0% of the animals, respectively. Adhesion to fibronectin and erythrophagocytosis were 28.7 and 37.5% inhibited by AaEaR, respectively. An ultrastructure analysis of AaEaR-treated trophozoites shows a decrease in the number of vacuoles but no apparent cell damage. Moreover, this extract affected the actin cytoskeleton structuration, and it prevented the formation of contractile rings by mechanism(s) that were independent of reactive oxygen species and RhoA activation pathways. 13C NMR data showed that the major compounds in the AaEaR extract are thiophenes. Our results suggest that AaEaR may be effective in treatments against amoebiasis, nevertheless, detailed toxicity studies on thiophenes, contained in AaEaR, are required to avoid misuse of this vegetal species. PMID:27445810

  3. CAPZA1 modulates EMT by regulating actin cytoskeleton remodelling in hepatocellular carcinoma.

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    Huang, Deng; Cao, Li; Zheng, Shuguo

    2017-01-16

    Epithelial-mesenchymal transition (EMT) elicits dramatic changes, including cytoskeleton remodelling as well as changes in gene expression and cellular phenotypes. During this process, actin filament assembly plays an important role in maintaining the morphology and movement of tumour cells. Capping protein, a protein complex referred to as CapZ, is an actin-binding complex that can regulate actin cytoskeleton remodelling. CAPZA1 is the α1 subunit of this complex, and we hypothesized that CAPZA1 regulates EMT through the regulation of actin filaments assembly, thus reducing the metastatic ability of hepatocellular carcinoma (HCC) cells. Immunohistochemistry was used to detect CAPZA1 expression in 129 HCC tissues. Western blotting and qPCR were used to detect CAPZA1, EMT markers and EMT transcription factors in HCC cells. Transwell migration and invasion assays were performed to observe the migration and invasion of HCC cells. Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of HCC cells. Immunoprecipitation was used to detect the interaction between CAPZA1 and actin filaments. Finally, a small animal magnetic resonance imager (MRI) was used to observe metastases in HCC cell xenografts in the liver. CAPZA1 expression levels were negatively correlated with the biological characteristics of primary HCC and patient prognosis. CAPZA1 expression was negatively correlated with the migration and invasion of HCC cells. CAPZA1 down regulation promoted the migration and invasion of HCC cells. Conversely, CAPZA1 overexpression significantly inhibited the migration and invasion of HCC cells. Moreover, CAPZA1 expression levels were correlated with the expression of the EMT markers E-cadherin, N-cadherin and Vimentin. Furthermore, the expression of Snail1 and ZEB1 were negatively correlated with CAPZA1 expression levels. Similarly, CAPZA1 significantly inhibited intrahepatic metastases of HCC cells in an orthotopic transplantation tumour model. CAPZA1 inhibits

  4. Actin cytoskeleton regulation of epithelial mesenchymal transition in metastatic cancer cells.

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    Jay Shankar

    Full Text Available Epithelial-mesenchymal transition (EMT is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells.

  5. Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

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    Rieder Gabriele

    2011-11-01

    Full Text Available Abstract Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS and the effector protein cytotoxin-associated gene A (CagA of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells.

  6. Effects of nitrogen ion implantation on lily pollen germination and the distribution of the actin cytoskeleton during pollen germination

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0±1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 1013 ions/cm2. Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.

  7. Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division.

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    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2002-01-01

    An Arabidopsis cDNA (AtCAP1) that encodes a predicted protein of 476 amino acids highly homologous with the yeast cyclase-associated protein (CAP) was isolated. Expression of AtCAP1 in the budding yeast CAP mutant was able to rescue defects such as abnormal cell morphology and random budding pattern. The C-terminal domain, 158 amino acids of AtCAP1 possessing in vitro actin binding activity, was needed for the regulation of cytoskeleton-related defects of yeast. Transgenic plants overexpressing AtCAP1 under the regulation of a glucocorticoid-inducible promoter showed different levels of AtCAP1 accumulation related to the extent of growth abnormalities, in particular size reduction of leaves as well as petioles. Morphological alterations in leaves were attributable to decreased cell size and cell number in both epidermal and mesophyll cells. Tobacco suspension-cultured cells (Bright Yellow 2) overexpressing AtCAP1 exhibited defects in actin filaments and were unable to undergo mitosis. Furthermore, an immunoprecipitation experiment suggested that AtCAP1 interacted with actin in vivo. Therefore, AtCAP1 may play a functional role in actin cytoskeleton networking that is essential for proper cell elongation and division.

  8. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

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    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  9. Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

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    Ogienko, Anna A; Karagodin, Dmitry A; Lashina, Valentina V; Baiborodin, Sergey I; Omelina, Eugeniya S; Baricheva, Elina M

    2013-02-01

    Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.

  10. Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti.

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    Yokota, Keisuke; Fukai, Eigo; Madsen, Lene H; Jurkiewicz, Anna; Rueda, Paloma; Radutoiu, Simona; Held, Mark; Hossain, Md Shakhawat; Szczyglowski, Krzysztof; Morieri, Giulia; Oldroyd, Giles E D; Downie, J Allan; Nielsen, Mette W; Rusek, Anna Maria; Sato, Shusei; Tabata, Satoshi; James, Euan K; Oyaizu, Hiroshi; Sandal, Niels; Stougaard, Jens

    2009-01-01

    Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

  11. From filaments to function:The role of the plant actin cytoskeleton in pathogen perception, signaling and immunity

    Institute of Scientific and Technical Information of China (English)

    Katie Porter; Brad Day

    2016-01-01

    The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, develop-ment and movement, gene expression and signal transduc-tion, and response to biotic and abiotic stress. In recent years, research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens, including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activa-tion of specific signaling responses following pathogen perception. B4ased on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens.

  12. Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

    Institute of Scientific and Technical Information of China (English)

    LI Guoping; HUANG Qunce; YANG Lusheng; QIN Guangyong

    2008-01-01

    Pollen grains of Pinus thunbergii Parl. (Japanese black pine) were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implanta-tion significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for con-tinuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam im-plantation and is involved in mediating certain subsequent cytological changes.

  13. The Drosophila planar polarity gene multiple wing hairs directly regulates the actin cytoskeleton.

    Science.gov (United States)

    Lu, Qiuheng; Schafer, Dorothy A; Adler, Paul N

    2015-07-15

    The evolutionarily conserved frizzled/starry night (fz/stan) pathway regulates planar cell polarity (PCP) in vertebrates and invertebrates. This pathway has been extensively studied in the Drosophila wing, where it is manifested by an array of distally pointing cuticular hairs. Using in vivo imaging we found that, early in hair growth, cells have multiple actin bundles and hairs that subsequently fuse into a single growing hair. The downstream PCP gene multiple wing hairs (mwh) plays a key role in this process and acts to antagonize the actin cytoskeleton. In mwh mutants hair initiation is not limited to a small region at the distal edge of pupal wing cells as in wild type, resulting in multiple hairs with aberrant polarity. Extra actin bundles/hairs are formed and do not completely fuse, in contrast to wild type. As development proceeded additional hairs continued to form, further increasing hair number. We identified a fragment of Mwh with in vivo rescue activity and that bound and bundled F-actin filaments and inhibited actin polymerization in in vitro actin assays. The loss of these activities can explain the mwh mutant phenotype. Our data suggest a model whereby, prior to hair initiation, proximally localized Mwh inhibits actin polymerization resulting in polarized activation of the cytoskeleton and hair formation on the distal side of wing cells. During hair growth Mwh is found in growing hairs, where we suggest it functions to promote the fusion of actin bundles and inhibit the formation of additional actin bundles that could lead to extra hairs.

  14. Effects of chondroitin sulfate on alteration of actin cytoskeleton in rats with acute necrotizing pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ye He; Ren-Xuan Guo

    2007-01-01

    BACKGROUND: In experimental acute pancreatitis, a large amount of reactive oxygen species are produced, and in turn cytoskeletal changes may be induced in pancreatic tissue. These changes contribute to an imbalance of digestive enzyme segregation, transport, exocytosis and activation, resulting in cell injury. In this study, we assessed the effects of chondroitin sulfate (CS) on attenuation of oxidative damage and protection of F-actin in rats with acute necrotizing pancreatitis (ANP). METHODS:Ninety male Wistar rats were divided randomly into three groups. Group A was infused with 5% sodium taurocholate; group B was treated with CS;and group C served as control. Rats from the three groups were killed at 1, 3 or 8 hours. The levels were measured of malonyl dialdehyde (MDA), total superoxide dismutase (SOD), glutathione synthetase (GSH), serum amylase (SAM) and adenosine triphosphate (ATP). F-actin immunostained with rhodamine-phalloidin was analyzed using a confocal laser scanning system and the content of F-actin protein was determined. RESULTS: The levels of SAM increased in groups A and B, whereas the levels of GSH, SOD and ATP in group A decreased markedly during pancreatitis, and MDA increased signiifcantly. The levels of GSH, SOD and ATP in group B were higher than those in group A, but the level of MDA was lower than in group A. At the same time, ANP resulted in early disruption of the cytoskeleton with dramatic changes and a loss of F-actin. Administration of CS moderated the damage to the actin cytoskeleton. CONCLUSIONS:Retrograde infusion of sodium taurocholate via the pancreatic duct may produce pancreatic necrosis and a marked increase in serum amylase activity, induce a severe depletion of ATP level, prime lipid peroxidation, and damage F-actin. Treatment with CS can ameliorate pancreatic cell conditions, limit cell membrane peroxidation, protect F-actin, and attenuate pancreatitis.

  15. Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues.

    Science.gov (United States)

    Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

    2016-01-01

    Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to

  16. Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1

    Science.gov (United States)

    Wei, Lei; Surma, Michelle; Gough, Gina; Shi, Stephanie; Lambert-Cheatham, Nathan; Chang, Jiang; Shi, Jianjian

    2015-01-01

    We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16–24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16–24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the

  17. ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function

    DEFF Research Database (Denmark)

    Kawaguchi, Nobuko; Sundberg, Christina; Kveiborg, Marie

    2003-01-01

    -100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary....... Moreover, ADAM12-expressing cells were more prone to apoptosis, which could be prevented by treating the cells with beta1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, beta1 integrin was more readily extracted with Triton X...

  18. Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion.

    Science.gov (United States)

    Zhang, Haitao; Ghai, Pooja; Wu, Huhehasi; Wang, Changhui; Field, Jeffrey; Zhou, Guo-Lei

    2013-07-19

    CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

  19. Jak3 enables chemokine-dependent actin cytoskeleton reorganization by regulating cofilin and Rac/Rhoa GTPases activation.

    Directory of Open Access Journals (Sweden)

    Xochitl Ambriz-Peña

    Full Text Available We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3⁻/⁻ lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3⁻/⁻ lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines.

  20. New EMBO members' review: actin cytoskeleton regulation through modulation of PI(4,5)P(2) rafts.

    Science.gov (United States)

    Caroni, P

    2001-08-15

    The phosphoinositide lipid PI(4,5)P(2) is now established as a key cofactor in signaling to the actin cytoskeleton and in vesicle trafficking. PI(4,5)P(2) accumulates at membrane rafts and promotes local co-recruitment and activation of specific signaling components at the cell membrane. PI(4,5)P(2) rafts may thus be platforms for local regulation of morphogenetic activity at the cell membrane. Raft PI(4,5)P(2) is regulated by lipid kinases (PI5-kinases) and lipid phosphatases (e.g. synaptojanin). In addition, GAP43-like proteins have recently emerged as a group of PI(4,5)P(2) raft-modulating proteins. These locally abundant proteins accumulate at inner leaflet plasmalemmal rafts where they bind to and co-distribute with PI(4,5)P(2), and promote actin cytoskeleton accumulation and dynamics. In keeping with their proposed role as positive modulators of PI(4,5)P(2) raft function, GAP43-like proteins confer competence for regulated morphogenetic activity on cells that express them. Their function has been investigated extensively in the nervous system, where their expression promotes neurite outgrowth, anatomical plasticity and nerve regeneration. Extrinsic signals and intrinsic factors may thus converge to modulate PI(4,5)P(2) rafts, upstream of regulated activity at the cell surface.

  1. p130Cas Couples the tyrosine kinase Bmx/Etk with regulation of the actin cytoskeleton and cell migration.

    Science.gov (United States)

    Abassi, Yama A; Rehn, Marko; Ekman, Niklas; Alitalo, Kari; Vuori, Kristiina

    2003-09-12

    Bmx/Etk, a member of the Tec/Btk family of nonreceptor kinases, has recently been shown to mediate cell motility in signaling pathways that become activated upon integrin-mediated cell adhesion (Chen, R., Kim, O., Li, M., Xiong, X., Guan, J. L., Kung, H. J., Chen, H., Shimizu, Y., and Qiu, Y. (2001) Nat Cell Biol. 3, 439-444). The molecular mechanisms of Bmx-induced cell motility have so far remained unknown. Previous studies by us and others have demonstrated that a complex formation between the docking protein p130Cas (Cas) and the adapter protein Crk is instrumental in connecting several stimuli to the regulation of actin cytoskeleton and cell motility. We demonstrate here that expression of Bmx leads to an interaction between Bmx and Cas at membrane ruffles, which are sites of active actin remodeling in motile cells. Expression of Bmx also enhances tyrosine phosphorylation of Cas and Cas.Crk complex formation, and coexpression of Bmx with Cas results in an enhanced membrane ruffling and haptotactic cell migration. Importantly, a mutant form of Bmx that fails to interact with Cas also fails to induce cell migration. Furthermore, expression of a dominant-negative form of Cas that is incapable of interacting with Crk inhibits Bmx-induced membrane ruffling and cell migration. These studies suggest that Bmx-Cas interaction, phosphorylation of Cas by Bmx, and subsequent Cas.Crk complex formation functionally couple Bmx to the regulation of actin cytoskeleton and cell motility.

  2. Interaction with mycorrhiza helper bacterium Streptomyces sp. AcH 505 modifies organisation of actin cytoskeleton in the ectomycorrhizal fungus Amanita muscaria (fly agaric).

    Science.gov (United States)

    Schrey, Silvia D; Salo, Vanamo; Raudaskoski, Marjatta; Hampp, Rüdiger; Nehls, Uwe; Tarkka, Mika T

    2007-08-01

    The actin cytoskeleton (AC) of fungal hyphae is a major determinant of hyphal shape and morphogenesis, implicated in controlling tip structure and secretory vesicle delivery. Hyphal growth of the ectomycorrhizal fungus Amanita muscaria and symbiosis formation with spruce are promoted by the mycorrhiza helper bacterium Streptomyces sp. AcH 505 (AcH 505). To investigate structural requirements of growth promotion, the effect of AcH 505 on A. muscaria hyphal morphology, AC and actin gene expression were studied. Hyphal diameter and mycelial density decreased during dual culture (DC), and indirect immunofluorescence microscopy revealed that the dense and polarised actin cap in hyphal tips of axenic A. muscaria changes to a loosened and dispersed structure in DC. Supplementation of growth medium with cell-free bacterial supernatant confirmed that reduction in hyphal diameter and AC changes occurred at the same stage of growth. Transcript levels of both actin genes isolated from A. muscaria remained unaltered, indicating that AC changes are regulated by reorganisation of the existing actin pool. In conclusion, the AC reorganisation appears to result in altered hyphal morphology and faster apical extension. The thus improved spreading of hyphae and increased probability to encounter plant roots highlights a mechanism behind the mycorrhiza helper effect.

  3. Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton.

    Science.gov (United States)

    Sultana, Hameeda; Rivero, Francisco; Blau-Wasser, Rosemarie; Schwager, Stephan; Balbo, Alessandra; Bozzaro, Salvatore; Schleicher, Michael; Noegel, Angelika A

    2005-10-01

    Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.

  4. Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton.

    Science.gov (United States)

    Winter, C G; Wang, B; Ballew, A; Royou, A; Karess, R; Axelrod, J D; Luo, L

    2001-04-06

    Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.

  5. Regulation of the actin cytoskeleton by an interaction of IQGAP related protein GAPA with filamin and cortexillin I.

    Directory of Open Access Journals (Sweden)

    Subhanjan Mondal

    Full Text Available Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

  6. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen

  7. The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten;

    2010-01-01

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocyto...

  8. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen m

  9. Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

    Science.gov (United States)

    Bottazzi, Maria Elena; Buzzai, Monica; Zhu, Xiaoyun; Desdouets, Chantal; Bréchot, Christian; Assoian, Richard K.

    2001-01-01

    Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G1-phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation. PMID:11604497

  10. Transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder.

    Science.gov (United States)

    Zhao, Z; Xu, J; Chen, J; Kim, S; Reimers, M; Bacanu, S-A; Yu, H; Liu, C; Sun, J; Wang, Q; Jia, P; Xu, F; Zhang, Y; Kendler, K S; Peng, Z; Chen, X

    2015-05-01

    Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of post-mortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q⩽0.01, 53 genes; q⩽0.05, 213 genes; q⩽0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (Pgenes show concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor-mediated phagocytosis, regulation of actin cytoskeleton pathways, along with several cancer pathways. Functional analyses of these genes revealed an interconnected pathway network centered on lysosomal function and the regulation of actin cytoskeleton. These pathways and their interacting network were principally confirmed by an independent transcriptome sequencing data set of the hippocampus. Dysregulation of lysosomal function and cytoskeleton remodeling has direct impacts on endocytosis, phagocytosis, exocytosis, vesicle trafficking, neuronal maturation and migration, neurite outgrowth and synaptic density and plasticity, and different aspects of these processes have been implicated in SCZ and BPD.

  11. Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD.

    Directory of Open Access Journals (Sweden)

    Christina E N Lord

    Full Text Available Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD. PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B, a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1 activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs. The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.

  12. The cell wall sensor Wsc1p is involved in reorganization of actin cytoskeleton in response to hypo-osmotic shock in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gualtieri, Tania; Ragni, Enrico; Mizzi, Luca; Fascio, Umberto; Popolo, Laura

    2004-10-15

    The cell wall is essential to preserve osmotic integrity of yeast cells. Some phenotypic traits of cell wall mutants suggest that, as a result of a weakening of the cell wall, hypo-osmotic stress-like conditions are created. Consequent expansion of the cell wall and stretching of the plasma membrane trigger a complex response to prevent cell lysis. In this work we examined two conditions that generate a cell wall and membrane stress: one is represented by the cell wall mutant gas1Delta and the other by a hypo-osmotic shock. We examined the actin cytoskeleton and the role of the cell wall sensors Wsc1p and Mid2p in these stress conditions. In the gas1 null mutant cells, which lack a beta(1,3)-glucanosyltransferase activity required for cell wall assembly, a constitutive marked depolarization of actin cytoskeleton was found. In a hypo-osmotic shock wild-type cells showed a transient depolarization of actin cytoskeleton. The percentage of depolarized cells was maximal at 30 min after the shift and then progressively decreased until cells reached a new steady-state condition. The maximal response was proportional to the magnitude of the difference in the external osmolarity before and after the shift within a given range of osmolarities. Loss of Wsc1p specifically delayed the repolarization of the actin cytoskeleton, whereas Wsc1p and Mid2p were essential for the maintenance of cell integrity in gas1Delta cells. The control of actin cytoskeleton is an important element in the context of the compensatory response to cell wall weakening. Wsc1p appears to be an important regulator of the actin network rearrangements in conditions of cell wall expansion and membrane stretching.

  13. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

    Science.gov (United States)

    Cui, Cheng; Chatterjee, Bishwanath; Lozito, Thomas P; Zhang, Zhen; Francis, Richard J; Yagi, Hisato; Swanhart, Lisa M; Sanker, Subramaniam; Francis, Deanne; Yu, Qing; San Agustin, Jovenal T; Puligilla, Chandrakala; Chatterjee, Tania; Tansey, Terry; Liu, Xiaoqin; Kelley, Matthew W; Spiliotis, Elias T; Kwiatkowski, Adam V; Tuan, Rocky; Pazour, Gregory J; Hukriede, Neil A; Lo, Cecilia W

    2013-11-01

    Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to

  14. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

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    Cheng Cui

    2013-11-01

    Full Text Available Planar cell polarity (PCP regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin

  15. [STRUCTURAL CHARACTERIZATION OF PLATELETS AND PLATELET-DERIVED MICROVESICLES].

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    Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Litvinov, R I

    2016-01-01

    Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states.

  16. Role of lipid raft components and actin cytoskeleton in fibronectin-binding, surface expression, and de novo synthesis of integrin subunits in PGE2- or 8-Br-cAMP-stimulated mastocytoma P-815 cells.

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    Okada, Yasuyo; Nishikawa, Jyun-ichi; Semma, Masanori; Ichikawa, Atsushi

    2014-04-01

    Integrins are heterodimeric adhesion receptors essential for adhesion of non-adherent cells to extracellular ligands such as extracellular matrix components. The affinity of integrins for ligands is regulated through a process termed integrin activation and de novo synthesis. Integrin activation is regulated by lipid raft components and the actin structure. However, there is little information on the relationship between integrin activation and its de novo synthesis. Cancerous mouse mast cells, mastocytoma P-815 cells (P-815 cells) are known to bind to fibronectin through de novo synthesis of integrin subtypes by prostaglandin (PG) E2 stimulation. The purpose of this study was to clarify the relationship between lipid raft components and the actin cytoskeleton, and PGE2-induced P-815 cells adhesion to fibronectin and the increase in surface expression and mRNA and protein levels of αvβ3 and αIIbβ3 integrins. Cholesterol inhibitor 6-O-α-maltosyl-β cyclodextrin, glycosylphosphatidylinositol-anchored proteins inhibitor phosphatidylinositol-specific phospholipase C and actin inhibitor cytochalasin D inhibited PGE2-induced cell adhesion to fibronectin, but did not regulate the surface expression and mRNA and protein levels of αv and αIIb, and β3 integrin subunits. In addition, inhibitor of integrin modulate protein CD47 had no effect on PGE2- and 8-Br-cAMP-induced cell adhesion. These results suggest that lipid raft components and the actin cytoskeleton are directly involved in increasing of adhesion activity of integrin αIIb, αv and β3 subunits to fibronectin but not in stimulating of de novo synthesis of them in PGE2-stimulated P-815 cells. The modulation of lipid rafts and the actin structure is essential for P-815 cells adhesion to fibronectin.

  17. The spreading process of Ehrlichia canis in macrophages is dependent on actin cytoskeleton, calcium and iron influx and lysosomal evasion.

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    Alves, R N; Levenhagen, M A; Levenhagen, M M M D; Rieck, S E; Labruna, M B; Beletti, M E

    2014-01-31

    Ehrlichia canis is an obligate intracellular microorganism and the etiologic agent of canine monocytic ehrlichiosis. The invasion process has already been described for some bacteria in this genus, such as E. muris and E. chaffeensis, and consists of four stages: adhesion, internalisation, intracellular proliferation and intercellular spreading. However, little is known about the spreading process of E. canis. The aim of this study was to analyse the role of the actin cytoskeleton, calcium, iron and lysosomes from the host cell in the spreading of E. canis in dog macrophages in vitro. Different inhibitory drugs were used: cytochalasin D (actin polymerisation inhibitor), verapamil (calcium channel blocker) and deferoxamine (iron chelator). Our results showed a decrease in the number of bacteria in infected cells treated with all drugs when compared to controls. Lysosomes in infected cells were cytochemically labelled with acid phosphatase to allow the visualisation of phagosome-lysosome fusion and were further analysed by transmission electron microscopy. Phagosome-lysosome fusion was rarely observed in vacuoles containing viable E. canis. These data suggest that the spreading process of E. canis in vitro is dependent on cellular components analysed and lysosomal evasion.

  18. Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections.

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    Moore, Chad L; Cheng, Delfine; Shami, Gerald J; Murphy, Christopher R

    2016-05-01

    In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro

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    Zhimei Lv

    2016-01-01

    Full Text Available Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation.

  20. Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentration.

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    Tiago, Teresa; Marques-da-Silva, Dorinda; Samhan-Arias, Alejandro K; Aureliano, Manuel; Gutierrez-Merino, Carlos

    2011-03-01

    Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1μM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.

  1. The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells

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    Li Li

    2017-09-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF, a potential therapeutic factor for Parkinson’s disease (PD, exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B and jasplakinolide (Jas to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret–F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.

  2. The Nebivolol action on vascular tone is dependent on actin cytoskeleton polymerization and Rho-A activity into ECs and SMCs.

    Science.gov (United States)

    Kadi, A; de Isla, N; Moby, V; Lacolley, P; Labrude, P; Stoltz, J F; Menu, P

    2014-01-01

    Nitric oxide is implicated in the target action of Nebivolol, a selective β1 adrenoceptor blocker used in hypertension treatment. As the Nitric Oxide (NO) production and the actin cytoskeleton are linked, the aim of this work was to study the involvement of actin cytoskeleton on mechanism of action of Nebivolol in cultured endothelial cells. We studied the effect of Nebivolol (200 μM) on actin filaments remodeling and its impact on NO production and eNOS activation. Results showed that Nebivolol perturbs actin filaments polymerization, increases NO production and eNOS activity between 30 minutes and 1 h. Stabilization of actin filaments with phalloïdine (50 μM) abolishes Nebivolol effects on eNOS activation and NO production. Furthermore, Rho-kinase activity decreased during the first hour of Nebivolol treatment, then increased after 3 h, while actin filaments repolymerized, eNOS activation and NO production decreased. In SMCs, Nebivolol induced a decrease in the Rho-kinase activity from 1 h until 24 h of incubation. In conclusion, we suggest that Nebivolol induced NO production in Endothelial Cells (ECs) via complementary actions between actin cytoskeleton remodeling inducing eNOS activation and Rho-kinase implication. The effect of Nebivolol on ECs occurs during the first hour, this effect on SMCs seems to be maintained until 24 h, explaining persisted action of Nebivolol observed in vivo.

  3. Dynamic reorganization of the actin cytoskeleton [version 1; referees: 2 approved

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    Gaëlle Letort

    2015-10-01

    Full Text Available Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations.

  4. EFFECTS OF ESTETROL ON MIGRATION AND INVASION IN T47-D BREAST CANCER CELLS THROUGH THE ACTIN CYTOSKELETON

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    Maria Silvia eGiretti

    2014-05-01

    Full Text Available Estetrol (E4 is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2 on T47-D estrogen receptor (ER positive breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, redistribution of actin fibers towards the cell membrane was observed. However, when E4 was added to E2, a inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin (ERM family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr558, which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of ERα. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring estrogen receptor modulator in the breast.

  5. Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

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    Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

    2010-09-01

    The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

  6. Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

    Science.gov (United States)

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Wang, Ching C.; Perez Rangel, Armando; Camacho Nuez, Minerva; Garcia Tovar, Carlos; Ayala-Sumuano, Jorge Tonatiuh; Luna-Arias, Juan Pedro; Hernandez, Jose Manuel

    2010-01-01

    Background Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. Methodology and Principal Findings In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. Conclusions and Significance Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and

  7. Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall.

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    Araceli Castillo-Romero

    Full Text Available BACKGROUND: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs and the transport of CWPs into encystation-specific vesicles (ESVs. Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and

  8. Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells

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    Bhanu Neupane

    2015-09-01

    Full Text Available Stimulated emission depletion (STED microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante and live rat chondrosarcoma cells (RCS transfected with actin-green fluorescent protein (GFP. Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

  9. Intracellular Theileria annulata promote invasive cell motility through kinase regulation of the host actin cytoskeleton.

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    Min Ma

    2014-03-01

    Full Text Available The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva or Tropical Theileriosis (T. annulata. These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell's dissemination capabilities.

  10. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens

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    Punsiri M Colonne

    2016-09-01

    Full Text Available Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, subversion of cell intrinsic immunity, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  11. Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

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    Kazuhiro eYamamoto

    2014-04-01

    Full Text Available Several missense mutations in the protein kinase Cγ (γPKC gene have been found to cause spinocerebellar ataxia type 14 (SCA14, an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca2+ and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate, a type of phorbol ester. Wild-type (WT γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in

  12. Myxoma virus oncolytic efficiency can be enhanced through chemical or genetic disruption of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Chad R Irwin

    Full Text Available Myxoma virus (MYXV is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV, MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L(+ MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L(+ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor. While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2. These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.

  13. Myxoma virus oncolytic efficiency can be enhanced through chemical or genetic disruption of the actin cytoskeleton.

    Science.gov (United States)

    Irwin, Chad R; Favis, Nicole A; Agopsowicz, Kate C; Hitt, Mary M; Evans, David H

    2013-01-01

    Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L(+)) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L(+) MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor. While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.

  14. Arabidopsis Vacuolar H+-ATPase (V-ATPase) B Subunits Are Involved in Actin Cytoskeleton Remodeling via Binding to, Bundling, and Stabilizing F-actin*

    OpenAIRE

    Ma, Binyun; Qian, Dong; Nan, Qiong; Tan, Chang; An, Lizhe; Xiang, Yun

    2012-01-01

    Vacuolar H+-ATPase (V-ATPase) is a membrane-bound multisubunit enzyme complex composed of at least 14 different subunits. The complex regulates the physiological processes of a cell by controlling the acidic environment, which is necessary for certain activities and the interaction with the actin cytoskeleton through its B and C subunits in both humans and yeast. Arabidopsis V-ATPase has three B subunits (AtVAB1, AtVAB2, and AtVAB3), which share 97.27% sequence identity and have two potential...

  15. Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

    Science.gov (United States)

    Ayling, Laura J.; Briddon, Stephen J.; Halls, Michelle L.; Hammond, Gerald R. V.; Vaca, Luis; Pacheco, Jonathan; Hill, Stephen J.; Cooper, Dermot M. F.

    2012-01-01

    The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub. PMID:22399809

  16. Prion Induction by the Short-lived Stress Induced Protein Lsb2 Is Regulated by Ubiquitination and Association with the Actin Cytoskeleton

    Science.gov (United States)

    Chernova, Tatiana A.; Romanyuk, Andrey V.; Karpova, Tatiana S.; Shanks, John R.; Ali, Moiez; Moffatt, Nela; Howie, Rebecca L.; O'Dell, Andrew; McNally, James G.; Liebman, Susan W.; Chernoff, Yury O.; Wilkinson, Keith D.

    2011-01-01

    SUMMARY Yeast prions are self-perpetuating QN-rich amyloids, that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s), and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form [PSI+]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI+] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion–inducing ability. These findings directly implicate the UPS and actin cytoskeleton in regulating prions via a stress-inducible QN-rich protein. PMID:21777813

  17. Recessive mutations in DOCK6, encoding the guanidine nucleotide exchange factor DOCK6, lead to abnormal actin cytoskeleton organization and Adams-Oliver syndrome.

    Science.gov (United States)

    Shaheen, Ranad; Faqeih, Eissa; Sunker, Asma; Morsy, Heba; Al-Sheddi, Tarfa; Shamseldin, Hanan E; Adly, Nouran; Hashem, Mais; Alkuraya, Fowzan S

    2011-08-12

    Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.

  18. [Actin cytoskeleton organization and spreading of bone marrow stromal cells and cartilage cells during their combined and independent cultivation on different extracellular matrix proteins].

    Science.gov (United States)

    Sakhenberg, E I; Nikolaenko, N S; Pinaev, G P

    2014-01-01

    To clarify the mutual influence of bone marrow stromal cells (BMSCs) and cartilage cells we studied the organization of their actin cytoskeleton and cell spreading on different extracellular matrix proteins--laminin 2/4, collagen type I or fibronectin. It has been shown that the most pronounced difference in morphological characteristics of the cells such as their form, size and actin cytoskeleton organization occur in the case of interaction with fibronectin. So, after separate brief incubation of both cell types on fibronectin, the average area of BMSCs spreading was about 4 times greater than the area of the cartilage cell spreading. However, in the co-culture of these cells in a ratio of 1:1, the average jointed spreading area on fibronctin was nearly 1.5 times less than the theoretically calculated. To determine the nature of exposure of the cells to each other we have studied spreading of these cells in the media conditioned by another cell type. We have found that the area of BMSC's spreading in the medium conditioned by cartilage cells is markedly smaller than the area of spreading of the same cells in the control medium. These data suggest that the cartilage cells secrete factors that reduce BMSC's spreading.

  19. Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein

    Directory of Open Access Journals (Sweden)

    Bewley J Derek

    2008-09-01

    Full Text Available Abstract Background The actin cytoskeleton responds quickly to diverse stimuli and plays numerous roles in cellular signalling, organelle motility and subcellular compartmentation during plant growth and development. Molecular and cell biological tools that can facilitate visualization of actin organization and dynamics in a minimally invasive manner are essential for understanding this fundamental component of the living cell. Results A novel, monomeric (m Eos-fluorescent protein derived from the coral Lobophyllia hemprichii was assessed for its green to red photo-convertibility in plant cells by creating mEosFP-cytosolic. mEosFP was fused to the F-(filamentous-Actin Binding Domain of the mammalian Talin gene to create mEosFP::FABDmTalin. Photo-conversion, visualization and colour quantification protocols were developed for EosFP targeted to the F-actin cytoskeleton. Rapid photo-conversion in the entire cell or in a region of interest was easily achieved upon illumination with an approximately 400 nm wavelength light beam using an epi-fluorescent microscope. Dual color imaging after photo-conversion was carried out using a confocal laser-scanning microscope. Time-lapse imaging revealed that although photo-conversion of single mEosFP molecules can be rapid in terms of live-cell imaging it involves a progressive enrichment of red fluorescent molecules over green species. The fluorescence of photo-converted cells thus progresses through intermediate shades ranging from green to red. The time taken for complete conversion to red fluorescence depends on protein expression level within a cell and the quality of the focusing lens used to deliver the illuminating beam. Three easily applicable methods for obtaining information on fluorescent intensity and colour are provided as a means of ensuring experimental repeatability and data quantification, when using mEosFP and similar photo-convertible proteins. Conclusion The mEosFP::FABD-mTn probe retains

  20. Involvement of Rac1 and the actin cytoskeleton in insulin- and contraction-stimulated intracellular signaling and glucose uptake in mature skeletal muscle

    DEFF Research Database (Denmark)

    Sylow, Lykke

    by exercise is therefore an important alternative way to maintain whole body glucose homeostasis in insulin resistant states such as Type 2 Diabetes. Although the insulin- and exercise-stimulated signaling pathways to glucose uptake have been studied extensively, the underlying mechanisms are not well...... understood. The aim of the current PhD was therefore to investigate the involvement of Rac1 and the actin cytoskeleton in the regulation of insulin- and contraction-stimulated glucose uptake in mature skeletal muscle. The central findings of this PhD thesis was that Rac1 was activated by both insulin...... and muscle contraction in mouse and human skeletal muscle. Most importantly, Rac1 was involved in the regulation of both insulin- and contraction-stimulated glucose uptake. Interestingly, Rac1 signaling was defective in skeletal muscle of insulin resistant obese and T2D human subjects as well as in obese...

  1. Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    G.D. Godbold

    1998-08-01

    Full Text Available It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

  2. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation

    DEFF Research Database (Denmark)

    Barres, Romain; Grémeaux, Thierry; Gual, Philippe

    2006-01-01

    a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma m......RNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport...... and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin...

  3. Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

    Energy Technology Data Exchange (ETDEWEB)

    Djuzenova, Cholpon S., E-mail: djuzenova_t@ukw.de [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Fiedler, Vanessa [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Memmel, Simon [Lehrstuhl für Biotechnologie und Biophysik, Universität Würzburg, Biozentrum Am Hubland, 97070 Würzburg (Germany); Katzer, Astrid; Hartmann, Susanne [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Krohne, Georg [Elektronenmikroskopie, Biozentrum, Universität Würzburg, Am Hubland, 97070 Würzburg (Germany); Zimmermann, Heiko [Hauptabteilung Biophysik and Kryotechnologie, Fraunhofer-Institut für Biomedizinische Technik, Lehrstuhl für Molekulare und Zelluläre Biotechnologie/Nanotechnologie, Universität des Saarlandes, Ensheimer Strasse 48, 66386 St. Ingbert (Germany); Scholz, Claus-Jürgen [Interdisciplinary Center for Clinical Research, University Hospital, Versbacher Strasse 7, 97078 Würzburg (Germany); Polat, Bülent; Flentje, Michael [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); and others

    2015-01-15

    Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed.

  4. Effect of the ulcerogenic agents ethanol, acetylsalicylic acid and taurocholate on actin cytoskeleton and cell motility in cultured rat gastric mucosal cells

    Institute of Scientific and Technical Information of China (English)

    Siamak Bidel; Harri Mustonen; Giti Khalighi-Sikaroudi; Eero Lehtonen; Pauli Puolakkainen; Tuula Kiviluoto; Eero Kivilaakso

    2005-01-01

    AIM: To assess the effects of ulcerogenic agents on actin cytoskeleton and cell motility and the contribution of oxidative stress.METHODS: Rat gastric mucosal cell monolayers were cultured on coverslips. The cells were exposed, with or without allopurinol (2 mmol/L), for 15 min to ethanol (10-150 mL/L), ASA (1-20 mmol/L) or taurocholate (1-20 mmol/L), then the cells were processed for actin and vinculin staining. Cell migration after wounding was also measured.RESULTS: Exposure to 10 mL/L ethanol caused divergence of zonula adherens-associated actin bundles of adjacent cells and decreased rate of migration. These actions were opposed by xanthine oxidase inhibitor allopurinol. Exposure to 50 mL/L ethanol induced degradation and divergence of zonula adherens-associated vinculin from adjacent cells,which was, again, partially reverted by allopurinol. With 1 mmol/L ASA actin filaments became shorter and thicker.However, higher concentrations (10, 20 mmol/L) of ASA returned microfilaments thinner and longer, and decreased rate of migration. Zonula adherens-associated actin bundles were moderately distorted with 10 mmol/L ASA and with 10 mmol/L taurocholate. Exposure to taurocholate provoked changes resembling those of ASA. Taurocholate 5-20 mmol/L decreased the rate of migration dose dependently. The effects of ASA and taurocholate were not prevented by allopurinol.CONCLUSION: All ulcerogenic agents decreased the rate of migration dose dependently and induced divergence of zonula adherens-associated actin bundles of adjacent cells.In addition, ethanol and ASA caused degradation of actin cytoskeleton. Oxidative stress seems to underlie ethanol,but not ASA or taurocholate, induced cytoskeletal damage.

  5. Restoration of responsiveness of phospholipase Cγ2-deficient platelets by enforced expression of phospholipase Cγ1.

    Directory of Open Access Journals (Sweden)

    Yongwei Zheng

    Full Text Available Receptor-mediated platelet activation requires phospholipase C (PLC activity to elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are classified into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2, which are critical for activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2. Although PLCγ2 has been shown to play a dominant role in platelet activation, the extent to which PLCγ1 contributes has not been evaluated. To ascertain the relative contributions of PLCγ1 and PLCγ2 to platelet activation, we generated conditionally PLCγ1-deficient, wild-type (WT, PLCγ2-deficient, and PLCγ1/PLCγ2 double-deficient mice and measured the ability of platelets to respond to different agonists. We found that PLCγ2 deficiency abrogated αIIbβ3-dependent platelet spreading, GPVI-dependent platelet aggregation, and thrombus formation on collagen-coated surfaces under shear conditions, which is dependent on both GPVI and αIIbβ3. Addition of exogenous ADP overcame defective spreading of PLCγ2-deficient platelets on immobilized fibrinogen, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistently, αIIbβ3-mediated release of granule contents was impaired in the absence of PLCγ2. In contrast, PLCγ1-deficient platelets spread and released granule contents normally on fibrinogen, exhibited normal levels of GPVI-dependent aggregation, and formed thrombi normally on collagen-coated surfaces. Interestingly, enforced expression of PLCγ1 fully restored GPVI-dependent aggregation and αIIbβ3-dependent spreading of PLCγ2-deficient platelets. We conclude that platelet activation through GPVI and αIIbβ3 utilizes PLCγ2 because PLCγ1 levels are insufficient to support responsiveness, but that PLCγ1 can restore responsiveness if expressed at levels normally achieved by PLCγ2.

  6. Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

    Directory of Open Access Journals (Sweden)

    Alleaume-Butaux A

    2013-07-01

    Full Text Available Aurélie Alleaume-Butaux,1,2 Caroline Dakowski,1,2 Mathéa Pietri,1,2 Sophie Mouillet-Richard,1,2 Jean-Marie Launay,3,4 Odile Kellermann,1,2 Benoit Schneider1,2 1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris Cité, UMR-S 747, 3Public Hospital of Paris, Department of Biochemistry, INSERM UMR-S 942, Lariboisière Hospital, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland Abstract: Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1 neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2 neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3 the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC, a glycosylphosphatidylinositol (GPI-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that Pr

  7. IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and re-polarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae.

    Science.gov (United States)

    Corbett, Mark; Xiong, Yulan; Boyne, James R; Wright, Daniel J; Munro, Ewen; Price, Clive

    2006-11-01

    In budding yeast the final stages of the cell division cycle, cytokinesis and cell separation, are distinct events that require to be coupled, both together and with mitotic exit. Here we demonstrate that mutations in genes of the mitotic exit network (MEN) prevent cell separation and are synthetically lethal in combination with both cytokinesis and septation defective mutations. Analysis of the synthetic lethal phenotypes reveals that Iqg1p functions in combination with the MEN components, Tem1p, Cdc15p Dbf20p and Dbf2p to govern the re-polarization of the actin cytoskeleton to either side of the bud neck. In addition phosphorylation of the conserved PCH protein, Hof1p, is dependent upon these activities and requires actin ring assembly. Recruitment of Dbf2p to the bud neck is dependent upon actin ring assembly and correlates with Hof1p phosphorylation. Failure to phosphorylate Hof1p results in the increased stability of the protein and its persistence at the bud neck. These data establish a mechanistic dependency of cell separation upon an intermediate step requiring actomyosin ring assembly.

  8. Roll, adhere, spread and contract: structural mechanics of platelet function.

    Science.gov (United States)

    Sorrentino, Simona; Studt, Jan-Dirk; Medalia, Ohad; Tanuj Sapra, K

    2015-01-01

    Platelets are involved in life-sustaining processes such as hemostasis, wound healing, atherothrombosis and angiogenesis. Mechanical trauma to blood vessels causes platelet activation resulting in their adherence and clot formation at the damaged site, culminating in clot retraction and tissue repair. Two of the major players underlying this process are the cytoskeleton, i.e., actin and microtubules, and the membrane integrin receptors. Rare congenital bleeding disorders such as Glanzmann thrombasthenia and Bernard-Soulier syndrome are associated with genetic alterations of platelet surface receptors, also affecting the platelet cytoskeletal structure. In this review, we summarize the current knowledge about platelet structure and adhesion, and delve into the mechanical aspects of platelet function. Platelets lack a nucleus, and can thus provide a minimal model of a biological cell. New biophysical tools may help to scrutinize platelets anew and to extend the existing knowledge on cell biology.

  9. Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

    OpenAIRE

    Barak, L S; Yocum, R R; Nothnagel, E A; Webb, W W

    1980-01-01

    An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin p...

  10. Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line.

    Science.gov (United States)

    Deyev, Igor E; Popova, Nadezhda V; Serova, Oxana V; Zhenilo, Svetlana V; Regoli, Marì; Bertelli, Eugenio; Petrenko, Alexander G

    2017-07-01

    Secretion of mildly alkaline (pH 8.0-8.5) juice to intestines is one of the key functions of the pancreas. Recent reports indicate that the pancreatic duct system containing the alkaline juice may adjoin the endocrine cells of pancreatic islets. We have previously identified the insulin receptor-related receptor (IRR) that is expressed in islets as a sensor of mildly alkaline extracellular media. In this study, we show that those islet cells that are in contact with the excretory ducts are also IRR-expressing cells. We further analyzed the effects of alkaline media on pancreatic beta cell line MIN6. Activation of endogenous IRR but not of the insulin receptor was detected that could be inhibited with linsitinib. The IRR autophosphorylation correlated with pH-dependent linsitinib-sensitive activation of insulin receptor substrate 1 (IRS-1), the primary adaptor in the insulin signaling pathway. However, in contrast with insulin stimulation, no protein kinase B (Akt/PKB) phosphorylation was detected as a result of alkali treatment. We observed overexpression of several early response genes (EGR2, IER2, FOSB, EGR1 and NPAS4) upon alkali treatment of MIN6 cells but those were IRR-independent. The alkaline medium but not insulin also triggered actin cytoskeleton remodeling that was blocked by pre-incubation with linsitinib. We propose that the activation of IRR by alkali might be part of a local loop of signaling between the exocrine and endocrine parts of the pancreas where alkalinization of the juice facilitate insulin release that increases the volume of secreted juice to control its pH and bicabonate content. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

    Science.gov (United States)

    Bordeleau, François; Myrand Lapierre, Marie-Eve; Sheng, Yunlong; Marceau, Normand

    2012-01-01

    Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells. PMID:22685604

  12. Keratin 8/18 regulation of cell stiffness-extracellular matrix interplay through modulation of Rho-mediated actin cytoskeleton dynamics.

    Directory of Open Access Journals (Sweden)

    François Bordeleau

    Full Text Available Cell mechanical activity generated from the interplay between the extracellular matrix (ECM and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18, hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells.

  13. Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

    Science.gov (United States)

    Aldinucci, Alessandra; Bonechi, Elena; Manuelli, Cinzia; Nosi, Daniele; Masini, Emanuela; Passani, Maria Beatrice; Ballerini, Clara

    2016-07-08

    Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1-4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.

  14. Transfer of a redox-signal through the cytosol by redox-dependent microcompartmentation of glycolytic enzymes at mitochondria and actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Joanna eWojtera-Kwiczor

    2013-01-01

    Full Text Available The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12, GapC plays an important role in glycolysis by providing the cell with ATP and NADH. Interestingly, despite its glycolytic function in the cytosol, GAPDH was reported to possess additional non-glycolytic activities, correlating with its nuclear or cytoskeletal localization in animal cells. In transiently transformed mesophyll protoplasts from Arabidopsis. thaliana colocalization and interaction of the glycolytic enzymes with the mitochondria and with the actin cytoskeleton was visualized by confocal laser scanning microscopy (cLSM using fluorescent protein fusions and by bimolecular fluorescence complementation (BiFC, respectively. Yeast two-hybrid screens, dot-blot overlay assays, and co-sedimentation assays were used to identify potential protein-protein interactions between two cytosolic GAPDH isoforms (GapC1, At3g04120; GapC2, At1g13440 from A. thaliana with the neighbouring glycolytic enzyme, fructose 1,6-bisphosphate aldolase (FBA6, At2g36460, the mitochondrial porin (VDAC3; At5g15090, and actin in vitro. From these experiments, a mitochondrial association is suggested for both glycolytic enzymes, GAPDH and aldolase, which appear to bind to the outer mitochondrial membrane, in a redox-dependent manner. In addition, both glycolytic enzymes were found to bind to F-actin in cosedimentation assays, and lead to bundling of purified rabbit actin, as visualized by cLSM. Actin binding and bundling occurred reversibly under oxidizing conditions. We speculate that such dynamic formation of microcompartments is part of a redox-dependent retrograde signal transduction network for adaptation upon oxidative stress.

  15. Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype.

    Science.gov (United States)

    Neuhuber, Birgit; Gallo, Gianluca; Howard, Linda; Kostura, Lisa; Mackay, Alastair; Fischer, Itzhak

    2004-07-15

    Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron-like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [2002] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time-lapse studies. Similar neurite-like processes were induced by treating MSC with DMSO only or with actin filament-depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages. Copyright 2004 Wiley-Liss, Inc.

  16. R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34(cdc2) expression in colonic adenocarcinoma SW480 cells.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Thorgeirsdottir, S; Broitman, S A

    1999-01-01

    Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K-ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from (14)C-mevalonate and decreased expression of the cell cycle regulatory kinase p34(cdc2). Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 microM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.

  17. Rho GTPases in platelet function.

    Science.gov (United States)

    Aslan, J E; McCarty, O J T

    2013-01-01

    The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low-molecular-weight or 'small' GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non-transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho-driven cellular processes. © 2012 International Society on Thrombosis and Haemostasis.

  18. Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors

    Science.gov (United States)

    Poggi, Marjorie; Canault, Matthias; Favier, Marie; Turro, Ernest; Saultier, Paul; Ghalloussi, Dorsaf; Baccini, Veronique; Vidal, Lea; Mezzapesa, Anna; Chelghoum, Nadjim; Mohand-Oumoussa, Badreddine; Falaise, Céline; Favier, Rémi; Ouwehand, Willem H.; Fiore, Mathieu; Peiretti, Franck; Morange, Pierre Emmanuel; Saut, Noémie; Bernot, Denis; Greinacher, Andreas; BioResource, NIHR; Nurden, Alan T.; Nurden, Paquita; Freson, Kathleen; Trégouët, David-Alexandre; Raslova, Hana; Alessi, Marie-Christine

    2017-01-01

    Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels. PMID:27663637

  19. Platelets

    Science.gov (United States)

    ... tiny fraction of the blood volume. The principal function of platelets is to prevent bleeding. Red blood cells are ... forming a long string. This illustrates the basic function of platelets, to stick to any foreign surface and then ...

  20. Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation.

    Science.gov (United States)

    Raslan, Zaher; Naseem, Khalid M

    2015-01-01

    Prostacyclin (PGI2) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI2. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI2 and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI2-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI2 were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI2. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.

  1. MiR-142-3p attenuates the migration of CD4⁺ T cells through regulating actin cytoskeleton via RAC1 and ROCK2 in arteriosclerosis obliterans.

    Science.gov (United States)

    Liu, Jiawei; Li, Wen; Wang, Siwen; Wu, Yidan; Li, Zilun; Wang, Wenjian; Liu, Ruiming; Ou, Jingsong; Zhang, Chunxiang; Wang, Shenming

    2014-01-01

    The migration of CD4+ T cells plays an important role in arteriosclerosis obliterans (ASO). However, the molecular mechanisms involved in CD4+ T cell migration are still unclear. The current study is aimed to determine the expression change of miR-142-3p in CD4+ T cells from patients with ASO and investigate its role in CD4+ T cell migration as well the potential mechanisms involved. We identified by qRT-PCR and in situ hybridization that the expression of miR-142-3p in CD4+ T cells was significantly down-regulated in patients with ASO. Chemokine (C-X-C motif) ligand 12 (CXCL12), a common inflammatory chemokine under the ASO condition, was able to down-regulate the expression of miR-142-3p in cultured CD4+ T cells. Up-regulation of miR-142-3p by lentivirus-mediated gene transfer had a strong inhibitory effect on CD4+ T cell migration both in cultured human cells in vitro and in mouse aortas and spleens in vivo. RAC1 and ROCK2 were identified to be the direct target genes in human CD4+ T cells, which are further confirmed by dual luciferase assay. MiR-142-3p had strong regulatory effects on actin cytoskeleton as shown by the actin staining in CD4+ T cells. The results suggest that the expression of miR-142-3p is down-regulated in CD4+ T cells from patients with ASO. The down-regulation of miR-142-3p could increase the migration of CD4+ T cells to the vascular walls by regulation of actin cytoskeleton via its target genes, RAC1 and ROCK2.

  2. MiR-142-3p attenuates the migration of CD4⁺ T cells through regulating actin cytoskeleton via RAC1 and ROCK2 in arteriosclerosis obliterans.

    Directory of Open Access Journals (Sweden)

    Jiawei Liu

    Full Text Available The migration of CD4+ T cells plays an important role in arteriosclerosis obliterans (ASO. However, the molecular mechanisms involved in CD4+ T cell migration are still unclear. The current study is aimed to determine the expression change of miR-142-3p in CD4+ T cells from patients with ASO and investigate its role in CD4+ T cell migration as well the potential mechanisms involved. We identified by qRT-PCR and in situ hybridization that the expression of miR-142-3p in CD4+ T cells was significantly down-regulated in patients with ASO. Chemokine (C-X-C motif ligand 12 (CXCL12, a common inflammatory chemokine under the ASO condition, was able to down-regulate the expression of miR-142-3p in cultured CD4+ T cells. Up-regulation of miR-142-3p by lentivirus-mediated gene transfer had a strong inhibitory effect on CD4+ T cell migration both in cultured human cells in vitro and in mouse aortas and spleens in vivo. RAC1 and ROCK2 were identified to be the direct target genes in human CD4+ T cells, which are further confirmed by dual luciferase assay. MiR-142-3p had strong regulatory effects on actin cytoskeleton as shown by the actin staining in CD4+ T cells. The results suggest that the expression of miR-142-3p is down-regulated in CD4+ T cells from patients with ASO. The down-regulation of miR-142-3p could increase the migration of CD4+ T cells to the vascular walls by regulation of actin cytoskeleton via its target genes, RAC1 and ROCK2.

  3. Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

    Directory of Open Access Journals (Sweden)

    F. Goulart-Silva

    2011-10-01

    Full Text Available The actions of thyroid hormone (TH on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a proinsulin mRNA expression, b proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c actin cytoskeleton arrangement, and d proinsulin mRNA poly(A tail length modulation in INS-1E cells cultured in different media containing: i normal fetal bovine serum - FBS (control; ii normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii FBS depleted of TH for 24 h (Tx. A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

  4. Effects of cyclic tensile strain on actin cytoskeleton rearrangement in annulus fibrosus cells%周期性牵张椎间盘纤维环细胞肌动蛋白骨架的重排

    Institute of Scientific and Technical Information of China (English)

    张德宏; 方鹏飞; 王兴盛; 赵继荣; 李晓娜

    2016-01-01

    BACKGROUND:When the intervertebral disc is under stress, the hydraulic pressure generated inside the nucleus pulposus makes the annulus fibrosus extend outward and expand, and the annulus colagen fibers are stretched so that the extracelular matrix of annulus fibrosus cels is also under the pressure. In the intervertebral disc, aggrecan is the main component of proteoglycans, matrix metaloproteinase-2 is a major enzyme for extracelular matrix degradation, and tissue inhibitor of metaloproteinase is a multifunctional specific inhibition factor for matrix metaloproteinase activity. There is a mutual regulation between the latter two to keep the homeostasis between them. OBJECTIVE: To investigate the mechanism of cyclic tensile strain in the metabolism of intervertebral disc annulus matrix. METHODS:Rat anulus fibrosus cels were subjected to 2% or 10% cyclic tensile strain at 1.0 Hz for 2 and 12 hours using Flexcel4000 tension system. Then cels were colected and cultured in conditioned medium for gene and protein detection. Real-time quantitative PCR was used to detect mRNA expression of aggrecan, matrix metaloproteinases-2 and tissue inhibitor of metaloproteinase-2. Gelatin zymography was used to detect matrix metaloproteinases-2 activity. RESULTS AND CONCLUSION:The use of 2% cyclic tensile strain had no obvious effect on the stress fiber of actin cytoskeleton, whereas actin cytoskeleton was depolymerized in response to 10% cyclic tensile strain. The 2% cyclic tensile strain raised the expression of Aggrecan at 12 hours; whereas raised the matrix metaloproteinases-2 and tissue inhibitor of metaloproteinase-2 at 2 hours, both of which were in homeostasis; matrix metaloproteinases-2 activity had no significant changes. 10% cyclic tensile strain had no effect on the mRNA expression of Aggrecan. No matter stretching 2 or 12 hours, the matrix metaloproteinases-2 was up-regulated, and the tissue inhibitor of metaloproteinase-2 was down-regulated, both of which were not in

  5. Direct Comparisons of the Morphology, Migration, Cell Adhesions, and Actin Cytoskeleton of Fibroblasts in Four Different Three-Dimensional Extracellular Matrices

    Science.gov (United States)

    Hakkinen, Kirsi M.; Harunaga, Jill S.; Doyle, Andrew D.

    2011-01-01

    Interactions between cells and the extracellular matrix are at the core of tissue engineering and biology. However, most studies of these interactions have used traditional two-dimensional (2D) tissue culture, which is less physiological than three-dimensional (3D) tissue culture. In this study, we compared cell behavior in four types of commonly used extracellular matrix under 2D and 3D conditions. Specifically, we quantified parameters of cell adhesion and migration by human foreskin fibroblasts in cell-derived matrix or hydrogels of collagen type I, fibrin, or basement membrane extract (BME). Fibroblasts in 3D were more spindle shaped with fewer lateral protrusions and substantially reduced actin stress fibers than on 2D matrices; cells failed to spread in 3D BME. Cell–matrix adhesion structures were detected in all matrices. Although the shapes of these cell adhesions differed, the total area per cell occupied by cell–matrix adhesions in 2D and 3D was nearly identical. Fibroblasts migrated most rapidly in cell-derived 3D matrix and collagen and migrated minimally in BME, with highest migration directionality in cell-derived matrix. This identification of quantitative differences in cellular responses to different matrix composition and dimensionality should help guide the development of customized 3D tissue culture and matrix scaffolds for tissue engineering. PMID:20929283

  6. Platelets recognize brain-specific glycolipid structures, respond to neurovascular damage and promote neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Ilya Sotnikov

    Full Text Available Platelets respond to vascular damage and contribute to inflammation, but their role in the neurodegenerative diseases is unknown. We found that the systemic administration of brain lipid rafts induced a massive platelet activation and degranulation resulting in a life-threatening anaphylactic-like response in mice. Platelets were engaged by the sialated glycosphingolipids (gangliosides integrated in the rigid structures of astroglial and neuronal lipid rafts. The brain-abundant gangliosides GT1b and GQ1b were specifically recognized by the platelets and this recognition involved multiple receptors with P-selectin (CD62P playing the central role. During the neuroinflammation, platelets accumulated in the central nervous system parenchyma, acquired an activated phenotype and secreted proinflammatory factors, thereby triggering immune response cascades. This study determines a new role of platelets which directly recognize a neuronal damage and communicate with the cells of the immune system in the pathogenesis of neurodegenerative diseases.

  7. The coordinate alteration of actin cytoskeleton, CD44 and matrix metalloproteinase-2 in the metastasis of breast cancer cells%转移相关分子链Actin-CD44-MMP-2在乳腺癌转移实验中的改变

    Institute of Scientific and Technical Information of China (English)

    赵威; 韩海勃; 林仲翔; 张志谦

    2011-01-01

    Objective To study the roles of actin and associated molecules in the control of human breast cancer cell malignant behaviors in vitro and in vivo.Methods A highly metastatic human breast cancer cell line BICR-H1 was compared with another breast cancer cell line MCF-7, which was well differentiated and non-metastatic.Western blot, immunofluorescence, gelatin zymography analysis and a chick embryonic chorioallantoic membrane (CAM) assay were used in this research.5~30 μg cisplatin or MMP-2 C terminal PEX domain were injected i.v.in CAM.Results BICR - H 1 expressed high level of CD44, which was closely associated with actin aggregates at the bottom side of attached cells.It was also shown with MMP-2 activity.On the contrary, MCF-7 cells showed weak disruption of actin cytoskeleton structures and a few actin aggregates.It expressed low or minimal level of CD44 and MMP-2.The expression of CD44 was down-regulated in cisplatin-treated BICR-H1 cells, and the activity of MMP-2 was also decreased upon PEX treatment.Both cell lines could form tumors in CAM, but only BICR-H1 cells could metastasize to distant tissues.Cisplatin inhibited the growth of BICR-H1 and MCF-7 cells in a time and dose dependent manner in CAM.The lung metastatic foci of BICR-H1 cells treated with 30 μg cisplatin were reduced from 30 ± 15/embryo (PBS group) to 8 ± 6/embryo, and the same dose of PEX could completely inhibit BICR-H1 metastasis.Conclusion It is concluded that actin cytoskeleton, CD44 and MMP-2 (ACM) molecular linkage is associated with breast cancer metastatic phenotypes, and both cisplatin and PEX can interfere with the ACM molecular linkage, resulting in the suppression of both tumor growth and metastasis.%目的 研究乳腺癌转移相关的分子机制及抑制体内外转移的作用和机制.方法 选择高、低转移性乳腺癌细胞系BICR-H1和MCF-7,用明胶底物非变性电泳分析法、Western blot和免疫荧光染色等方法,观察肌动蛋白、CD44

  8. Real-Time Visualization of Platelet Interaction With Micro Structured Surfaces.

    Science.gov (United States)

    Gester, Kathrin; Birtel, Stephan; Clauser, Johanna; Steinseifer, Ulrich; Sonntag, Simon Johannes

    2016-02-01

    Improving the hemocompatibility of artificial implants by micro structuring their surfaces has shown promising results, but the mechanisms which lead to this improvement are not yet understood. Therefore, we built a test setup for real-time visualization of platelet interaction with a plain and two micro structured surfaces. The micro structures, defined by the distance of the plain surface area between the structures, were chosen to be 3 and 30 μm, representing a positive and a negative effect on the hemocompatibility. The main part of the test setup was a flow chamber containing films of low density polyethylene (LDPE) with the differently structured surfaces. For different wall shear stresses, no considerable differences were observed in the platelet-surface interaction for all surface types. Whereas, major differences in flow behavior were observed when comparing the surfaces to each other. The platelets "rolled" along the smooth surface, being in constant contact with the surface material. Although the platelets "rolled" over the surface with small structures as well, they were only in contact with the tips of the structure and therefore had less surface contact with the foreign material. The increased distance and height of the structures of the last surface led to a trapping of platelets between the structures. This resulted in a longer contact time with the foreign material as well as a larger contact area, which both increase the risk of platelet activation, adhesion, and finally clotting. Our results showed the mechanisms which lead to these effects and thus revealed why micro structuring of surfaces impacts the hemocompatibility. Furthermore, we established a test setup which can be used for future investigations on the platelet-structure interactions.

  9. Effects of low dose of X-ray radiation on the actin cytoskeleton of osteoblasts%低剂量X线照射对成骨细胞纤维肌动蛋白的影响

    Institute of Scientific and Technical Information of China (English)

    黄群; 董启榕; 陈明; 徐炜; 王创利; 史高龙

    2015-01-01

    Objective To observe the effects of different doses of X-ray irradiation on the morphology,mierostructure changes and actin cytoskeleton of osteoblasts.The findings of this research will provide evidence for further study of low dose X-ray irradiation biological effects.Methods MC3T3-E1 cells were exposed to irradiation of 0.5,5.0 Gy.We investigated cellular morphological changes by phase contrast microscope and transmission electron microscopy.The organization of actin microfilaments was determined by immunofluorescence.Results After 2 h exposure to irradiation,the F-actin fluorescence intensity of ceils in 0.5,5.0 Gy group were significantly lower than non-irradiated group (25.329 ± 12.209,27.021 ± 13.049 vs.29.107 ± 13.296,P < 0.05).But 24 hours later,the fluorescence intensity of F-actin in 0.5,5.0 Gy group increased gradually and the fiber stress also increased.The most significant changes appeared in the third day after X-ray irrddiation (38.687 ± 18.072,36.039 ± 12.128 vs.35.645 ± 17.213).However,these changes gradually returned to normal in the fifth day,close to 0 Gy group (28.527 ±14.107,27.258 ±13.322 vs.27.309±15.039).Conclusion Thecytoskeleton of MC3TE-E cells were destroyed after 2 hours,exposure to X-ray irradiation.Howerver,0.5,5.0 Gy X-ray irradiation induced reorganization of actin filaments of MC3T3 cells 1 d later.%目的 观察不同剂量X线照射成骨细胞后,细胞形态、胞内微结构及纤维肌动蛋白的变化.方法 采用医用直线加速器以0、0.5、5.0Gy作用成骨细胞(MC3T3-E1)后,用倒置相差显微镜观察细胞形态变化,透射电镜观察细胞内微结构变化以及异硫酸氢荧光素-鬼笔环肽(FITC-phalloidin)对各实验组细胞的纤维肌动蛋白(F-actin)进行染色,荧光显微镜下观察各实验组F-actin细胞骨架的变化.结果 X线照射后2h,0.5、5.0Gy组细胞F-actin绿色荧光强度明显低于未照射组(25.329±12.209、27.021±13.049比29.107±13.296),

  10. Structural plasticity with preserved topology in the postsynaptic protein network

    OpenAIRE

    Blanpied, Thomas A.; Kerr, Justin M.; Ehlers, Michael D.

    2008-01-01

    The size, shape, and molecular arrangement of the postsynaptic density (PSD) determine the function of excitatory synapses in the brain. Here, we directly measured the internal dynamics of scaffold proteins within single living PSDs, focusing on the principal scaffold protein PSD-95. We found that individual PSDs undergo rapid, continuous changes in morphology driven by the actin cytoskeleton and regulated by synaptic activity. This structural plasticity is accompanied by rapid fluctuations i...

  11. Platelet lipidomic.

    Science.gov (United States)

    Dolegowska, B; Lubkowska, A; De Girolamo, L

    2012-01-01

    Lipids account for 16-19 percent dry platelet matter and includes 65 percent phospholipids, 25 percent neutral lipids and about 8 percent glycosphingolipids. The cell membrane that surrounds platelets is a bilayer that contains different types phospholipids symmetrically distributed in resting platelets, such as phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine, and sphingomyelin. The collapse of lipid asymmetry is exposure of phosphatidylserine in the external leaflet of the plasma bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. During activation, this asymmetrical distribution becomes disrupted, and PS and PE become exposed on the cell surface. The transbilayer movement of phosphatidylserine is responsible for the platelet procoagulant activity. Exposure of phosphatidylserine is a flag for macrophage recognition and clearance from the circulation. Platelets, stored at room temperature for transfusion for more than 5 days, undergo changes collectively known as platelet storage lesions. Thus, the platelet lipid composition and its possible modifications over time are crucial for efficacy of platelet rich plasma therapy. Moreover, a number of substances derived from lipids are contained into platelets. Eicosanoids are lipid signaling mediators generated by the action of lipoxygenase and include prostaglandins, thromboxane A2, 12-hydroxyeicosatetraenoic acid. Isoprostanes have a chemical structure similar to this of prostanoids, but are differently produced into the particle, and are ligands for prostaglandins receptors, exhibiting biological activity like thromboxane A2. Endocannabinoids are derivatives from arachidonic acid which could reduce local pain. Phospholipids growth factors (sphingolipids, lysophosphatidic acid, platelet-activating factor) are involved in tissue

  12. Fibrin clot structure and platelet aggregation in patients with aspirin treatment failure.

    Directory of Open Access Journals (Sweden)

    Søs Neergaard-Petersen

    Full Text Available BACKGROUND: Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF. This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF. METHODS: We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66% had ATF defined as myocardial infarction (MI whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics. RESULTS: Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222 vs. 97 (60; 1776 AU*min, p = 0.005 and collagen 1.0 µg/mL (293 (198; 427 vs. 220 (165; 370 AU*min, p = 0.03. Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52 vs. 0.42 (0.38; 0.50, p = 0.02, and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003 and prolonged lysis time (552 (498; 756 vs. 519 (468; 633 seconds; p = 0.02. Patients with ATF also had increased levels of C-reactive protein (CRP (1.34 (0.48; 2.94 and 0.88 (0.32; 1.77 mg/L, p = 0.01 compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31-0.35, p-values<0.001 and CRP levels (r = 0.60, p<0.001. CONCLUSIONS: Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet

  13. Fibrin clot structure and platelet aggregation in patients with aspirin treatment failure.

    Science.gov (United States)

    Neergaard-Petersen, Søs; Ajjan, Ramzi; Hvas, Anne-Mette; Hess, Katharina; Larsen, Sanne Bøjet; Kristensen, Steen Dalby; Grove, Erik Lerkevang

    2013-01-01

    Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF. We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics. Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31-0.35, p-valuesaspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.

  14. Structural basis for quinine-dependent antibody binding to platelet integrin αIIbβ3.

    Science.gov (United States)

    Zhu, Jianghai; Zhu, Jieqing; Bougie, Daniel W; Aster, Richard H; Springer, Timothy A

    2015-10-29

    Drug-induced immune thrombocytopenia (DITP) is caused by antibodies that react with specific platelet-membrane glycoproteins when the provoking drug is present. More than 100 drugs have been implicated as triggers for this condition, quinine being one of the most common. The cause of DITP in most cases appears to be a drug-induced antibody that binds to a platelet membrane glycoprotein only when the drug is present. How a soluble drug promotes binding of an otherwise nonreactive immunoglobulin to its target, leading to platelet destruction, is uncertain, in part because of the difficulties of working with polyclonal human antibodies usually available only in small quantities. Recently, quinine-dependent murine monoclonal antibodies were developed that recognize a defined epitope on the β-propeller domain of the platelet integrin αIIb subunit (GPIIb) only when the drug is present and closely mimic the behavior of antibodies found in human patients with quinine-induced thrombocytopenia in vitro and in vivo. Here, we demonstrate specific, high-affinity binding of quinine to the complementarity-determining regions (CDRs) of these antibodies and define in crystal structures the changes induced in the CDR by this interaction. Because no detectable binding of quinine to the target integrin could be demonstrated in previous studies, the findings indicate that a hybrid paratope consisting of quinine and reconfigured antibody CDR plays a critical role in recognition of its target epitope by an antibody and suggest that, in this type of drug-induced immunologic injury, the primary reaction involves binding of the drug to antibody CDRs, causing it to acquire specificity for a site on a platelet integrin.

  15. A Study on the radiation effects for the function and structure of rabbit blood platelets in various dose rates

    Energy Technology Data Exchange (ETDEWEB)

    Okumura, Kohichi (Nippon Dental Univ., Tokyo (Japan))

    1991-12-01

    Mature peripheral platelets in rabbits were irradiated with a total 10 Gy of {sup 60}Co-{gamma} rays at the average dose rates of 0.2, 0.5, 1.0, 1.5 and 1.7 Gy/min. The effects was evaluated from the functional aspect by determining the ability of platelets to aggregate and replease, and the metabolic aspect by examining the kinetics of prostaglandin in platelets. In addition, platelet structure was compared using an electron microscope. The ability of platelets to aggregate and release was accelerated in all irradiated groups, compared with a non-irradiated group, especially in groups with average dose rates of 0.5 Gy/min and 1.0 Gy/min. The amount of MDA, a final product of prostaglandin in platelets, increased in all irradiated groups in comparison with the non-irradiated group, especially in the 0.5 Gy/min, 1.0 Gy/min and 1.5 Gy/min groups. Observation with a scanning electron microscope revealed a clear rock-like appearance of the surface of aggregates of platelets and a larger number of pseudopodia with longer projections in the 1.0 Gy/min group than in the non-irradiated group. Moreover, the surfaces of the aggregates in the 1.7 Gy/min group, but the adhension between psudopodia of the platelet aggregates was weaker than that of 1.0 Gy/min group. In observation with a transmission electron microscope, dense bodies that released their contents were noticed in platelet aggregates, and a stenopeic appearance between psudopodia and between platelets, and density aggregated platelets were observed in the 1.0 Gy/min irradiated group. Vacuolation of granules in platelets was more marked in aggregates of 1.7 Gy/min group than in that of the non-irradiated group, and large numbers of platelets with uneven surfaces were observed. Therefore, the effects of dose rates were found to be closely related to changes in structures, as well as to the inner function of platelets. (author).

  16. Structural Basis for Platelet Collagen Responses by the Immune-type Receptor Glycoprotein VI

    Energy Technology Data Exchange (ETDEWEB)

    Horii,K.; Kahn, M.; Herr, A.

    2006-01-01

    Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immunetype receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.

  17. Micro-structuring of polycarbonate-urethane surfaces in order to reduce platelet activation and adhesion.

    Science.gov (United States)

    Clauser, Johanna; Gester, Kathrin; Roggenkamp, Jan; Mager, Ilona; Maas, Judith; Jansen, Sebastian V; Steinseifer, Ulrich

    2014-01-01

    In the development of new hemocompatible biomaterials, surface modification appears to be a suitable method in order to reduce the thrombogenetic potential of such materials. In this study, polycarbonate-urethane (PCU) tubes with different surface microstructures to be used for aortic heart valve models were investigated with regard to the thrombogenicity. The surface structures were produced by using a centrifugal casting process for manufacturing PCU tubes with defined casting mold surfaces which are conferred to the PCU surface during the process. Tubes with different structures defined by altering groove widths were cut into films and investigated under dynamic flow conditions in contact with porcine blood. The analysis was carried out by laser scanning microscopy which allowed for counting various morphological types of platelets with regard to the grade of activation. The comparison between plain and shaped PCU samples showed that the surface topography led to a decline of the activation of the coagulation cascade and thus to the reduction of the fibrin synthesis. Comparing different types of structures revealed that smooth structures with a small groove width (d ~ 3 μm) showed less platelet activation as well as less adhesion in contrast to a distinct wave structure (d ~ 90 μm). These results prove surface modification of polymer biomaterials to be a suitable method for reducing thrombogenicity and hence give reason for further alterations and improvements.

  18. A functional interplay between the small GTPase Rab11a and mitochondria-shaping proteins regulates mitochondrial positioning and polarization of the actin cytoskeleton downstream of Src family kinases.

    Science.gov (United States)

    Landry, Marie-Claude; Champagne, Claudia; Boulanger, Marie-Chloé; Jetté, Alexandra; Fuchs, Margit; Dziengelewski, Claire; Lavoie, Josée N

    2014-01-24

    It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

  19. Platelets and hemostasis

    Directory of Open Access Journals (Sweden)

    M. A. Panteleev

    2014-09-01

    Full Text Available Platelets are anuclear cell fragments playing important role in hemostasis, termination of bleeding after damage, as well as in pathological thrombus formation. The main action of platelets is the formation of aggregates, overlapping the injury. They obtained the ability to aggregate by the transition process called activation. Despite the relatively simple and definite function platelet structure is very difficult: they have almost a full set of organelles, including the endoplasmic reticulum, mitochondria and other entities. When activated platelets secrete various granules interact with plasma proteins and red blood cells and other tissues. Their activation is controlled by multiple receptors and complex signaling cascades. In this review platelet structure, mechanisms of its functioning in health and disease, diagnostic methods of platelet function and approaches to their correction were considered. Particular attention will be given to those areas of the science of platelets, which still lay hidden mysteries.

  20. Defining Platelet Function During Polytrauma

    Science.gov (United States)

    2013-02-01

    using calibrated automated thrombography ( CAT ). 3. Platelet-induced clot contraction and using viscoelastic measures such as TEG with Platelet Mapping...using calibrated automated thrombography ( CAT ) in platelet-rich plasma. 3. Platelet-induced clot contraction and effect on clot structure by platelet...if injury with stable vital signs on initial evaluation.  Pregnancy (confirmed with urine pregnancy testing)  Documented do not resuscitate order

  1. Actin cytoskeleton: putting a CAP on actin polymerization.

    Science.gov (United States)

    Stevenson, V A; Theurkauf, W E

    2000-10-05

    Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

  2. Control of the actin cytoskeleton in plant cell growth

    NARCIS (Netherlands)

    Hussey, P.J.; Ketelaar, M.J.; Deeks, M.J.

    2006-01-01

    Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the a

  3. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

    Science.gov (United States)

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

  4. Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba

    Science.gov (United States)

    Negrete, Jose; Pumir, Alain; Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Beta, Carsten; Bodenschatz, Eberhard

    2016-09-01

    Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

  5. Thermal-structural analysis of the platelet heat-pipe-cooled leading edge of hypersonic vehicle

    Science.gov (United States)

    Hongpeng, Liu; Weiqiang, Liu

    2016-10-01

    One of the main challenges for the hypersonic vehicle is its thermal protection, more specifically, the cooling of its leading edge. To investigate the feasibility of a platelet heat-pipe-cooled leading edge structure, thermal/stress distributions for steady-state flight conditions are calculated numerically. Studies are carried on for IN718/Na, C-103/Na and T-111/Li compatible material combinations of heat pipe under nominal operations and a central heat pipe failure cases, and the influence of wall thickness on the design robustness is also investigated. And the heat transfer limits (the sonic limit, the capillary limit and the boiling limit) are also computed to check the operation of platelet heat pipes. The results indicate that, with a 15 mm leading edge radius and a wall thickness of 0.5 mm, C-103/Na and T-111/Li combinations of heat pipe is capable of withstanding both nominal and failure conditions for Mach 8 and Mach 10 flight respectively.

  6. The Structure and Regulation of Human Muscle α-Actinin

    OpenAIRE

    Ribeiro, Euripedes de Almeida; Pinotsis, Nikos; Holt, Mark R.; Aachmann, Finn L.; Žagrović, Bojan; Bordignon, Enrica; Pirker, Katharina F.; Svergun, Dmitri; Gautel, Mathias; Djinović-Carugo, Kristina; Ghisleni, Andrea; Salmazo, Anita; Konarev, Petr; Kostan, Julius; Sjöblom, Björn

    2014-01-01

    Summary The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer f...

  7. Universal composition-structure-property maps for natural and biomimetic platelet-matrix composites and stacked heterostructures.

    Science.gov (United States)

    Sakhavand, Navid; Shahsavari, Rouzbeh

    2015-03-16

    Many natural and biomimetic platelet-matrix composites--such as nacre, silk, and clay-polymer-exhibit a remarkable balance of strength, toughness and/or stiffness, which call for a universal measure to quantify this outstanding feature given the structure and material characteristics of the constituents. Analogously, there is an urgent need to quantify the mechanics of emerging electronic and photonic systems such as stacked heterostructures. Here we report the development of a unified framework to construct universal composition-structure-property diagrams that decode the interplay between various geometries and inherent material features in both platelet-matrix composites and stacked heterostructures. We study the effects of elastic and elastic-perfectly plastic matrices, overlap offset ratio and the competing mechanisms of platelet versus matrix failures. Validated by several 3D-printed specimens and a wide range of natural and synthetic materials across scales, the proposed universally valid diagrams have important implications for science-based engineering of numerous platelet-matrix composites and stacked heterostructures.

  8. Structures of a platelet-derived growth factor/propeptide complex and a platelet-derived growth factor/receptor complex

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Ann Hye-Ryong; Liu, Heli; Focia, Pamela J.; Chen, Xiaoyan; Lin, P. Charles; He, Xiaolin (Vanderbilt); (NWU)

    2010-07-13

    Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are prototypic growth factors and receptor tyrosine kinases which have critical functions in development. We show that PDGFs share a conserved region in their prodomain sequences which can remain noncovalently associated with the mature cystine-knot growth factor domain after processing. The structure of the PDGF-A/propeptide complex reveals this conserved, hydrophobic association mode. We also present the structure of the complex between PDGF-B and the first three Ig domains of PDGFR{beta}, showing that two PDGF-B protomers clamp PDGFR{beta} at their dimerization seam. The PDGF-B:PDGFR{beta} interface is predominantly hydrophobic, and PDGFRs and the PDGF propeptides occupy overlapping positions on mature PDGFs, rationalizing the need of propeptides by PDGFs to cover functionally important hydrophobic surfaces during secretion. A large-scale structural organization and rearrangement is observed for PDGF-B upon receptor binding, in which the PDGF-B L1 loop, disordered in the structure of the free form, adopts a highly specific conformation to form hydrophobic interactions with the third Ig domain of PDGFR{beta}. Calorimetric data also shows that the membrane-proximal homotypic PDGFR{alpha} interaction, albeit required for activation, contributes negatively to ligand binding. The structural and biochemical data together offer insights into PDGF-PDGFR signaling, as well as strategies for PDGF-antagonism.

  9. Effect of construction of TiO2 nanotubes on platelet behaviors: Structure-property relationships.

    Science.gov (United States)

    Huang, Qiaoling; Yang, Yun; Zheng, Dajiang; Song, Ran; Zhang, Yanmei; Jiang, Pinliang; Vogler, Erwin A; Lin, Changjian

    2017-03-15

    Blood compatibility of TiO2 nanotubes (TNTs) has been assessed in rabbit platelet-rich plasma (PRP), which combines activation of both blood plasma coagulation and platelets. We find that (i) amorphous TiO2 nanotubes (TNTs) with relatively larger outer diameters led to reduced platelet adhesion/activation, (ii) TNTs with relatively smaller outer diameters in a predominately rutile phase also inhibited platelet adhesion and activation, and (iii) a pervasive fibrin network formed on larger outer diameter TNTs in a predominately anatase phase. Thus, this study suggests that combined effect of crystalline phase and surface chemistry controls blood-contact behavior of TNTs. A more comprehensive mechanism is proposed for understanding hemocompatibility of TiO2 which might prove helpful as a guide to prospective design of TiO2-based biomaterials.

  10. Electrical Transport and Network Percolation in Graphene and Boron Nitride Mixed-Platelet Structures.

    Science.gov (United States)

    Debbarma, Rousan; Behura, Sanjay; Nguyen, Phong; Sreeprasad, T S; Berry, Vikas

    2016-04-06

    Percolating network of mixed 2D nanomaterials (2DNs) can leverage the unique electronic structures of different 2DNs, their interfacial doping, manipulable conduction pathways, and local traps. Here, we report on the percolation mechanism and electro-capacitive transport pathways of mixed-platelet network of hexagonal boron nitride (hBN) and reduced graphene oxide (rGO), two isostructural and isoelectronic 2DNs. The transport mechanism is explained in terms of electron hopping through isolated hBN defect traps between rGO (possibly via electron tunneling/hopping through "funneling" points). With optical bandgaps of 4.57 and 4.08 eV for the hBN-domains and 2.18 eV for the rGO domains, the network of hBN with rGO exhibits Poole-Frenkel emission-based transport with mean hopping gap of 1.12 nm (∼hBN trilayer) and an activation barrier of ∼15 ± 0.7 meV. Further, hBN (1.7 pF) has a 6-fold lower capacitance than 1:1 hBN:rGO, which has a resistance 2 orders of magnitude higher than that of rGO (1.46 MΩ). These carrier transport results can be applied to other multi-2DN networks for development of next-generation functional 2D-devices.

  11. Platelet mimicry

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Hunter, Alan Christy; Peer, Dan

    2016-01-01

    Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities...

  12. Platelet Count

    Science.gov (United States)

    ... their spleen removed surgically Use of birth control pills (oral contraceptives) Some conditions may cause a temporary (transitory) increased ... increased platelet counts include estrogen and birth control pills (oral contraceptives). Mildly decreased platelet counts may be seen in ...

  13. Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis

    DEFF Research Database (Denmark)

    Prats, Clara; Cadefau, Joan A; Cussó, Roser;

    2005-01-01

    . Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed...... structures that were not present in basal muscle, and we present evidence that indicate that they are products of actin cytoskeleton remodeling. Furthermore, for the first time, we show a phosphorylation-dependent intracellular distribution of GS. Here, we present evidence of a new regulatory mechanism...

  14. Platelet proteomics.

    Science.gov (United States)

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  15. Structure formation in active networks

    CERN Document Server

    Köhler, Simone; Bausch, Andreas R

    2011-01-01

    Structure formation and constant reorganization of the actin cytoskeleton are key requirements for the function of living cells. Here we show that a minimal reconstituted system consisting of actin filaments, crosslinking molecules and molecular-motor filaments exhibits a generic mechanism of structure formation, characterized by a broad distribution of cluster sizes. We demonstrate that the growth of the structures depends on the intricate balance between crosslinker-induced stabilization and simultaneous destabilization by molecular motors, a mechanism analogous to nucleation and growth in passive systems. We also show that the intricate interplay between force generation, coarsening and connectivity is responsible for the highly dynamic process of structure formation in this heterogeneous active gel, and that these competing mechanisms result in anomalous transport, reminiscent of intracellular dynamics.

  16. Platelet antigens and antibodies. Literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2013-01-01

    Full Text Available Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.

  17. Platelet antigens and antibodies. Literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2014-07-01

    Full Text Available Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.

  18. Reduced platelet adhesion on the surface of polyurethane bearing structure of sulfobetaine.

    Science.gov (United States)

    Yuan, J; Zhang, J; Zhu, J; Shen, J; Lin, S C; Zhu, W; Fang, J L

    2003-10-01

    Poly(etherurethane)s are widely used as blood-contacting biomaterials due to their good biocompatibility and mechanical properties. Nevertheless, their blood compatibility is still not adequate for the more demanding applications. Surface modification is an effective way to improve the blood compatibility and retain the bulk properties of biomaterials. The purpose of present study was to design and synthesize a novel nonthrombogenic biomaterial by modifying the surface of poly(etherurethane) with zwitterionic monomer. Films of polyurethane were grafted with sulfobetaine by a three-step procedure. In the first step, the film surfaces were treated with hexamethylene diisocyanate (HDI) in toluene at 50 degrees C in the presence of di-n-butyl tin dilaurate (DBTDL) as a catalyst. The extent of the reaction was measured by ATR-IR spectra; a maximum number of free NCO group was obtained after a reaction time of 90 min. In the second step, the hydroxyl group of 4-dimethylamino-1-butanol (DMAB) was allowed to react in toluene with isocyanate groups bound on the surface. In the third step, sulfobetaine was formed on the surface through the ring-opening reaction between tertiary amine of DMAB and 1,3- propane-sultone (PS). It was characterized by ATR-IR, XPS. The data showed that the grafted surfaces were composed of sulfobetaine. The results of the contact angle measurements showed that they were strongly hydrophilic. The state of platelet adhesion and shape variation for the attached platelets was described. The modified surface shows excellent blood compatibility feature by the low platelet adhesion.

  19. Ubiquitin/proteasome-rich particulate cytoplasmic structures (PaCSs) in the platelets and megakaryocytes of ANKRD26-related thrombo-cytopenia.

    Science.gov (United States)

    Necchi, Vittorio; Balduini, Alessandra; Noris, Patrizia; Barozzi, Serena; Sommi, Patrizia; di Buduo, Christian; Balduini, Carlo L; Solcia, Enrico; Pecci, Alessandro

    2013-02-01

    ANKRD26-related thrombocytopenia (ANKRD26-RT) is an autosomal-dominant thrombocytopenia caused by mutations in the 5'UTR of the ANKRD26 gene. ANKRD26-RT is characterised by dysmegakaryopoiesis and an increased risk of leukaemia. PaCSs are novel particulate cytoplasmic structures with selective immunoreactivity for polyubiquitinated proteins and proteasome that have been detected in a number of solid cancers, in the epithelia of Helicobacter pylori gastritis and related preneoplastic lesions, and in the neutrophils of Schwachman-Diamond syndrome, a genetic disease with neutropenia and increased leukaemia risk. We searched for PaCSs in blood cells from 14 consecutive patients with ANKRD26-RT. Electron microscopy combined with immunogold staining for polyubiquitinated proteins, 20S and 19S proteasome showed PaCSs in most ANKRD26-RT platelets, as in a restricted minority of platelets from healthy controls and from subjects with other inherited or immune thrombocytopenias. In ANKRD26-RT platelets, the PaCS amount exceeded that of control platelets by a factor of 5 (p<0.0001). Immunoblotting showed that the higher PaCS number was associated with increased amounts of polyubiquitinated proteins and proteasome in ANKRD26-RT platelets. PaCSs were also extensively represented in ANKRD26-RT megakaryocytes, but not in healthy control megakaryocytes, and were absent in other ANKRD26-RT and control blood cells. Therefore, large amounts of PaCSs are a characteristic feature of ANKRD26-RT platelets and megakaryocytes, although these novel cell components are also present in a small subpopulation of normal platelets. The widespread presence of PaCSs in inherited diseases with increased leukaemia risk, as well as in solid neoplasms and their preneoplastic lesions, suggests a link of these structures with oncogenesis.

  20. Molecular cloning, genomic structure, chromosomal localization, and alternative splice forms of the platelet collagen receptor glycoprotein VI.

    Science.gov (United States)

    Ezumi, Y; Uchiyama, T; Takayama, H

    2000-10-14

    Glycoprotein VI (GPVI) is the major collagen receptor underlying platelet activation. We cloned the full-length cDNA for GPVI (GPVI-1) and its two isoforms (GPVI-2 and -3) from phorbol-ester-stimulated CMK cells. The GPVI-1 cDNA was identical in the coding region with the cDNA that has recently been reported to belong to the immunoglobulin superfamily. The GPVI gene consisted of 8 exons spanning over 23 kbp and was mapped on the chromosome 19q13. 4. The promoter of GPVI gene lacked TATA and CAAT boxes and had multiple transcription start sites like other megakaryocytic genes. When COS-7 cells were cotransfected with the GPVI isoforms and Fc receptor gamma chain, Fc receptor gamma chain was associated with GPVI-1 and -2 but did not affect the GPVI expression levels. GPVI-1 and -2 could bind the collagen-related peptide, which exhibits triple-helical and polymeric structure of collagen to activate platelets via GPVI. Copyright 2000 Academic Press.

  1. Acquired platelet function defect

    Science.gov (United States)

    Acquired qualitative platelet disorders; Acquired disorders of platelet function ... blood clotting. Disorders that can cause problems in platelet function include: Idiopathic thrombocytopenic purpura Chronic myelogenous leukemia Multiple ...

  2. Platelet Donation

    Science.gov (United States)

    ... of gratitude that washed over me when I saw those platelets going into my husband’s body. I ... Needles LGBTQ+ Donors Blood Donor Community SleevesUp Games Facebook Avatars and Badges Banners eCards Red Cross Information ...

  3. Probing cell structure responses through a shear and stretching mechanical stimulation technique.

    Science.gov (United States)

    Steward, Robert L; Cheng, Chao-Min; Wang, Danny L; LeDuc, Philip R

    2010-04-01

    Cells are complex, dynamic systems that respond to various in vivo stimuli including chemical, mechanical, and scaffolding alterations. The influence of mechanics on cells is especially important in physiological areas that dictate what modes of mechanics exist. Complex, multivariate physiological responses can result from multi-factorial, multi-mode mechanics, including tension, compression, or shear stresses. In this study, we present a novel device based on elastomeric materials that allowed us to stimulate NIH 3T3 fibroblasts through uniaxial strip stretching or shear fluid flow. Cell shape and structural response was observed using conventional approaches such as fluorescent microscopy. Cell orientation and actin cytoskeleton alignment along the direction of applied force were observed to occur after an initial 3 h time period for shear fluid flow and static uniaxial strip stretching experiments although these two directions of alignment were oriented orthogonal relative to each other. This response was then followed by an increasingly pronounced cell and actin cytoskeleton alignment parallel to the direction of force after 6, 12, and 24 h, with 85% of the cells aligned along the direction of force after 24 h. These results indicate that our novel device could be implemented to study the effects of multiple modes of mechanical stimulation on living cells while probing their structural response especially with respect to competing directions of alignment and orientation under these different modes of mechanical stimulation. We believe that this will be important in a diversity of fields including cell mechanotransduction, cell-material interactions, biophysics, and tissue engineering.

  4. Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling.

    Science.gov (United States)

    Pakhomov, Andrei G; Xiao, Shu; Pakhomova, Olga N; Semenov, Iurii; Kuipers, Marjorie A; Ibey, Bennett L

    2014-12-01

    Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.

  5. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    Science.gov (United States)

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-02-24

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. Evidence for Lipid Packaging in the Crystal Structure of the GM2-Activator Complex with Platelet Activating Factor

    Energy Technology Data Exchange (ETDEWEB)

    Wright, Christine S.; Mi, Li-Zhi; Rastinejad, Fraydoon (Virginia)

    2010-11-16

    GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2 {angstrom} crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.

  7. Fine structure of synapses on dendritic spines

    Directory of Open Access Journals (Sweden)

    Michael eFrotscher

    2014-09-01

    Full Text Available Camillo Golgi’s Reazione Nera led to the discovery of dendritic spines, small appendages originating from dendritic shafts. With the advent of electron microscopy (EM they were identified as sites of synaptic contact. Later it was found that changes in synaptic strength were associated with changes in the shape of dendritic spines. While live-cell imaging was advantageous in monitoring the time course of such changes in spine structure, EM is still the best method for the simultaneous visualization of all cellular components, including actual synaptic contacts, at high resolution. Immunogold labeling for EM reveals the precise localization of molecules in relation to synaptic structures. Previous EM studies of spines and synapses were performed in tissue subjected to aldehyde fixation and dehydration in ethanol, which is associated with protein denaturation and tissue shrinkage. It has remained an issue to what extent fine structural details are preserved when subjecting the tissue to these procedures. In the present review, we report recent studies on the fine structure of spines and synapses using high-pressure freezing (HPF, which avoids protein denaturation by aldehydes and results in an excellent preservation of ultrastructural detail. In these studies, HPF was used to monitor subtle fine-structural changes in spine shape associated with chemically induced long-term potentiation (cLTP at identified hippocampal mossy fiber synapses. Changes in spine shape result from reorganization of the actin cytoskeleton. We report that cLTP was associated with decreased immunogold labeling for phosphorylated cofilin (p-cofilin, an actin-depolymerizing protein. Phosphorylation of cofilin renders it unable to depolymerize F-actin, which stabilizes the actin cytoskeleton. Decreased levels of p-cofilin, in turn, suggest increased actin turnover, possibly underlying the changes in spine shape associated with cLTP. The findings reviewed here establish HPF as

  8. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro.

    Science.gov (United States)

    Mangalpally, Kiran Kumar R; Siqueiros-Garcia, Alan; Vaduganathan, Muthiah; Dong, Jing-Fei; Kleiman, Neal S; Guthikonda, Sasidhar

    2010-10-01

    Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 μM arachidonic acid (AA), 10 μM ADP or 25 μM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbβ3) were assessed. Platelet thromboxane B(2) (TxB(2)) synthesis following AA stimulation was measured in vitro before and after incubation with 265 μM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbβ3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin. Platelets exhibit heterogeneity in size and structure. Whether this translates into platelet function and efficacy of antiplatelet therapy is unclear. We evaluated platelet functional properties and the effects of aspirin on separated platelet subpopulations in an in vitro setting. Platelets were sorted into the largest and smallest size quintiles using flow cytometry forward scatter. Alpha-granule protein release, dense granule content

  9. Determination of structural changes of dispersed clay platelets in a polymer blend during solid-state rheological property measurement by small-angle X-ray scattering

    CSIR Research Space (South Africa)

    Bandyopadhyay, J

    2011-05-01

    Full Text Available -1 Polymer Volume 52, Issue 12, 26 May 2011, Pages 2628?2642 Determination of structural changes of dispersed clay platelets in a polymer blend during solid-state rheological property measurement by small-angle X-ray scattering ? Jayita Bandyopadhyaya... frequency and temperature sweep tests. Graphical abstract Keywords ? Blend composites; ? Small-angle X-ray scattering; ? Solid-state rheology ...

  10. Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen*

    OpenAIRE

    Herr, Andrew B.; Farndale, Richard W.

    2009-01-01

    Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α2β1, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of ...

  11. Blood platelets in the progression of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Nina S Gowert

    Full Text Available Alzheimer's disease (AD is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA. Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

  12. Platelet Function Tests

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Platelet Function Tests Share this page: Was this page helpful? ... their patients by ordering one or more platelet function tests. Platelet function testing may include one or more of ...

  13. Congenital platelet function defects

    Science.gov (United States)

    ... storage pool disorder; Glanzmann's thrombasthenia; Bernard-Soulier syndrome; Platelet function defects - congenital ... Congenital platelet function defects are bleeding disorders that ... function, even though there are normal platelet numbers. Most ...

  14. Platelet Function Tests in Bleeding Disorders.

    Science.gov (United States)

    Lassila, Riitta

    2016-04-01

    Functional disorders of platelets can involve any aspect of platelet physiology, with many different effects or outcomes. These include platelet numbers (thrombocytosis or thrombocytopenia); changes in platelet production or destruction, or capture to the liver (Ashwell receptor); altered adhesion to vascular injury sites and/or influence on hemostasis and wound healing; and altered activation or receptor functions, shape change, spreading and release reactions, procoagulant and antifibrinolytic activity. Procoagulant membrane alterations, and generation of thrombin and fibrin, also affect platelet aggregation. The above parameters can all be studied, but standardization and quality control of assay methods have been limited despite several efforts. Only after a comprehensive clinical bleeding assessment, including family history, information on drug use affecting platelets, and exclusion of coagulation factor, and tissue deficits, should platelet function testing be undertaken to confirm an abnormality. Current diagnostic tools include blood cell counts, platelet characteristics according to the cell counter parameters, peripheral blood smear, exclusion of pseudothrombocytopenia, whole blood aggregometry (WBA) or light transmission aggregometry (LTA) in platelet-rich plasma, luminescence, platelet function analysis (PFA-100) for platelet adhesion and deposition to collagen cartridges under blood flow, and finally transmission electron microscopy to exclude rare structural defects leading to functional deficits. The most validated test panels are included in WBA, LTA, and PFA. Because platelets are isolated from their natural environment, many simplifications occur, as circulating blood and interaction with vascular wall are omitted in these assays. The target to reach a highly specific platelet disorder diagnosis in routine clinical management can be exhaustive, unless needed for genetic counseling. The elective overall assessment of platelet function disorder

  15. Actin: Structure, Function, Dynamics, and Interactions with Bacterial Toxins.

    Science.gov (United States)

    Kühn, Sonja; Mannherz, Hans Georg

    Actin is one of the most abundant proteins in any eukaryotic cell and an indispensable component of the cytoskeleton. In mammalian organisms, six highly conserved actin isoforms can be distinguished, which differ by only a few amino acids. In non-muscle cells, actin polymerizes into actin filaments that form actin structures essential for cell shape stabilization, and participates in a number of motile activities like intracellular vesicle transport, cytokinesis, and also cell locomotion. Here, we describe the structure of monomeric and polymeric actin, the polymerization kinetics, and its regulation by actin-binding proteins. Probably due to its conserved nature and abundance, actin and its regulating factors have emerged as prefered targets of bacterial toxins and effectors, which subvert the host actin cytoskeleton to serve bacterial needs.

  16. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors.

    Science.gov (United States)

    Takayama, Naoya; Nishikii, Hidekazu; Usui, Joichi; Tsukui, Hiroko; Sawaguchi, Akira; Hiroyama, Takashi; Eto, Koji; Nakauchi, Hiromitsu

    2008-06-01

    Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

  17. Detection of microbial contamination in platelets

    Science.gov (United States)

    Berg, Tracy L.; Leparc, German; Huffman, Debra E.; Gennaccaro, Angela L.; Garcia-Lopez, Alicia; Klungness, Greta; Stephans, Christie; Garcia-Rubio, Luis H.

    2005-03-01

    In the United States, approximately 100 patients develop fatal sepsis associated with platelet transfusions every year. Current culture methods take 24-48 hours to acquire results, which in turn decrease the shelf life of platelets. Many of the microorganisms that contaminate platelets can replicate easily at room temperature, which is the necessary storage temperature to keep platelets functional. Therefore, there is a need for in-situ quality control assessment of the platelet quality. For this purpose, a real time spectrophotometric technique has been developed. The Spectral Acquisition Processing Detection (SAPD) method, comprised of a UV-vis spectrophotometer and modeling algorithms, is a rapid method that can be performed prior to platelet transfusion to decrease the risk of bacterial infection to patients. The SAPD method has been used to determine changes in cell suspensions, based on size, shape, chemical composition and internal structure. Changes in these cell characteristics can in turn be used to determine microbial contamination, platelet aging and other physiologic changes. Detection limits of this method for platelet suspensions seeded with bacterial contaminants were identified to be less than 100 cfu/ml of sample. Bacterial counts below 1000 cfu/ml are not considered clinically significant. The SAPD method can provide real-time identification of bacterial contamination of platelets affording patients an increased level of safety without causing undue strain on laboratory budgets or personnel while increasing the time frame that platelets can be used by dramatically shortening contaminant detection time.

  18. Platelet matching for alloimmunized patients

    Institute of Scientific and Technical Information of China (English)

    S H.Hsu

    2010-01-01

    @@ Platelets play an essential role in blood coagulation,hemostasis and maintenance of vascular integrity.Platelets are utilized primarily to prevent or treat bleeding in thrombocytopenic patients and patients with impaired platelet production in the bone marrow and/or with dysfunctional platelets.In current practice,platelet transfusion begins with randomly selected platelet products:either pooled platelets prepared from whole blood derived platelets; or single donor platelets prepared by apheresis procedures.

  19. Structure-activity relationship studies of 1-substituted 3-dodecanoylindole-2-carboxylic acids as inhibitors of cytosolic phospholipase A2-mediated arachidonic acid release in intact platelets.

    Science.gov (United States)

    Griessbach, Klaus; Klimt, Monika; Schulze Elfringhoff, Alwine; Lehr, Matthias

    2002-01-01

    A series of 3-dodecanoylindole-2-carboxylic acid derivatives with varied carboxylic acid substituents at the indole 1-position were synthesized and evaluated for their ability to inhibit arachidonic acid release in human platelets mediated by the cytosolic phospholipase A(2). Structure-activity relationship studies revealed that increasing the polarity of these substituents by the introduction of additional polar groups in the proximity of the carboxylic acid moiety reduced activity. Conformational restriction of the indole-1-carboxylic acid substituents in distinct positions as well as extending the length of these residues led to compounds which did not substantially differ in their potencies.

  20. Resveratrol preserves the function of human platelets stored for transfusion.

    Science.gov (United States)

    Lannan, Katie L; Refaai, Majed A; Ture, Sara K; Morrell, Craig N; Blumberg, Neil; Phipps, Richard P; Spinelli, Sherry L

    2016-03-01

    Stored platelets undergo biochemical, structural and functional changes that lead to decreased efficacy and safety of platelet transfusions. Not only do platelets acquire markers of activation during storage, but they also fail to respond normally to agonists post-storage. We hypothesized that resveratrol, a cardioprotective antioxidant, could act as a novel platelet storage additive to safely prevent unwanted platelet activation during storage, while simultaneously preserving normal haemostatic function. Human platelets treated with resveratrol and stored for 5 d released less thromboxane B2 and prostaglandin E2 compared to control platelets. Resveratrol preserved the ability of platelets to aggregate, spread and respond to thrombin, suggesting an improved ability to activate post-storage. Utilizing an in vitro model of transfusion and thromboelastography, clot strength was improved with resveratrol treatment compared to conventionally stored platelets. The mechanism of resveratrol's beneficial actions on stored platelets was partly mediated through decreased platelet apoptosis in storage, resulting in a longer half-life following transfusion. Lastly, an in vivo mouse model of transfusion demonstrated that stored platelets are prothrombotic and that resveratrol delayed vessel occlusion time to a level similar to transfusion with fresh platelets. We show resveratrol has a dual ability to reduce unwanted platelet activation during storage, while preserving critical haemostatic function.

  1. Platelet lipidomics: a modern day perspective on lipid discovery and characterization in platelets

    OpenAIRE

    O’Donnell, Valerie B.; Murphy, Robert C.; Watson, Steve P.

    2014-01-01

    Lipids are diverse families of biomolecules that perform essential structural and signaling roles in platelets. Their formation and metabolism is tightly controlled by enzymes and signal transduction pathways, and their dysregulation leads to significant defects in platelet function and disease. Platelet activation is associated with significant changes to membrane lipids, and formation of diverse bioactive lipids that play essential roles in hemostasis. In recent years, new generation mass s...

  2. Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

    Science.gov (United States)

    Reed, G. L.; Matsueda, G. R.; Haber, E.

    1992-01-01

    Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

  3. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

    DEFF Research Database (Denmark)

    Shupliakov, Oleg; Bloom, Ona; Gustafsson, Jenny S

    2002-01-01

    at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons...

  4. Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.

    Science.gov (United States)

    Bradamante, Silvia; Villa, Alessandro; Versari, Silvia; Barenghi, Livia; Orlandi, Ivan; Vai, Marina

    2010-12-01

    A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).

  5. Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig

    NARCIS (Netherlands)

    Zijlstra, C.; Kidson, A.; Schoevers, E.J.; Daemen, A.J.J.M.; Tharasanit, T.; Kuijk, E.W.; Hazeleger, W.; Ducro-Steverink, D.W.B.; Colenbrander, B.; Roelen, B.A.J.

    2008-01-01

    Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to seve

  6. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.

    Science.gov (United States)

    Ren, L; Hong, S H; Cassavaugh, J; Osborne, T; Chou, A J; Kim, S Y; Gorlick, R; Hewitt, S M; Khanna, C

    2009-02-12

    Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

  7. Drosophila Kelch functions with Cullin-3 to organize the ring canal actin cytoskeleton

    OpenAIRE

    Hudson, Andrew M.; Cooley, Lynn

    2010-01-01

    Drosophila melanogaster Kelch (KEL) is the founding member of a diverse protein family defined by a repeated sequence motif known as the KEL repeat (KREP). Several KREP proteins, including Drosophila KEL, bind filamentous actin (F-actin) and contribute to its organization. Recently, a subset of KREP proteins has been shown to function as substrate adaptor proteins for cullin-RING (really interesting new gene) ubiquitin E3 ligases. In this study, we demonstrate that association of Drosophila K...

  8. N-cadherin negatively regulates collective Drosophila glial migration through actin cytoskeleton remodeling.

    Science.gov (United States)

    Kumar, Arun; Gupta, Tripti; Berzsenyi, Sara; Giangrande, Angela

    2015-03-01

    Cell migration is an essential and highly regulated process. During development, glia cells and neurons migrate over long distances - in most cases collectively - to reach their final destination and build the sophisticated architecture of the nervous system, the most complex tissue of the body. Collective migration is highly stereotyped and efficient, defects in the process leading to severe human diseases that include mental retardation. This dynamic process entails extensive cell communication and coordination, hence, the real challenge is to analyze it in the entire organism and at cellular resolution. We here investigate the impact of the N-cadherin adhesion molecule on collective glial migration, by using the Drosophila developing wing and cell-type specific manipulation of gene expression. We show that N-cadherin timely accumulates in glial cells and that its levels affect migration efficiency. N-cadherin works as a molecular brake in a dosage-dependent manner, by negatively controlling actin nucleation and cytoskeleton remodeling through α/β catenins. This is the first in vivo evidence for N-cadherin negatively and cell autonomously controlling collective migration.

  9. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    NARCIS (Netherlands)

    P. Dhonukshe (Pankaj); I. Grigoriev (Ilya); R. Fischer (Rainer); M. Tominaga (Motoki); D.G. Robinson (David); J. Hašek (Jiří); T. Paciorek (Tomasz); J. Petrášek (Jan); D. Seifertová (Daniela); R. Tejos (Ricardo); L.A. Meisel (Lee); E. Zažímalová (Eva); T.W.J. Gadella (Theodorus); Y.D. Stierhof; T. Ueda (Takashi); K. Oiwa (Kazuhiro); A.S. Akhmanova (Anna); R. Brock (Roland); A. Spang (Anne); J. Friml (Jiří)

    2008-01-01

    textabstractMany aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating

  10. Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses.

    Science.gov (United States)

    Dustin, Michael L

    2007-10-01

    The immunological synapse is a stable adhesive junction between a polarized immune effector cell and an antigen-bearing cell. Immunological synapses are often observed to have a striking radial symmetry in the plane of contact with a prominent central cluster of antigen receptors surrounded by concentric rings of adhesion molecules and actin-rich projections. There is a striking similarity between the radial zones of the immunological synapse and the dynamic actinomyosin modules employed by migrating cells. Breaking the symmetry of an immunological synapse generates a moving adhesive junction that can be defined as a kinapse, which facilitates signal integration by immune cells while moving over the surface of antigen-presenting cells.

  11. Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Walmod, P S; Skladchikova, G; Kawa, A

    1999-01-01

    The antiepileptic drug valproic acid (VPA) and teratogenic VPA analogues have been demonstrated to inhibit cell motility and affect cell morphology. We here show that disruption of microtubules or of microfilaments by exposure to nocodazole or cytochalasin D had different effects on morphology...... of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased...

  12. Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton

    NARCIS (Netherlands)

    Tudor, C.; Riet, J. te; Eich, C.; Harkes, R.; Smisdom, N.; Bouhuijzen-Wenger, J.; Ameloot, M.; Holt, M.; Kanger, J.S.; Figdor, C.G.; Cambi, A.; Subramaniam, V.

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both

  13. Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

    Science.gov (United States)

    Collart-Dutilleul, Pierre-Yves; Panayotov, Ivan; Secret, Emilie; Cunin, Frédérique; Gergely, Csilla; Cuisinier, Frédéric; Martin, Marta

    2014-10-01

    The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions.

  14. Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeleton.

    Science.gov (United States)

    Chen, Xing Judy; Squarr, Anna Julia; Stephan, Raiko; Chen, Baoyu; Higgins, Theresa E; Barry, David J; Martin, Morag C; Rosen, Michael K; Bogdan, Sven; Way, Michael

    2014-09-01

    Ena/VASP proteins and the WAVE regulatory complex (WRC) regulate cell motility by virtue of their ability to independently promote actin polymerization. We demonstrate that Ena/VASP and the WRC control actin polymerization in a cooperative manner through the interaction of the Ena/VASP EVH1 domain with an extended proline rich motif in Abi. This interaction increases cell migration and enables VASP to cooperatively enhance WRC stimulation of Arp2/3 complex-mediated actin assembly in vitro in the presence of Rac. Loss of this interaction in Drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of Ena to the tips of filopodia-like extensions. Rescue experiments of abi mutants also reveals a physiological requirement for the Abi:Ena interaction in photoreceptor axon targeting and oogenesis. Our data demonstrate that the activities of Ena/VASP and the WRC are intimately linked to ensure optimal control of actin polymerization during cell migration and development.

  15. Androgens Regulate T47D Cells Motility and Invasion through Actin Cytoskeleton Remodeling

    Science.gov (United States)

    Montt-Guevara, Maria Magdalena; Shortrede, Jorge Eduardo; Giretti, Maria Silvia; Giannini, Andrea; Mannella, Paolo; Russo, Eleonora; Genazzani, Alessandro David; Simoncini, Tommaso

    2016-01-01

    The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgen receptor (AR) is expressed in approximately 70 to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple-negative breast cancers. Recent studies have associated the actin-binding proteins of the ezrin–radixin–moesin (ERM) family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) may regulate breast cancer cell motility and invasion through the control of actin remodeling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER), while the non-aromatizable androgen – DHT – only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer and, eventually, to develop new strategies for breast cancer treatment. PMID:27746764

  16. CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

    Directory of Open Access Journals (Sweden)

    Elena P Moiseeva

    Full Text Available CADM1 is a major receptor for the adhesion of mast cells (MCs to fibroblasts, human airway smooth muscle cells (HASMCs and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM. Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

  17. ANDROGENS REGULATE T47D CELLS MOTILITY AND INVASION THROUGH ACTIN CYTOSKELETON REMODELLING

    Directory of Open Access Journals (Sweden)

    Maria Magdalena Montt-Guevara

    2016-09-01

    Full Text Available The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgens receptor (AR is expressed in approximately 70% to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple negative breast cancers. Recent studies have associated the actin-binding proteins of the Ezrin-Radixin-Moesin (ERM family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T, dihydrotestosterone (DHT and dehydroepiandrosterone (DHEA may regulate breast cancer cell motility and invasion through the control of actin remodelling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER, while the non aromatizable androgen – DHT only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer, and eventually to develop new strategies for treatment of breast cancer.

  18. N-terminal Slit2 inhibits HIV-1 replication by regulating the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Anand Appakkudal R

    2013-01-01

    Full Text Available Abstract Background Slit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus-1 pathogenesis. Results In the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N (~120 kDa inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization. Conclusions Taken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.

  19. Clinical application of radiolabelled platelets

    Energy Technology Data Exchange (ETDEWEB)

    Kessler, C. (Medical Univ. Lubeck, Lubeck (DE))

    1990-01-01

    This book presents papers on the clinical applications of radiolabelled platelets. The papers are grouped into six sections on platelet labelling techniques, radiolabelled platelets in cardiology, monitoring of antiplatelet therapy, platelet scintigraphy in stroke patients, platelet scintigraphy in angiology, and platelet scintigraphy in hematology and other clinical applications, including renal transplant rejection.

  20. Fractal and Euclidean descriptors of platelet shape.

    Science.gov (United States)

    Kraus, Max-Joseph; Neeb, Heiko; Strasser, Erwin F

    2014-01-01

    Platelet shape change is a dynamic membrane surface process that exhibits remarkable morphological heterogeneity. Once the outline of an irregular shape is identified and segmented from a digital image, several mathematical descriptors can be applied to numerical characterize the irregularity of the shapes surface. 13072 platelet outlines (PLO) were segmented automatically from 1928 microscopic images using a newly developed algorithm for the software product Matlab R2012b. The fractal dimension (FD), circularity, eccentricity, area and perimeter of each PLO were determined. 972 PLO were randomly assigned for computer-assisted manual measurement of platelet diameter as well as number, width and length of filopodia per platelet. FD can be used as a surrogate parameter for determining the roughness of the PLO and circularity can be used as a surrogate to estimate the number and length of filopodia. The relationship between FD and perimeter of the PLO reveals the existence of distinct groups of platelets with significant structural differences which may be caused by platelet activation. This new method allows for the standardized continuous numerical classification of platelet shape and its dynamic change, which is useful for the analysis of altered platelet activity (e.g. inflammatory diseases, contact activation, drug testing).

  1. Erythrocyte-platelet interaction in uncomplicated pregnancy.

    Science.gov (United States)

    Swanepoel, Albe C; Pretorius, Etheresia

    2014-12-01

    Maternal and fetal requirements during uncomplicated pregnancy are associated with changes in the hematopoietic system. Platelets and erythrocytes [red blood cells (RBCs)], and especially their membranes, are involved in coagulation, and their interactions may provide reasons for the changed hematopoietic system during uncomplicated pregnancy. We review literature regarding RBC and platelet membrane structure and interactions during hypercoagulability and hormonal changes. We then study interactions between RBCs and platelets in uncomplicated pregnancy, as their interactions may be one of the reasons for increased hypercoagulability during uncomplicated pregnancy. Scanning electron microscopy was used to study whole blood smears from 90 pregnant females in different phases of pregnancy. Pregnancy-specific interaction was seen between RBCs and platelets. Typically, one or more platelets interacted through platelet spreading and pseudopodia formation with a single RBC. However, multiple interactions with RBCs were also shown for a single platelet. Specific RBC-platelet interaction seen during uncomplicated pregnancy may be caused by increased estrogen and/or increased fibrinogen concentrations. This interaction may contribute to the hypercoagulable state associated with healthy and uncomplicated pregnancy and may also play a fundamental role in gestational thrombocytopenia.

  2. Effect of annealing temperature on the structural and magnetic properties of CTAB-capped SrFe12O19 platelets

    Science.gov (United States)

    Harikrishnan, V.; Saravanan, P.; Ezhil Vizhi, R.; Babu, D. Rajan; Vinod, V. T. P.; Kejzlar, Pavel; Černík, Miroslav

    2016-03-01

    The use of surfactant such as cetyl-trimethyl ammonium bromide (CTAB) in producing highly coercive SrFe12O19 platelets is presented in this study. The synthesis of SrFe12O19 was accomplished by co-precipitation in presence of 1 wt% CTAB. The CTAB-coated precipitant thus obtained was subjected to annealing at different temperatures: 700, 800, 900 and 1000 °C. The annealed counterparts were characterized with respect to their structural and magnetic properties and the results are compared with that of those processed without CTAB. Thermogravimetry analysis was employed to study the thermo-chemical behavior for the SrFe12O19 samples. The evolution of crystalline phases as a function of annealing temperature was studied using x-ray diffraction. For the SrFe12O19 samples without CTAB, formation of α-Fe2O3 secondary phases are noticed at annealing temperatures of 700 and 800 °C; while such a secondary phase formation is not evident for the CTAB-capped SrFe12O19. Fourier transform infrared spectroscopy of the samples annealed at 1000 °C showed deformation in the structure due to the splitting of the bands. Both morphology and composition of the samples were examined by a field-emission scanning electron microscope attached with energy dispersive x-ray analysis. The morphology of CTAB-capped SrFe12O19 samples showed the presence of hexagonal platelets at higher annealing temperatures. The magnetic parameters such as saturation magnetization, MS and coercivity, HC were evaluated from the magnetic hysteresis loops obtained by vibrating sample magnetometer. Maximum values of HC (6.3 kOe) and MS (42.7 emu/g) were obtained for the CTAB-capped SrFe12O19 samples annealed at 900 °C. The possible mechanism on the formation of M-type hexagonal phase with platelet morphology using minimal amount of CTAB (1 wt%) in achieving high the HC values for the SrFe12O19 is discussed.

  3. Structural basis for distinctive recognition of fibrinogen [gamma]C peptide by the platelet integrin [alpha][subscript IIb][beta]3

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Timothy A.; Zhu, Jianghai; Xiao, Tsan (Harvard-Med)

    2009-01-12

    Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin {alpha}{sub IIb}{beta}{sub 3} on platelets, resulting in platelet aggregation. {alpha}{sub v}{beta}{sub 3} binding fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's {alpha} subunit. {alpha}{sub IIb}{beta}{sub 3} also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the {gamma} subunit ({gamma}C peptide). These distinct modes of fibrinogen binding enable {alpha}{sub IIb}{beta}{sub 3} and {alpha}{sub v}{beta}{sub 3} to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin {alpha}{sub IIb}{beta}{sub 3}-{gamma}C peptide interface, and, for comparison, integrin {alpha}{sub IIb}{beta}{sub 3} bound to a lamprey {gamma}C primordial RGD motif. Compared with RGD, the GAKQAGDV motif in {gamma}C adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg{sup 2+} ion binds the {gamma}C Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca{sup 2+} ion binds the {gamma}C C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered {gamma}C peptide enhances our understanding of the involvement of {gamma}C peptide and integrin {alpha}{sub IIb}{beta}{sub 3} in hemostasis and thrombosis.

  4. Platelet derivatives in regenerative medicine: an update.

    Science.gov (United States)

    De Pascale, Maria Rosaria; Sommese, Linda; Casamassimi, Amelia; Napoli, Claudio

    2015-01-01

    Prior preclinical and clinical studies support the use of platelet-derived products for the treatment of soft and hard tissue lesions. These regenerative effects are controlled by autocrine and paracrine biomolecules including growth factors and cytokines contained in platelet alpha granules. Each growth factor is involved in a phase of the healing process, such as inflammation, collagen synthesis, tissue granulation, and angiogenesis collectively promoting tissue restitution. Platelet derivatives have been prepared as platelet-rich plasma, platelet gel, platelet-rich fibrin, and platelet eye drops. These products vary in their structure, growth factors, composition, and cytokine concentrations. Here, we review the current use of platelet-derived biological products focusing on the rationale for their use and the main requirements for their preparation. Variation in the apparent therapeutic efficacy may have resulted from a lack of reproducible, standardized protocols for preparation. Despite several individual studies showing favorable treatment effects, some randomized controlled trials as well as meta-analyses have found no constant clinical benefit from the application of platelet-derived products for prevention of tissue lesions. Recently, 3 published studies in dentistry showed an improvement in bone density. Seven published studies showed positive results in joint regeneration. Five published studies demonstrated an improvement in the wound healing, and an improvement of eye epithelial healing was observed in 2 reports. Currently, at least 14 ongoing clinical trials in phase 3 or 4 have been designed with large groups of treated patients (n > 100). Because the rationale of the therapy with platelet-derived compounds is still debated, a definitive insight can be acquired only when these large randomized trials will be completed.

  5. Human platelet antigen genotyping of platelet donors in southern Brazil.

    Science.gov (United States)

    Merzoni, J; Fagundes, I S; Lunardi, L W; Lindenau, J D-R; Gil, B C; Jobim, M; Dias, V G; Merzoni, L; Sekine, L; Onsten, T G H; Jobim, L F

    2015-10-01

    Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

  6. Platelet alloimmunization after transfusion

    DEFF Research Database (Denmark)

    Taaning, E; Simonsen, A C; Hjelms, E;

    1997-01-01

    BACKGROUND AND OBJECTIVES: The frequency of platelet-specific antibodies after one series of blood transfusions has not been reported, and in multiply transfused patients is controversial. MATERIALS AND METHODS: We studied the frequency of alloimmunization against platelet antigens in 117 patient...

  7. Flavanols and Platelet Reactivity

    Directory of Open Access Journals (Sweden)

    Debra A. Pearson

    2005-01-01

    Full Text Available Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.

  8. Platelet activation and aggregation

    DEFF Research Database (Denmark)

    Jensen, Maria Sander; Larsen, O H; Christiansen, Kirsten

    2013-01-01

    This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated...

  9. Gasotransmitters and platelets.

    Science.gov (United States)

    Truss, Nicola J; Warner, Timothy D

    2011-11-01

    Platelets are essential to prevent blood loss and promote wound healing. Their activation comprises of several complex steps which are regulated by a range of mediators. Over the last few decades there has been intense interest in a group of gaseous mediators known as gasotransmitters; currently comprising nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H(2)S). Here we consider the action of gasotransmitters on platelet activity. NO is a well established platelet inhibitor which mediates its effects predominantly through activation of soluble guanylyl cyclase leading to a decrease in intraplatelet calcium. More recently CO has been identified as a gasotransmitter with inhibitory actions on platelets; CO acts through the same mechanism as NO but is less potent. The in vivo and platelet functions of the most recently identified gasotransmitter, H(2)S, are still the subject of investigations, but they appear generally inhibitory. Whilst there is evidence for the individual action of these mediators, it is also likely that combinations of these mediators are more relevant regulators of platelets. Furthermore, current evidence suggests that these mediators in combination alter the production of each other, and so modify the circulating levels of gasotransmitters. The use of gasotransmitters as therapeutic agents is also being explored for a range of indications. In conclusion, the importance of NO in the regulation of vascular tone and platelet activity has long been understood. Other gasotransmitters are now establishing themselves as mediators of vascular tone, and recent evidence suggests that these other gasotransmitters may also modulate platelet function.

  10. Alloimmune refractoriness to platelet transfusions.

    Science.gov (United States)

    Sandler, S G

    1997-11-01

    Patients who are transfused on multiple occasions with red cells or platelets may develop platelet-reactive alloantibodies and experience decreased clinical responsiveness to platelet transfusion. This situation, conventionally described as "refractoriness to platelet transfusions," is defined by an unsatisfactory low post-transfusion platelet count increment. If antibodies to HLAs are detected, improved clinical outcomes may result from transfusions of HLA-matched or donor-recipient cross-matched platelets. Because refractoriness is an expected, frequently occurring phenomenon, prevention of HLA alloimmunization is an important management strategy. Prevention strategies include efforts to decrease the number of transfusions, filtration of cellular components to reduce the number of HLA-bearing leukocytes, or pretransfusion ultraviolet B irradiation of cellular components to decrease their immunogenicity. Other investigational approaches include reducing the expression of HLAs on transfused platelets, inducing a transient reticuloendothelial system blockade by infusions of specialized immunoglobulin products, or transfusing semisynthetic platelet substitutes (thromboerythrocytes, thrombospheres) or modified platelets (infusible platelet membranes, lyophilized platelets).

  11. Platelet function in dogs

    DEFF Research Database (Denmark)

    Nielsen, Line A.; Zois, Nora Elisabeth; Pedersen, Henrik D.

    2007-01-01

    Cairn Terriers, 10 Boxers, and 11 Labrador Retrievers) were included in the study. Platelet function was assessed by whole-blood aggregation with ADP (1, 5, 10, and 20 µM) as agonist and by PFA-100 using collagen and epinephrine (Col + Epi) and Cpæ + ADP as agonists. Plasma thromboxane B2 concentration......Background: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. Objectives: The objective of this study was to investigate platelet function in clinically healthy dogs of 4...... different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Methods: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12...

  12. Cisplatin triggers platelet activation.

    Science.gov (United States)

    Togna, G I; Togna, A R; Franconi, M; Caprino, L

    2000-09-01

    Clinical observations suggest that anticancer drugs could contribute to the thrombotic complications of malignancy in treated patients. Thrombotic microangiopathy, myocardial infarction, and cerebrovascular thrombotic events have been reported for cisplatin, a drug widely used in the treatment of many solid tumours. The aim of this study is to explore in vitro cisplatin effect on human platelet reactivity in order to define the potentially active role of platelets in the pathogenesis of cisplatin-induced thrombotic complications. Our results demonstrate that cisplatin increases human platelet reactivity (onset of platelet aggregation wave and thromboxane production) to non-aggregating concentrations of the agonists involving arachidonic acid metabolism. Direct or indirect activation of platelet phospholipase A(2) appears to be implicated. This finding contributes to a better understanding of the pathogenesis of thrombotic complications occurring during cisplatin-based chemotherapy.

  13. Platelet function in dogs

    DEFF Research Database (Denmark)

    Nielsen, Line A.; Zois, Nora Elisabeth; Pedersen, Henrik D.

    2007-01-01

    Background: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. Objectives: The objective of this study was to investigate platelet function in clinically healthy dogs of 4...... different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Methods: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12...... applied. However, the importance of these breed differences remains to be investigated. The PFA-100 method with Col + Epi as agonists, and ADP-induced platelet aggregation appear to be sensitive to ASA in dogs....

  14. Increased platelet aggregability following an atherogenic diet in rabbits

    OpenAIRE

    Velkovski Saško D.; Mazić Sanja; Nešić Dejan M.; Igrački Iva; Milošević Verica L.; Starčević Vesna P.

    2002-01-01

    In atherosclerosis researches different animal models are used but the most common is the rabbit, because of the easy development of atherosclerotic lesions. Atherosclerosis is a multicellular process and platelets play an important role in atherogenesis. Excessive plasma lipids stimulate platelet aggregability and thus atherosclerosis development. The effects of an atherogenic diet on lipid status, abdominal aorta wall structure, and platelet aggregability were studied in rabbits. Adult male...

  15. Measurement of platelet aggregation, independently of patient platelet count

    DEFF Research Database (Denmark)

    Vinholt, P. J.; Frederiksen, H.; Hvas, A.M.

    2017-01-01

    platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Summary: Background: Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective: To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count......, and examine the association of aggregation with a bleeding history in thrombocytopenic patients. Patients/methods: We established a flow-cytometric assay of platelet aggregation, and measured samples from healthy individuals preincubated with antiplatelet drugs, and samples from two patients with inherited...... platelets at platelet counts of > 10 × 109 L-1; otherwise, platelet isolation was required. The platelet aggregation percentage decreased with increasing antiplatelet drug concentration. Platelet aggregation in patients was reduced as compared with healthy individuals: 42% (interquartile range [IQR] 27...

  16. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    Energy Technology Data Exchange (ETDEWEB)

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA) State Univ. of New York, Buffalo (USA))

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  17. In-vitro model for the ultrastructural study of the formation of thrombi in human platelets.

    Science.gov (United States)

    Cerecedo, Doris; González, Sirenia; Mondragón, Mónica; Reyes, Elba; Mondragón, Ricardo

    2006-03-01

    Platelets are cell fragments with dynamic properties involved in clot formation after tissue damage. Platelet activation causes a change in shape, secretion of intracellular granules and aggregation with each other through the cytoskeleton components and biochemical changes. Platelet adhesion, considered as the major event in haemostasis, has been studied in several in-vitro and in-vivo models to evaluate the feasible thrombogenicity of some materials, the dynamics of specific receptors, as well as the effect of different buffers and inhibitors in this process. In spite of the numerous reports about platelet activation, to date there is no information available about the fine structure of the platelet-platelet and platelet-substrate interactions. In the present report we describe an in-vitro system that allows the visualization of these interactions: platelets are adhered to an inert substrate, and interactions with suspended platelets as a process to initiate the formation of thrombi was followed by ultramicrotomy and transmission electron microscopy.

  18. Platelet concentration in platelet concentrates and periodontal regeneration-unscrambling the ambiguity

    Directory of Open Access Journals (Sweden)

    A Suchetha

    2015-01-01

    Full Text Available Context: Platelet-rich-plasma (PRP and Platelet-rich-fibrin (PRF are extensively used autologous platelet concentrates in periodontal regeneration, and PRF has a better efficacy as compared to PRP. The rationale for this difference has often been attributed to the difference in the structure of the fibrin matrix. However, the effect of concentration of platelets on the regenerative potential of these concentrates is obscure. Aims: The study was conducted to evaluate and compare, clinically and radiographically, the efficacy of PRF and PRP in the treatment of periodontal endosseous defects and to assess the effect of platelet concentration on periodontal regeneration. Materials and Methods: Twenty intrabony defects were selected and divided into two groups randomly by the coin toss method. Group I received PRP and Group II subjects were treated with PRF. The platelet counts in PRP and PRF were analyzed. Clinical and radiological parameters were assessed at baseline and 3, 6, and 9 months postoperatively. Statistical Analysis: Kruskal–Wallis Chi-square test, Wilcoxon signed rank test, t-test, and Spearman's rank correlation were used for statistical analysis of data. Results: There was statistically significant improvement in all the parameters in the two groups except in relation to gingival recession. There was a statistically significant difference between the platelet count in Group I and Group II (P = 0.002. Conclusion: PRP and PRF appear to have nearly comparable effects in terms of periodontal regeneration. The concentration of platelets appears to play a paradoxical role in regeneration. The regenerative potential of platelets appears to be optimal within a limited range.

  19. Platelet concentration in platelet concentrates and periodontal regeneration-unscrambling the ambiguity

    Science.gov (United States)

    Suchetha, A.; Lakshmi, P.; Bhat, Divya; Mundinamane, Darshan B.; Soorya, K. V.; Bharwani, G. Ashit

    2015-01-01

    Context: Platelet-rich-plasma (PRP) and Platelet-rich-fibrin (PRF) are extensively used autologous platelet concentrates in periodontal regeneration, and PRF has a better efficacy as compared to PRP. The rationale for this difference has often been attributed to the difference in the structure of the fibrin matrix. However, the effect of concentration of platelets on the regenerative potential of these concentrates is obscure. Aims: The study was conducted to evaluate and compare, clinically and radiographically, the efficacy of PRF and PRP in the treatment of periodontal endosseous defects and to assess the effect of platelet concentration on periodontal regeneration. Materials and Methods: Twenty intrabony defects were selected and divided into two groups randomly by the coin toss method. Group I received PRP and Group II subjects were treated with PRF. The platelet counts in PRP and PRF were analyzed. Clinical and radiological parameters were assessed at baseline and 3, 6, and 9 months postoperatively. Statistical Analysis: Kruskal–Wallis Chi-square test, Wilcoxon signed rank test, t-test, and Spearman's rank correlation were used for statistical analysis of data. Results: There was statistically significant improvement in all the parameters in the two groups except in relation to gingival recession. There was a statistically significant difference between the platelet count in Group I and Group II (P = 0.002). Conclusion: PRP and PRF appear to have nearly comparable effects in terms of periodontal regeneration. The concentration of platelets appears to play a paradoxical role in regeneration. The regenerative potential of platelets appears to be optimal within a limited range. PMID:26681857

  20. Synthesis, characterizations and applications of some nanomaterials (TiO2 and SiC nanostructured films, organized CNT structures, ZnO structures and CNT{blood platelet clusters)

    Indian Academy of Sciences (India)

    O N Srivastava; A Srivastava; D Dash; D P Singh; R M Yadava; P R Mishra; J Singh

    2005-10-01

    TiO2 nanostructured films have been synthesized by the hydrolysis of Ti[OCH(CH3)2]4 as the precursor. These films have been utilized for the dissociation of phenol contaminant in water. Free-standing nanostructured film of silicon carbide (SiC) has been synthesized, employing a simple and new route of spray pyrolysis technique utilizing a slurry of Si in hexane. Another study is done on organized carbon nanotube (CNT) structures. These are made in the form of hollow cylinders (50 mm length, 4 mm diameter and 1.5 mm wall thickness). These CNT-based cylinders are made of conventional CNT and bamboo-shaped CNT. The filtrations of heavy hydrocarbons and . coli bacteria from water have been carried out. In addition to this, ZnO nanostructures have also been studied. Another study concerns CNT-blood platelet clusters.

  1. Clinical application of radiolabelled platelets

    Energy Technology Data Exchange (ETDEWEB)

    Kessler, C. (Medical University Luebeck (Federal Republic of Germany). Department of Neurology); Hardeman, M.R. (Amsterdam Univ. (Netherlands). Academisch Ziekenhuis); Henningsen, H. (Heidelberg Univ. (Germany, F.R.). Neurologische Klinik); Petrovici, J.-N. (Cologne-Merheim Hospital (Federal Republic of Germany). Department of Neurology) (eds.)

    1990-01-01

    The increasing number of therapeutic modalities available for the management of patients with thromboembolic complications, such as fibrinolytic treatment or vascular surgery, require the development of new imaging techniques to provide more information on the xtent, age and activity of the thromboembolic material causing clinical symptoms. Since the introduction of radiolabelling of platelets with indium-111, platelet scintigraphy (PSC) has been used as a tool in the diagnosis of various thromboembolic diseases. During the International Symposium on Radiolabelled Platelets scientists from a variety of medical backgrounds presented their results on the clinical applictions of radiolabelled platelets. The papers presented there have been updated to take account of the latest results before publication in this volume. The papers are grouped into six sections on platelet labelling techniques, radiolabelled platelets in cardiology, monitoring of antiplatelet therapy, platelet scintigraphy in stroke patients, platelet scintigraphy in angiology, and platelet scintigraphy in hematology and other clinical applications, including renal transplant rejection. refs.; figs.; tabs.

  2. Modifications of blood platelet proteins of patients with schizophrenia.

    Science.gov (United States)

    Dietrich-Muszalska, Anna; Olas, Beata

    2009-03-01

    Oxidative damage to lipids in plasma, blood platelets and neurons in patients with schizophrenia was described. The aim of our present study was to evaluate oxidative/nitrative modifications of blood platelets proteins by measurement the level of biomarkers of oxidative stress such as carbonyl groups, thiol groups and 3-nitrotyrosine in proteins in patients with schizophrenia and compare with a control group. Levels of carbonyl groups and 3-nitrotyrosine residues in platelet proteins were measured by ELISA and competition ELISA, respectively. The method with 5,5'-dithio-bis(2-nitro-benzoic acid) has been used to analyse thiol groups in platelet proteins. We demonstrated for the first time in platelet proteins from patients with schizophrenia a statistically significant increase of the level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine; in schizophrenic patients the amount of thiol groups in platelet proteins was lower than in platelets from healthy subjects. Our results strongly indicate that in patients with schizophrenia reactive oxygen species and reactive nitrogen species induce not only peroxidation of lipids, but also may stimulate oxidative/nitrative modifications of platelet proteins. The consequence of these modifications may be the alteration of platelet protein structure and function.

  3. Platelet preservation: agitation and containers.

    Science.gov (United States)

    van der Meer, Pieter F; de Korte, Dirk

    2011-06-01

    For platelets to maintain their in vitro quality and in vivo effectiveness, they need to be stored at room temperature with gentle agitation in gas-permeable containers. The mode of agitation affects the quality of the platelets, and a gentle method of agitation, either a circular or a flat bed movement, provides the best results. Tumblers or elliptical agitators induce platelet activation and subsequent damage. As long as the platelets remain in suspension, the agitation speed is not important. Agitation of the platelet concentrates ensures that the platelets are continuously oxygenated, that sufficient oxygen can enter the storage container and that excess carbon dioxide can be expelled. During transportation of platelet concentrates, nowadays over long distances where they are held without controlled agitation, platelets may tolerate a certain period without agitation. However, evidence is accumulating that during the time without agitation, local hypoxia surrounding the platelets may induce irreversible harm to the platelets. Over the decades, more gas-permeable plastics have been used to manufacture platelet containers. The use of different plastics and their influence on the platelet quality both in vitro and in vivo is discussed. The improved gas-permeability has allowed the extension of platelet storage from 3 days in the early 1980s, to currently at least 7 days. In the light of new developments, particularly the introduction of pathogen reduction techniques, the use of platelet additive solutions and the availability of improved automated separators, further (renewed) research in this area is warranted.

  4. Platelets and cardiac arrhythmia

    Directory of Open Access Journals (Sweden)

    Jonas S De Jong

    2010-12-01

    Full Text Available Sudden cardiac death remains one of the most prevalent modes of death in industrialized countries, and myocardial ischemia due to thrombotic coronary occlusion is its primary cause. The role of platelets in the occurrence of SCD extends beyond coronary flow impairment by clot formation. Here we review the substances released by platelets during clot formation and their arrhythmic properties. Platelet products are released from three types of platelet granules: dense core granules, alpha-granules, and platelet lysosomes. The physiologic properties of dense granule products are of special interest as a potential source of arrhythmic substances. They are released readily upon activation and contain high concentrations of serotonin, histamine, purines, pyrimidines, and ions such as calcium and magnesium. Potential arrhythmic mechanisms of these substances, e.g. serotonin and high energy phosphates, include induction of coronary constriction, calcium overloading, and induction of delayed after-depolarizations. Alpha-granules produce thromboxanes and other arachidonic acid products with many potential arrhythmic effects mediated by interference with cardiac sodium, calcium and potassium channels. Alpha-granules also contain hundreds of proteins that could potentially serve as ligands to receptors on cardiomyocytes. Lysosomal products probably do not have an important arrhythmic effect. Platelet products and ischemia can induce coronary permeability, thereby enhancing interaction with surrounding cardiomyocytes. Antiplatelet therapy is known to improve survival after myocardial infarction. Although an important part of this effect results from prevention of coronary clot formation, there is evidence to suggest that antiplatelet therapy also induces anti-arrhythmic effects during ischemia by preventing the release of platelet activation products.

  5. The Platelet and Platelet Function Testing in Liver Disease

    NARCIS (Netherlands)

    Hugenholtz, Greg G. C.; Porte, Robert J.; Lisman, Ton

    2009-01-01

    Patients who have liver disease commonly present with alterations in platelet number and function. Recent data have questioned the contribution of these changes to bleeding complications in these patients. Modern tests of platelet function revealed compensatory mechanisms for the decreased platelet

  6. Investigation of platelet function and platelet disorders using flow cytometry.

    Science.gov (United States)

    Rubak, Peter; Nissen, Peter H; Kristensen, Steen D; Hvas, Anne-Mette

    2016-01-01

    Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

  7. Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

    Directory of Open Access Journals (Sweden)

    Zainab S Al-Hosni

    2016-11-01

    Full Text Available Objectives: Manual platelet estimation is one of the methods used when automated platelet estimates are very low. However, the reproducibility of manual platelet estimation has not been adequately studied. We sought to assess the reproducibility of manual platelet estimation following automated low platelet counts and to evaluate the impact of the level of experience of the person counting on the reproducibility of manual platelet estimates. Methods: In this cross-sectional study, peripheral blood films of patients with platelet counts less than 100 × 109/L were retrieved and given to four raters to perform manual platelet estimation independently using a predefined method (average of platelet counts in 10 fields using 100× objective multiplied by 20. Data were analyzed using intraclass correlation coefficient (ICC as a method of reproducibility assessment. Results: The ICC across the four raters was 0.840, indicating excellent agreement. The median difference of the two most experienced raters was 0 (range: -64 to 78. The level of platelet estimate by the least-experienced rater predicted the disagreement (p = 0.037. When assessing the difference between pairs of raters, there was no significant difference in the ICC (p = 0.420. Conclusions: The agreement between different raters using manual platelet estimation was excellent. Further confirmation is necessary, with a prospective study using a gold standard method of platelet counts.

  8. The role of platelets in hemostasis and the effects of snake venom toxins on platelet function.

    Science.gov (United States)

    de Queiroz, Mayara Ribeiro; de Sousa, Bruna Barbosa; da Cunha Pereira, Déborah Fernanda; Mamede, Carla Cristine Neves; Matias, Mariana Santos; de Morais, Nadia Cristina Gomes; de Oliveira Costa, Júnia; de Oliveira, Fábio

    2017-07-01

    The human body has a set of physiological processes, known as hemostasis, which keeps the blood fluid and free of clots in normal vessels; in the case of vascular injury, this process induces the local formation of a hemostatic plug, preventing hemorrhage. The hemostatic system in humans presents complex physiological interactions that involve platelets, plasma proteins, endothelial and subendothelial structures. Disequilibrium in the regulatory mechanisms that control the growth and the size of the thrombus is one of the factors that favors the development of diseases related to vascular disorders such as myocardial infarction and stroke, which are among the leading causes of death in the western world. Interfering with platelet function is a strategy for the treatment of thrombotic diseases. Antiplatelet drugs are used mainly in cases related to arterial thrombosis and interfere in the formation of the platelet plug by different mechanisms. Aspirin (acetylsalicylic acid) is the oldest and most widely used antithrombotic drug. Although highly effective in most cases, aspirin has limitations compared to other drugs used in the treatment of homeostatic disorders. For this reason, research related to molecules that interfere with platelet aggregation are of great relevance. In this regard, snake venoms are known to contain a number of molecules that interfere with hemostasis, including platelet function. The mechanisms by which snake venom components inhibit or activate platelet aggregation are varied and can be used as tools for the diagnosis and the treatment of several hemostatic disorders. The aim of this review is to present the role of platelets in hemostasis and the mechanisms by which snake venom toxins interfere with platelet function. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Mean platelet volume and mean platelet volume/platelet count ratio ...

    African Journals Online (AJOL)

    Amira M. Elsayed

    2016-03-30

    Mar 30, 2016 ... Abstract The mean platelet volume (MPV) is a laboratory marker associated with platelet func- tion and activity. .... the first 24 h of presentation to the emergency department. Severity of ..... J Neurol Neurosurg Psychiatry.

  10. Role of reactive nitrogen species in blood platelet functions.

    Science.gov (United States)

    Olas, Beata; Wachowicz, Barbara

    2007-12-01

    Blood platelets, in analogy to other circulating blood cells, can generate reactive oxygen/nitrogen species (ROS/RNS) that may behave as second messengers and may regulate platelet functions. Accumulating evidence suggest a role of ROS/RNS in platelet activation. On the other hand, an increased production of ROS/RNS causes oxidative stress, and thus, may contribute to the development of different diseases, including vascular complications, inflammatory and psychiatric illnesses. Oxidative stress in platelets leads to chemical changes in a wide range of their components, and platelet proteins may be initial targets of ROS/RNS action. It has been demonstrated that reaction of proteins with ROS/RNS results in the oxidation and nitration of some amino acid residues, formation of aggregates or fragmentation of proteins. In oxidized proteins new carbonyl groups and protein hydroperoxides are also formed. In platelets, low molecular weight thiols such as glutathione (GSH), cysteine and cysteinylglycine and protein thiols may be also target for ROS/RNS action. This review describes the chemical structure and biological activities of reactive nitrogen species, mainly nitric oxide ((*)NO) and peroxynitrite (ONOO(-)) and their effects on blood platelet functions, and the mechanisms involved in their action on platelets.

  11. Annexin A2 Heterotetramer: Structure and Function

    Directory of Open Access Journals (Sweden)

    David Waisman

    2013-03-01

    Full Text Available Annexin A2 is a pleiotropic calcium- and anionic phospholipid-binding protein that exists as a monomer and as a heterotetrameric complex with the plasminogen receptor protein, S100A10. Annexin A2 has been proposed to play a key role in many processes including exocytosis, endocytosis, membrane organization, ion channel conductance, and also to link F-actin cytoskeleton to the plasma membrane. Despite an impressive list of potential binding partners and regulatory activities, it was somewhat unexpected that the annexin A2-null mouse should show a relatively benign phenotype. Studies with the annexin A2-null mouse have suggested important functions for annexin A2 and the heterotetramer in fibrinolysis, in the regulation of the LDL receptor and in cellular redox regulation. However, the demonstration that depletion of annexin A2 causes the depletion of several other proteins including S100A10, fascin and affects the expression of at least sixty-one genes has confounded the reports of its function. In this review we will discuss the annexin A2 structure and function and its proposed physiological and pathological roles.

  12. Prophylactic platelets in dengue

    DEFF Research Database (Denmark)

    Whitehorn, James; Rodriguez Roche, Rosmari; Guzman, Maria G

    2012-01-01

    of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity...

  13. The ultrastructure of camel blood platelets: a comparative study with human, bovine, and equine cells.

    Science.gov (United States)

    Gader, Abdel Galil M Abdel; Ghumlas, Abeer K Al; Hussain, Mansour F; Haidari, Ahmed Al; White, James G

    2008-02-01

    Previous studies indicated that the camel has a very active haemostatic mechanism with a short bleeding time and thrombocytosis. However, platelet function, when tested by agonist-induced aggregation and PFA 100 closure time, showed marked inhibition compared to humans. Since camels are also far more resistant to long exposure to excessive heat and high body temperature than humans, it seemed worthwhile to explore fundamental morphological differences between human and camel platelets and those from other species. The present study has examined the ultrastructure of camel platelets and compared them with the fine structures of human, bovine and equine thrombocytes. Camel platelets, like bovine and equine cells, are discoid in shape and about two-thirds the size of human platelets. A circumferential coil of microtubular supports the disk-like form of camel platelets. Their cytoplasm, like bovine and equine platelets, is filled with alpha granule twice as large as those in human platelets, but lacking the organized matrix of equine alpha granules. Dense bodies are present in camel platelets with whip-like extensions not present on bovine or equine thrombocytes, but found on occasional human platelet dense bodies. Camel platelets, like bovine and equine thrombocytes, lack an open canalicular system (OCS) and must secrete granule products by fusion with the cell wall rather than an OCS. Future studies will determine if the differences in ultrastructural anatomy protect camel platelets from heat more than human thrombocytes.

  14. Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan

    OpenAIRE

    Liu, Zhi-Jian; Hoffmeister, Karin M.; Hu, Zhongbo; Mager, Donald E.; Ait-Oudhia, Sihem; Debrincat, Marlyse A.; Pleines, Irina; Josefsson, Emma C.; Benjamin T Kile; Italiano, Joseph; Ramsey, Haley; Grozovsky, Renata; Veng-Pedersen, Peter; Chavda, Chaitanya; Sola-Visner, Martha

    2014-01-01

    Rapid growth and rising platelet counts result in a significant expansion of platelet mass during neonatal life.The rise in platelet counts is mediated by a prolongation in the neonatal platelet lifespan.

  15. PREGNANCY WITH PLATELET FUNCTION DISORDER

    Directory of Open Access Journals (Sweden)

    Sheila K

    2014-01-01

    Full Text Available latelets play a vital role in haemostasis . Antenatal patients with platelet function disorders should be managed in tertiary care centres that are well equipped to tackle any obstetric haemorrhage that can ensue during labour and delivery . Primi gravida was admitted for safe confinement . She had been evaluated earlier for complaints of multiple episodes of mucosal bleeding . On evaluation she had nor mal platelet counts and coagulation factor assay was normal . Platelet aggregometry revealed mild disorder of platelet aggregation . She was planned for induction of labour after arranging enough blood and blood products . She got into active labour and was p ut on syntocinon augmentation . She had emergency Caesarean section for foetal distress . Oxytocics were given proactively . Intraoperatively platelet transfusions and tranexamic acid infusion were given . Complete haemostasis was achieved . She had an uneventf ul postoperative period . Patients with functional platelet disorders can be successfully managed with local application of antifibrinolytic agents like tranexamic acid , in case of minor bleeds . Platelet transfusions are very effective in tackling major ble eds , especially during surgeries and for obstetric indications . If a patient has the history of clinically significant bleeding suggestive of platelet dysfunction , appropriate platelet function tests should be obtained so that the risk of bleeding can be adequately assessed and therapy chosen more rationally . . In obstetric practice the response of such patients to platelet transfusions has been excellent

  16. Effects of hormones on platelet aggregation.

    Science.gov (United States)

    Farré, Antonio López; Modrego, Javier; Zamorano-León, José J

    2014-04-01

    Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

  17. the role of the actin cytoskeleton and lipid rafts in the localization and function of the ABCC1 transporter

    NARCIS (Netherlands)

    Kok, Jan; Klappe, Katharina; Hummel, Ina

    2014-01-01

    ATP-binding cassette (ABC) transporters are known to be important factors in multidrug resistance of tumor cells. Lipid rafts have been implicated in their localization in the plasma membrane, where they function as drug efflux pumps. This specific localization in rafts may support the activity of A

  18. Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

    Science.gov (United States)

    Mouneimne, Ghassan; Hansen, Scott D; Selfors, Laura M; Petrak, Lara; Hickey, Michele M; Gallegos, Lisa L; Simpson, Kaylene J; Lim, James; Gertler, Frank B; Hartwig, John H; Mullins, R Dyche; Brugge, Joan S

    2012-11-13

    Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

  19. Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Jensen, Morten Østergaard; Hansen, Gry Aune; Vorum, Henrik

    2006-01-01

    cytoskeleton and is involved in cytokinesis. Labeling of S phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin possesses a paracrine role on the cells in its vicinity and therefore may be involved...... in the pathophysiology of thrombosis or in wound healing....

  20. Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

    LENUS (Irish Health Repository)

    Vlahou, Georgia

    2009-07-14

    Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

  1. Girdin-mediated interactions between cadherin and the actin cytoskeleton are required for epithelial morphogenesis in Drosophila.

    Science.gov (United States)

    Houssin, Elise; Tepass, Ulrich; Laprise, Patrick

    2015-05-15

    E-cadherin-mediated cell-cell adhesion is fundamental for epithelial tissue morphogenesis, physiology and repair. E-cadherin is a core transmembrane constituent of the zonula adherens (ZA), a belt-like adherens junction located at the apicolateral border in epithelial cells. The anchorage of ZA components to cortical actin filaments strengthens cell-cell cohesion and allows for junction contractility, which shapes epithelial tissues during development. Here, we report that the cytoskeletal adaptor protein Girdin physically and functionally interacts with components of the cadherin-catenin complex during Drosophila embryogenesis. Fly Girdin is broadly expressed throughout embryonic development and enriched at the ZA in epithelial tissues. Girdin associates with the cytoskeleton and co-precipitates with the cadherin-catenin complex protein α-Catenin (α-Cat). Girdin mutations strongly enhance adhesion defects associated with reduced DE-cadherin (DE-Cad) expression. Moreover, the fraction of DE-Cad molecules associated with the cytoskeleton decreases in the absence of Girdin, thereby identifying Girdin as a positive regulator of adherens junction function. Girdin mutant embryos display isolated epithelial cell cysts and rupture of the ventral midline, consistent with defects in cell-cell cohesion. In addition, loss of Girdin impairs the collective migration of epithelial cells, resulting in dorsal closure defects. We propose that Girdin stabilizes epithelial cell adhesion and promotes morphogenesis by regulating the linkage of the cadherin-catenin complex to the cytoskeleton.

  2. The Role of the Actin Cytoskeleton and Lipid Rafts in the Localization and Function of the ABCC1 Transporter

    Directory of Open Access Journals (Sweden)

    Jan Willem Kok

    2014-01-01

    Full Text Available ATP-binding cassette (ABC transporters are known to be important factors in multidrug resistance of tumor cells. Lipid rafts have been implicated in their localization in the plasma membrane, where they function as drug efflux pumps. This specific localization in rafts may support the activity of ABC/Abc transporters. This raises questions regarding the nature and composition of the lipid rafts that harbor ABC/Abc transporters and the dependence of ABC/Abc transporters—concerning their localization and activity—on lipid raft constituents. Here we review our work of the past 10 years aimed at evaluating whether ABC/Abc transporters are dependent on a particular membrane environment for their function. What is the nature of this membrane environment and which of the lipid raft constituents are important for this dependency? It turns out that cortical actin is of major importance for stabilizing the localization and function of the ABC/Abc transporter, provided it is localized in an actin-dependent subtype of lipid rafts, as is the case for human ABCC1/multidrug resistance-related protein 1 (MRP1 and rodent Abcc1/Mrp1 but not human ABCB1/P-glycoprotein (PGP. On the other hand, sphingolipids do not appear to be modulators of ABCC1/MRP1 (or Abcc1/Mrp1, even though they are coregulated during drug resistance development.

  3. Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Jensen, Morten Østergaard; Hansen, Gry Aune; Vorum, Henrik

    2006-01-01

    cytoskeleton and is involved in cytokinesis. Labeling of S phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin possesses a paracrine role on the cells in its vicinity and therefore may be involved...... in the pathophysiology of thrombosis or in wound healing....... but not in normal vasculature. In order to study the possible effects of calumenin extracellularly we used proteomic profiling of fibroblasts cultured in absence as well as in presence of calumenin. Using two-dimensional gel electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) we show that normal fibroblasts...

  4. Proteomic profiling in Drosophila reveals potential Dube3a regulation of the actin cytoskeleton and neuronal homeostasis.

    Directory of Open Access Journals (Sweden)

    Laura Jensen

    Full Text Available The molecular defects associated with Angelman syndrome (AS and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. Here we used Drosophila melanogaster to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS or increased (as in dup(15 levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing of proteins from whole fly head extracts we identified a total of 50 proteins that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. We analyzed head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins. Our results indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins were >50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. We investigated one autism-associated protein, ATPα, and found that it can be ubiquitinated in a Dube3a dependent manner. We also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism.

  5. Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena A Goncharova

    Full Text Available TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1(-/- MEFs have decreased migration compared to littermate-derived Tsc1(+/+ MEFs. Migration of Tsc1(-/- MEFs with re-expressed TSC1 was comparable to Tsc1(+/+ MEF migration. In contrast, Tsc2(-/- MEFs showed an increased migration compared to Tsc2(+/+ MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1(-/- MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2(-/- MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1(-/- or Tsc2(-/- MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2(-/- MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2-null cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.

  6. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light

  7. Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

    DEFF Research Database (Denmark)

    Jorgensen, Nanna K; Pedersen, Stine F; Rasmussen, Hanne B;

    2003-01-01

    Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125......%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced...... by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling...

  8. Regulation of cell shape, wing hair initiation and the actin cytoskeleton by Trc/Fry and Wts/Mats complexes.

    Science.gov (United States)

    Fang, Xiaolan; Adler, Paul N

    2010-05-15

    The two NDR kinase family genes in Drosophila are tricornered (trc) and warts (wts). Previous studies on trc have focused on its role in the morphogenesis of extensions of epidermal cells and in dendrite branching and tiling. Studies on wts have focused on its roles as a tumor suppressor, in controlling photoreceptor type and in the maintenance of dendrites. Here we examine and compare the function of these genes in wing cells prior to their terminal differentiation. Mutations in these genes lead to changes in cell shape, cellular levels of F-actin, the timing of differentiation, and the expression of multiple wing hairs and DE-Cadherin. We showed that the effects of wts on all of these processes appear to be mediated by its regulation of the Yorkie transcription factor. We also provide evidence that trc regulates the expression of DE-cadherin and mwh. In addition, we showed that the effects on cell shape and the timing of differentiation appear to be not linked to changes in relative growth rate of cells compared to their neighbors.

  9. Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

    OpenAIRE

    Lahne, Manuela; Li, Jingling; Marton, Rebecca M.; Hyde, David R.

    2015-01-01

    Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cel...

  10. Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade body trafficking and exocytosis.

    Science.gov (United States)

    Conte, Ianina L; Hellen, Nicola; Bierings, Ruben; Mashanov, Gregory I; Manneville, Jean-Baptiste; Kiskin, Nikolai I; Hannah, Matthew J; Molloy, Justin E; Carter, Tom

    2016-02-01

    Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.

  11. Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches

    Science.gov (United States)

    Byrne, Kate M.; Monsefi, Naser; Dawson, John C.; Degasperi, Andrea; Bukowski-Wills, Jimi-Carlo; Volinsky, Natalia; Dobrzyński, Maciej; Birtwistle, Marc R.; Tsyganov, Mikhail A.; Kiyatkin, Anatoly; Kida, Katarzyna; Finch, Andrew J.; Carragher, Neil O.; Kolch, Walter; Nguyen, Lan K.; von Kriegsheim, Alex; Kholodenko, Boris N.

    2016-01-01

    Summary Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches. PMID:27136688

  12. A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

    Science.gov (United States)

    Bhajun, Ricky; Guyon, Laurent; Pitaval, Amandine; Sulpice, Eric; Combe, Stéphanie; Obeid, Patricia; Haguet, Vincent; Ghorbel, Itebeddine; Lajaunie, Christian; Gidrol, Xavier

    2015-02-01

    MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues

  13. SYP73 Anchors the ER to the Actin Cytoskeleton for Maintenance of ER Integrity and Streaming in Arabidopsis.

    Science.gov (United States)

    Cao, Pengfei; Renna, Luciana; Stefano, Giovanni; Brandizzi, Federica

    2016-12-05

    The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments.

  14. Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

    OpenAIRE

    1996-01-01

    GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchor...

  15. NADPH oxidase complex-derived reactive oxygen species, the actin cytoskeleton, and rho GTPases in cell migration

    DEFF Research Database (Denmark)

    Stanley, Alanna; Thompson, Kerry; Hynes, Ailish

    2014-01-01

    , these data will contribute significantly to our understanding of this intricate network under physiological conditions. Based on this, in vivo and in vitro studies can then be combined to elucidate the signaling pathways involved and their targets. Antioxid. Redox Signal. 20, 2026-2042....

  16. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    1993-01-01

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light c

  17. AFAP-1L1-mediated actin filaments crosslinks hinder Trypanosoma cruzi cell invasion and intracellular multiplication.

    Science.gov (United States)

    de Araújo, Karine Canuto Loureiro; Teixeira, Thaise Lara; Machado, Fabrício Castro; da Silva, Aline Alves; Quintal, Amanda Pifano Neto; da Silva, Claudio Vieira

    2016-10-01

    Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection.

  18. L-carnitine effectively improves the metabolism and quality of platelet concentrates during storage.

    Science.gov (United States)

    Deyhim, Mohammad Reza; Mesbah-Namin, Seyed Alireza; Yari, Fatemeh; Taghikhani, Mohammad; Amirizadeh, Naser

    2015-04-01

    Human platelets undergo structural and biochemical alternations during storage which are collectively called platelet storage lesion (PSL). PSL is characterized as metabolic and functionally changes. It causes decrease in platelet recovery and survival. Here, we evaluated the effect of L-carnitine (LC) on the metabolism, function, and mitochondrial metabolic activity of platelet during storage. Platelet-rich plasma was used to prepare platelet concentrate (PC) in Iranian Blood Transfusion Organization. For this purpose, ten PC bags from healthy donors were stored at 22 °C with gentle agitation in the presence or absence of LC. The effects of LC (15 mM) on the platelet quality were assessed by analyzing the levels of glucose, lactate, ATP, and lactate dehydrogenase (LDH) activity. Platelet aggregations induced by arachidonate and ristocetin were analyzed by aggregometer. Platelet mitochondrial melablolic activity was measured by tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay; platelet count and mean platelet volume were also determined by a hematology analyzer during 5 days of PC storage. The results indicated that LC could significantly decrease lactate concentration and glucose consumption accompanied with the increased oxygen consumption in stored PC. LDH activity also less significantly increased in LC-treated PC on days 2 and 5 of storage. Platelet aggregation in response to the ristocetin and arachidonate was significantly higher in LC-treated PC than that in untreated PC on day 5 of storage. Finally, platelet mitochondrial metabolic activity less significantly decreased in LC-treated PC compared to the control group on days 2 and 5 of storage. It seems that LC would be a good additive to reduce PSL and improve the platelet metabolism and quality of the stored PC for platelet transfusion therapy.

  19. Platelet aggregation following trauma

    DEFF Research Database (Denmark)

    Windeløv, Nis A; Sørensen, Anne M; Perner, Anders

    2014-01-01

    We aimed to elucidate platelet function in trauma patients, as it is pivotal for hemostasis yet remains scarcely investigated in this population. We conducted a prospective observational study of platelet aggregation capacity in 213 adult trauma patients on admission to an emergency department (ED......). Inclusion criteria were trauma team activation and arterial cannula insertion on arrival. Blood samples were analyzed by multiple electrode aggregometry initiated by thrombin receptor agonist peptide 6 (TRAP) or collagen using a Multiplate device. Blood was sampled median 65 min after injury; median injury...... severity score (ISS) was 17; 14 (7%) patients received 10 or more units of red blood cells in the ED (massive transfusion); 24 (11%) patients died within 28 days of trauma: 17 due to cerebral injuries, four due to exsanguination, and three from other causes. No significant association was found between...

  20. A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

    Directory of Open Access Journals (Sweden)

    Mustafa Tunalı

    2014-01-01

    Full Text Available We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF. The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun’s leukocyte- and platelet-rich fibrin (L-PRF method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF was processed with a scanning electron microscope (SEM. The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  1. A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

    Science.gov (United States)

    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

    2014-01-01

    We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  2. Platelets in inflammation and immunity.

    Science.gov (United States)

    Herter, J M; Rossaint, J; Zarbock, A

    2014-11-01

    The paradigm of platelets as mere mediators of hemostasis has long since been replaced by a dual role: hemostasis and inflammation. Now recognized as key players in innate and adaptive immune responses, platelets have the capacity to interact with almost all known immune cells. These platelet-immune cell interactions represent a hallmark of immunity, as they can potently enhance immune cell functions and, in some cases, even constitute a prerequisite for host defense mechanisms such as NETosis. In addition, recent studies have revealed a new role for platelets in immunity: They are ubiquitous sentinels and rapid first-line immune responders, as platelet-pathogen interactions within the vasculature appear to precede all other host defense mechanisms. Here, we discuss recent advances in our understanding of platelets as inflammatory cells, and provide an exemplary review of their role in acute inflammation.

  3. Estrogen, inflammation, and platelet phenotype.

    Science.gov (United States)

    Miller, Virginia M; Jayachandran, Muthuvel; Hashimoto, Kazumori; Heit, John A; Owen, Whyte G

    2008-01-01

    Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain. This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk. To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study. Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine. Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

  4. Guidance of mesenchymal stem cells on fibronectin structured hydrogel films.

    Directory of Open Access Journals (Sweden)

    Annika Kasten

    Full Text Available Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN that was homogeneously immmobilized to NCO-sP(EO-stat-PO, which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.

  5. Complement Activation Alters Platelet Function

    Science.gov (United States)

    2015-12-01

    Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet Function PRINCIPAL INVESTIGATOR: George Tsokos, M.D. CONTRACTING...Activation Alters Platelet Function 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0523 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) George Tsokos, M.D...a decreased level of disease. Further studies will expand upon these observations better outlining the function of platelets in the injury associated

  6. An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

    Directory of Open Access Journals (Sweden)

    Andrey Skripchenko

    Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

  7. Human platelet aggregation inhibitors from thyme (Thymus vulgaris L.).

    Science.gov (United States)

    Okazaki, Kenji; Kawazoe, Kazuyoshi; Takaishi, Yoshihisa

    2002-06-01

    Two antiaggregant compounds, thymol (compound 1) and 3,4,3',4'-tetrahydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (compound 2) were isolated from the leaves of thyme (Thymus vulgaris L.). The structures were determined by (1)H-, (13)C-NMR and mass spectra (MS) studies. These compounds inhibited platelet aggregation induced by collagen, ADP, arachidonic acid (AA) and thrombin except that compound 2 did not inhibit platelet aggregation induced by thrombin.

  8. Platelet effects on ovarian cancer

    Science.gov (United States)

    Davis, Ashley; Afshar-Kharghan, Vahid; Sood, Anil K.

    2014-01-01

    Growing understanding of the role of thrombocytosis, high platelet turnover, and the presence of activated platelets in the circulation in cancer progression and metastasis has brought megakaryocytes into focus. Platelet biology is essential to hemostasis, vascular integrity, angiogenesis, inflammation, innate immunity, wound healing, and cancer biology. However, before megakaryocyte/platelet-directed therapies can be considered for clinical use, understanding of the mechanism and biology of paraneoplastic thrombocytosis in malignancy is required. Here, we provide an overview of the clinical implications, biological significance, and mechanisms of paraneoplastic thrombocytosis in the context of ovarian cancer. PMID:25023353

  9. Novel aspects of platelet aggregation

    Directory of Open Access Journals (Sweden)

    Roka-Moya Y. M.

    2014-01-01

    Full Text Available The platelet aggregation is an important process, which is critical for the hemostatic plug formation and thrombosis. Recent studies have shown that the platelet aggregation is more complex and dynamic than it was previously thought. There are several mechanisms that can initiate the platelet aggregation and each of them operates under specific conditions in vivo. At the same time, the influence of certain plasma proteins on this process should be considered. This review intends to summarize the recent data concerning the adhesive molecules and their receptors, which provide the platelet aggregation under different conditions.

  10. Overview of platelet physiology and laboratory evaluation of platelet function.

    Science.gov (United States)

    Rodgers, G M

    1999-06-01

    Appropriate laboratory testing for the platelet-type bleeding disorders hinges on an adequate assessment in the history and physical examination. Patients with histories and screening laboratory results consistent with coagulation disorders (hemophilia, disseminated intravascular coagulation) are not appropriate candidates for platelet function testing. In contrast, patients with a lifelong history of platelet-type bleeding symptoms and perhaps a positive family history of bleeding would be appropriate for testing. Figure 6 depicts one strategy to evaluate these patients. Platelet morphology can easily be evaluated to screen for two uncommon qualitative platelet disorders: Bernard-Soulier syndrome (associated with giant platelets) and gray platelet syndrome, a subtype of storage pool disorder in which platelet granulation is morphologically abnormal by light microscopy. If the bleeding disorder occurred later in life (no bleeding with surgery or trauma early in life), the focus should be on acquired disorders of platelet function. For those patients thought to have an inherited disorder, testing for vWD should be done initially because approximately 1% of the population has vWD. The complete vWD panel (factor VIII coagulant activity, vWf antigen, ristocetin cofactor activity) should be performed because many patients will have abnormalities of only one particular panel component. Patients diagnosed with vWD should be classified using multimeric analysis to identify the type 1 vWD patients likely to respond to DDAVP. If vWD studies are normal, platelet aggregation testing should be performed, ensuring that no antiplatelet medications have been ingested at least 1 week before testing. If platelet aggregation tests are normal and if suspicion for an inherited disorder remains high, vWD testing should be repeated. The evaluation of thrombocytopenia may require bone marrow examination to exclude primary hematologic disorders. If future studies with thrombopoietin assays

  11. External quality assessment of platelet disorder investigations: results of international surveys on diagnostic tests for dense granule deficiency and platelet aggregometry interpretation.

    Science.gov (United States)

    Hayward, Catherine P M; Moffat, Karen A; Plumhoff, Elizabeth; Timleck, Marnie; Hoffman, Suzanne; Spitzer, Ernie; Van Cott, Elizabeth M; Meijer, Piet

    2012-09-01

    The quality of platelet aggregation and dense granule deficiency testing is important for diagnosing platelet function disorders. After a successful pilot exercise on diagnosing platelet dense granule deficiency by electron microscopy (EM), the North American Specialized Coagulation Laboratory Association (NASCOLA) has launched regular external quality assurance (EQA) for dense granule EM, as well as for the interpretation of platelet aggregation findings. EQA records were analyzed to assess performance. For EM EQA, between 2009 and 2011, there was excellent performance in distinguishing normal from dense granule-deficient samples and good (>70%) agreement on classifying most electron dense structures in platelets. For aggregation EQA, some normal variants were misclassified and overall case interpretations were more acceptable for rare disorders than for common findings. NASCOLA experiences with these EQAs indicate that there is a need to improve the quality of platelet disorder evaluations. For aggregometry interpretations, deficits in performance could be addressed by translating guideline recommendations into practice.

  12. Platelet Concentrates: Past, Present and Future

    OpenAIRE

    2011-01-01

    Platelets play a crucial role in hemostasis and wound healing, platelet growth factors are well known source of healing cytokines. Numerous techniques of autologous platelet concentrates have been developed and applied in oral and maxillofacial surgery. This review describes the evolution of the first and second generation of platelet concentrates (platelet rich plasma and platelet rich fibrin respectively) from their fore runner-fibrin sealants.

  13. Studies on megakaryopoiesis and platelet function

    OpenAIRE

    Meinders, M.

    2015-01-01

    Platelets are blood circulating specialized subcellular fragments, which are produced by megakaryocytes. Platelets are essential for hemostasis and wound healing but also play a role in non-hemostatic processes such as the immune response or cancer metastasis. Considering the immediate precursors of platelets, normal megakaryocyte development is essential for normal platelet function. Although much is known about platelet development, some aspects of platelet production remain poorly understo...

  14. The effects of platelet-rich fibrin extract (PRFe) on osteoblast MC3T3-E1 cells%富血小板纤维蛋白提取液对成骨细胞影响的实验研究

    Institute of Scientific and Technical Information of China (English)

    董凯; 柳忠豪; 张晓洁; 许丰伟

    2013-01-01

    目的:探讨富血小板纤维蛋白提取液(PRFe)对成骨细胞增殖、分化及细胞骨架的影响.方法:将培养中的小鼠成骨细胞MC3T3-E1分为2组,实验组采用50%浓度PRFe,对照组为正常αt-MEM培养液.MTr法检测细胞增殖;碱性磷酸酶(ALP)试剂盒检测ALP活性;茜素红染色观察细胞矿化功能,并用图像分析软件进行半定量分析;激光共聚焦显微镜观察细胞骨架形态.结果:MTT实验显示随时间延长,细胞数目明显增加(P<0.05),在各时间点,实验组细胞数量明显高于对照组(P<0.05);ALP检测显示随时间延长,ALP活性明显增大(P<0.05),在各时间点,实验组A值均显著大于对照组(P<0.05);茜素红染色显示随时间延长,钙结节染色的积分吸光度值逐渐增大(P<0.05),每一时间点,实验组的钙结节积分吸光度值大于对照组(P<0.05);细胞骨架观察显示在各时间点,实验组细胞骨架较对照组更加伸展.结论:PRFe能促进MC3T3-E1细胞的增殖、分化,对细胞骨架的排列和伸展有促进作用.%Objective:To evaluate the effects of platelet-rich fibrin extract (PRFe) on the proliferation and differentiation of osteoblast MC3T3-E1 cells.Methods:MC3T3-E1 cells were cultured in 50% PRFe (test group) and normal α-MEM respectively (controlgroup).The proliferation,alkaline phosphatase (ALP) activity and mineralization were examined by MTT assay,ALP Kit and Alizarin red dye staining respectively; the F-actin cytoskeleton was observed by confocal laser scaning microscopy (CLSM).Results:PRFe treatment increased the proliferation(P < 0.05),ALP activity(P < 0.05),and calcium nodus formation of MC3T3-E1 cells(P <0.05) in a time-dependant manner.At each time point,filaments in PRFe treated cells were more well spread than those in the untreated.Conclusion:PRFe may stimulate the proliferation and differentiation of osteoblasts and can promote the spread of F-actin cytoskeleton.

  15. Inhibition of smooth muscle contraction and platelet aggregation by peptide 204–212 of lipocortin 5: an attempt to define some structure requirements

    Directory of Open Access Journals (Sweden)

    K. G. Mugridge

    1993-01-01

    Full Text Available Peptide 204–212 of lipocortin (LC 5 inhibited porcine pancreatic phospholipase A2 (PLA2 induced rat stomach strip contractions and ADP induced rabbit platelet aggregation in a concentration dependent manner (IC30 of 10 μM and 400 μM, respectively. The first two amino acids are not necessary since the eptapeptide 206–212 was equipotent in both assays (IC30 of 12.5 μM and 420 μM. Of the two pentapeptides 204–208 and 208–212 only the latter showed inhibitory activity in both models although the potency was much reduced (IC30 of 170 μM and 630 μM compared with that of the parent nonapeptide. Comparison of peptide 204–212 effects with those of its analogues on LC1 and LC2 indicate that lysine 208 and aspartic acid 211 are essential in order to maintain a fully active nonapeptide.

  16. Platelets, inflammation and tissue regeneration.

    Science.gov (United States)

    Nurden, Alan T

    2011-05-01

    Blood platelets have long been recognised to bring about primary haemostasis with deficiencies in platelet production and function manifesting in bleeding while upregulated function favourises arterial thrombosis. Yet increasing evidence indicates that platelets fulfil a much wider role in health and disease. First, they store and release a wide range of biologically active substances including the panoply of growth factors, chemokines and cytokines released from a-granules. Membrane budding gives rise to microparticles (MPs), another active participant within the blood stream. Platelets are essential for the innate immune response and combat infection (viruses, bacteria, micro-organisms). They help maintain and modulate inflammation and are a major source of pro-inflammatory molecules (e.g. P-selectin, tissue factor, CD40L, metalloproteinases). As well as promoting coagulation, they are active in fibrinolysis; wound healing, angiogenesis and bone formation as well as in maternal tissue and foetal vascular remodelling. Activated platelets and MPs intervene in the propagation of major diseases. They are major players in atherosclerosis and related diseases, pathologies of the central nervous system (Alzheimers disease, multiple sclerosis), cancer and tumour growth. They participate in other tissue-related acquired pathologies such as skin diseases and allergy, rheumatoid arthritis, liver disease; while, paradoxically, autologous platelet-rich plasma and platelet releasate are being used as an aid to promote tissue repair and cellular growth. The above mentioned roles of platelets are now discussed.

  17. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

    Science.gov (United States)

    Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

    2014-05-01

    Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

  18. Structure of the active N-terminal domain of Ezrin. Conformational and mobility changes identify keystone interactions.

    Science.gov (United States)

    Smith, William James; Nassar, Nicolas; Bretscher, Anthony; Cerione, Richard A; Karplus, P Andrew

    2003-02-14

    Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and also may function in signaling cascades that regulate the assembly of actin stress fibers. Here, we report a crystal structure for the free (activated) FERM domain (residues 2-297) of recombinant human ezrin at 2.3 A resolution. Structural comparison among the dormant moesin FERM domain structure and the three known active FERM domain structures (radixin, moesin, and now ezrin) allows the clear definition of regions that undergo structural changes during activation. The key regions affected are residues 135-150 and 155-180 in lobe F2 and residues 210-214 and 235-267 in lobe F3. Furthermore, we show that a large increase in the mobilities of lobes F2 and F3 accompanies activation, suggesting that their integrity is compromised. This leads us to propose a new concept that we refer to as keystone interactions. Keystone interactions occur when one protein (or protein part) contributes residues that allow another protein to complete folding, meaning that it becomes an integral part of the structure and would rarely dissociate. Such interactions are well suited for long-lived cytoskeletal protein interactions. The keystone interactions concept leads us to predict two specific docking sites within lobes F2 and F3 that are likely to bind target proteins.

  19. Analyzing the platelet proteome.

    Science.gov (United States)

    García, Angel; Zitzmann, Nicole; Watson, Steve P

    2004-08-01

    During the last 10 years, mass spectrometry (MS) has become a key tool for protein analysis and has underpinned the emerging field of proteomics. Using high-throughput tandem MS/MS following protein separation, it is potentially possible to analyze hundreds to thousands of proteins in a sample at a time. This technology can be used to analyze the protein content (i.e., the proteome) of any cell or tissue and complements the powerful field of genomics. The technology is particularly suitable for platelets because of the absence of a nucleus. Cellular proteins can be separated by either gel-based methods such as two-dimensional gel electrophoresis or one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by liquid chromatography (LC) -MS/MS or by multidimensional LC-MS/MS. Prefractionation techniques, such as subcellular fractionations or immunoprecipitations, can be used to improve the analysis. Each method has particular advantages and disadvantages. Proteomics can be used to compare the proteome of basal and diseased platelets, helping to reveal information on the molecular basis of the disease.

  20. Platelet Rich Plasma and Knee Surgery

    Directory of Open Access Journals (Sweden)

    Mikel Sánchez

    2014-01-01

    Full Text Available In orthopaedic surgery and sports medicine, the knee joint has traditionally been considered the workhorse. The reconstruction of every damaged element in this joint is crucial in achieving the surgeon’s goal to restore the knee function and prevent degeneration towards osteoarthritis. In the last fifteen years, the field of regenerative medicine is witnessing a boost of autologous blood-derived platelet rich plasma products (PRPs application to effectively mimic and accelerate the tissue healing process. The scientific rationale behind PRPs is the delivery of growth factors, cytokines, and adhesive proteins present in platelets and plasma, as well as other biologically active proteins conveyed by the plasma such as fibrinogen, prothrombin, and fibronectin; with this biological engineering approach, new perspectives in knee surgery were opened. This work describes the use of PRP to construct and repair every single anatomical structure involved in knee surgery, detailing the process conducted in ligament, meniscal, and chondral surgery.

  1. Differential proteomic analysis of platelets suggested possible signal cascades network in platelets treated with salvianolic acid B.

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    Chao Ma

    Full Text Available BACKGROUND: Salvianolic acid B (SB is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, a differential proteomic analysis (two-dimensional electrophoresis was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70, LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca²+ and reactive oxygen species (ROS were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets. CONCLUSIONS/SIGNIFICANCE: Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca²+ level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

  2. Differential Proteomic Analysis of Platelets Suggested Possible Signal Cascades Network in Platelets Treated with Salvianolic Acid B

    Science.gov (United States)

    Ma, Chao; Yao, Yan; Yue, Qing-Xi; Zhou, Xin-Wen; Yang, Peng-Yuan; Wu, Wan-Ying; Guan, Shu-Hong; Jiang, Bao-Hong; Yang, Min; Liu, Xuan; Guo, De-An

    2011-01-01

    Background Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear. Methodology/Principal Findings In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca(2+) and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets. Conclusions/Significance Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets. PMID:21379382

  3. Helicobacter pylori urease activates blood platelets through a lipoxygenase-mediated pathway.

    Science.gov (United States)

    Wassermann, German E; Olivera-Severo, Deiber; Uberti, Augusto F; Carlini, Célia R

    2010-07-01

    The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase-derived eicosanoids. We investigated whether H. pylori urease displays platelet-activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein-labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED(50) 0.28 microM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12-lipoxygenase inhibitor) and enhanced approximately 3-fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12-lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet-activating factor, but required activation of verapamil-inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di- or tri-chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.

  4. Platelets in inflammation and infection.

    Science.gov (United States)

    Jenne, Craig N; Kubes, Paul

    2015-01-01

    Although platelets are traditionally recognized for their central role in hemostasis, many lines of research clearly demonstrate these rather ubiquitous blood components are potent immune modulators and effectors. Platelets have been shown to directly recognize, sequester and kill pathogens, to activated and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behavior, enhancing their ability to phagocytose and kill pathogens and inducing unique effector functions, such as the production of Neutrophil Extracellular Traps (NETs). This multifaceted response to infection and inflammation is due, in part, to the huge array of soluble mediators and cell surface molecules expressed by platelets. From their earliest origins as primordial hemocytes in invertebrates to their current form as megakaryocyte-derived cytoplasts, platelets have evolved to be one of the key regulators of host intravascular immunity and inflammation. In this review, we present the diverse roles platelets play in immunity and inflammation associated with autoimmune diseases and infection. Additionally, we highlight recent advances in our understanding of platelet behavior made possible through the use of advanced imaging techniques that allow us to visualize platelets and their interactions, in real-time, within the intact blood vessels of a living host.

  5. [Murine models of platelet diseases].

    Science.gov (United States)

    Lanza, F

    2007-05-01

    Platelet-related diseases correspond to functional defects or abnormal production (thrombopoiesis) of hereditary and immunological origins. Recent progress in the manipulation of the mouse genome (transgenesis, gene inactivation or insertion) has resulted in the generation of numerous strains exhibiting defective platelet function or production. Some strains reproduce known hereditary diseases affecting haemostasis (Glanzmann thrombasthenia, Bernard-Soulier syndrome (BSS) or thrombopoiesis (Wiscott-Aldrich or May-Hegglin syndrome). More often the mutated strains have no human equivalent and represent useful models to study: (i) the role of adhesive or signalling receptors or of signalling proteins in platelet-dependent haemostasis and thrombosis or; (ii) to study the poorly characterized mechanisms of thrombopoiesis, which implicate transcription factors (GATA, Fli1), growth factors and receptors (TPO, cMPL), and cytoskeletal or contractile proteins (tubulin, myosin). Additional mouse strains result from the selection of spontaneous mutants many of which affect intracellular platelet granules, representing models of storage pool diseases (SPD) such as the Gray platelet syndrome (alphaSPD) or Hermansky-Pudlack syndrome (deltaSPD). More recently, a systematic chemical mutagenesis approach has also identified genes involved in thrombopoiesis and platelet survival. Finally, mouse models of auto- or allo-immune thrombocytopenia have been developed to study the mechanisms of platelet destruction or removal.

  6. Platelet scintigraphy in atherothrombotic disease

    Energy Technology Data Exchange (ETDEWEB)

    Isaka, Yoshinari (Osaka National Hospital (Japan))

    1993-01-01

    Indium-111 platelet scintigraphy for the measurement of in vivo thrombogenicity is a useful noninvasive technique with a number of applications. From 1982 to 1989, we explored clinical relevance of this method for 576 consecutive patients with atherothrombotic disease. There was a disease-related difference in the percentage of positive platelet accumulation; 85% in patients with Dacron bifurcation graft, 75% in abdominal or thoracic aneurysm, 40% in intra-cardiac thrombi, 33% in arteriosclerosis obliterans and 25% in ischemic cerebrovascular disease. Labelled platelets accumulated frequently in the lesion with severe arteriographic abnormality. Aspirin clearly inhibited platelet accumulation on carotid atheroma but the effect of ticlopidine has been less conclusive. Short-term orally active PGI[sub 2] analogue had inhibitory effects on platelet accumulation in carotid atheroma and platelet aggregability, but did not cause significant reduction in plaque size. The results suggest the usefulness of platelet scintigraphy for monitoring the thrombogenicity in various atherothrombotic diseases. It will be necessary, however, to simplify the labelling procedures and to develop a new [sup 99m]Tc-labelled thrombus imaging agent, if thrombus imaging is to be considered for more generall use for patients with atherosclerosis. (author).

  7. Cyclosporine A enhances platelet aggregation.

    Science.gov (United States)

    Grace, A A; Barradas, M A; Mikhailidis, D P; Jeremy, J Y; Moorhead, J F; Sweny, P; Dandona, P

    1987-12-01

    In view of the reported increase in thromboembolic episodes following cyclosporine A (CyA) therapy, the effect of this drug on platelet aggregation and thromboxane A2 release was investigated. The addition of CyA, at therapeutic concentrations to platelet rich plasma from normal subjects in vitro was found to increase aggregation in response to adrenaline, collagen and ADP. Ingestion of CyA by healthy volunteers was also associated with enhanced platelet aggregation. The CyA-mediated enhancement of aggregation was further enhanced by the addition in vitro of therapeutic concentrations of heparin. Platelets from renal allograft recipients treated with CyA also showed hyperaggregability and increased thromboxane A2 release, which were most marked at "peak" plasma CyA concentration and less so at "trough" concentrations. Platelet hyperaggregability in renal allograft patients on long-term CyA therapy tended to revert towards normal following the replacement of CyA with azathioprine. Hypertensive patients with renal allografts on nifedipine therapy had normal platelet function and thromboxane release in spite of CyA therapy. These observations suggest that CyA-mediated platelet activation may contribute to the pathogenesis of the thromboembolic phenomena associated with the use of this drug. The increased release of thromboxane A2 (a vasoconstrictor) may also play a role in mediating CyA-related nephrotoxicity.

  8. PLATELET RICH FIBRIN: A PROMISING INNOVATION IN REGENERATIVE THERAPY

    Directory of Open Access Journals (Sweden)

    Arun

    2015-04-01

    Full Text Available Platelets can play a crucial role in regenerative therapy as they are reservoirs of growth factors and cytokines which are the key factors for regeneration of the bone and maturation of the soft tissue. Platelet - rich fibr in (PRF was first described by Choukroun et al. in France. It has been referred to as a second - generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Platelet - rich fibrin (PRF is autologous plate let concentrates prepared from patient’s own blood. It is a natural fibrin - based biomaterial prepared from an anticoagulant - free blood harvest without any artificial biochemical modification that allows obtaining fibrin membranes enriched with platelets a nd growth factors. Evidence from the literature suggests the potential role of PRF in regeneration and tissue engineering. The slow polymerisation during centrifugation and fibrin - based structure makes PRF a better healing biomaterial than PRP and other fi brin adhesives. The purpose of this review article is to describe the novel second - generation platelet concentrate PRF, which is an improvement over the traditionally prepared PRP for use in regenerative dentistry.

  9. Platelet enzyme abnormalities in leukemias

    Directory of Open Access Journals (Sweden)

    S Sharma

    2011-01-01

    Full Text Available Aim of the Study: The aim of this study was to evaluate platelet enzyme activity in cases of leukemia. Materials and Methods: Platelet enzymes glucose-6-phosphate dehydrogenase (G6PD, pyruvate kinase (PK and hexokinase (HK were studied in 47 patients of acute and chronic leukemia patients, 16 patients with acute myeloid leukemia (AML(13 relapse, three in remission, 12 patients with acute lymphocytic leukemia (ALL (five in relapse, seven in remission, 19 patients with chronic myeloid leukemia (CML. Results: The platelet G6PD activity was significantly low in cases of AML, ALL and also in CML. G6PD activity was normalized during AML remission. G6PD activity, although persistently low during ALL remission, increased significantly to near-normal during remission (P < 0.05 as compared with relapse (P < 0.01. Platelet PK activity was high during AML relapse (P < 0.05, which was normalized during remission. Platelet HK however was found to be decreased during all remission (P < 0.05. There was a significant positive correlation between G6PD and PK in cases of AML (P < 0.001 but not in ALL and CML. G6PD activity did not correlate with HK activity in any of the leukemic groups. A significant positive correlation was however seen between PK and HK activity in cases of ALL remission (P < 0.01 and CML (P < 0.05. Conclusions: Both red cell and platelet enzymes were studied in 36 leukemic patients and there was no statistically significant correlation between red cell and platelet enzymes. Platelet enzyme defect in leukemias suggests the inherent abnormality in megakaryopoiesis and would explain the functional platelet defects in leukemias.

  10. Platelet surface glutathione reductase-like activity.

    Science.gov (United States)

    Essex, David W; Li, Mengru; Feinman, Richard D; Miller, Anna

    2004-09-01

    We previously found that reduced glutathione (GSH) or a mixture of GSH/glutathione disulfide (GSSG) potentiated platelet aggregation. We here report that GSSG, when added to platelets alone, also potentiates platelet aggregation. Most of the GSSG was converted to GSH by a flavoprotein-dependent platelet surface mechanism. This provided an appropriate redox potential for platelet activation. The addition of GSSG to platelets generated sulfhydryls in the beta subunit of the alpha(IIb)beta(3) fibrinogen receptor, suggesting a mechanism for facilitation of agonist-induced platelet activation.

  11. MST Kinases Monitor Actin Cytoskeletal Integrity and Signal via c-Jun N-Terminal Kinase Stress-Activated Kinase To Regulate p21Waf1/Cip1 Stability

    OpenAIRE

    Densham, R. M.; E'Neill, Eric; Munro, J; et al, ...

    2009-01-01

    As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pa...

  12. Quantifying platelet gel coagulation using Sonoclot and Thrombelastograph hemostasis analyzer.

    Science.gov (United States)

    Cassidy, Lynsay K; Finney, Angela S; Ellis, William Cory; Spiwak, Allison J; Riley, Jeffrey B

    2005-03-01

    Little in vitro research exists discussing platelet gel composition and the resulting strength and degradation characteristics using point-of-care technologies. There must be a quantifiable way of determining the structural integrity of the resulting formed platelet gel thrombus. The Thrombelastograph Hemostasis Analyzer (TEG) and Sonoclot measure the elasticity of a clot as it forms and subsequently degrades naturally. The objective of this study was to determine the application of TEG and Sonoclot technologies as point-of-care devices for technicians using platelet gel therapy. The collected bovine blood was anticoagulated with CPD and processed using a previously published plasma sequestration protocol, using normal saline as a wash solution. The resulting platelet-rich plasma was stored in a sequestration bag in a water bath to maintain the blood temperature at 37 degrees C. Sequestered bovine platelet-rich plasma was made into platelet gel using three different thrombin concentrations. A total of 30 experiments were performed on the platelet gel product using both the TEG and the Sonoclot. We discovered that 6 of the Sonoclot tests and 15 of the TEG tests were valid. None of the TEG clot signatures and nine of the Sonoclot signatures were discovered to be invalid. A chi2 test was performed on the resultant data. The value of the chi2 test was calculated to be 12.86, which translated into a p value of less than 0.001. Despite the vast use and growing popularity of platelet gels, a method in which to quantify platelet gels has yet to be reported. There remains a possibility that gels formed with different concentrations of components may prove useful in different areas of surgery or their uses may expand to a broader spectrum of medicine. However, technology to quantify platelet gels must first be standardized. On the basis of the data collected in this study, it was determined that the TEG and the Sonoclot are not equally capable of analyzing platelet gel clots

  13. Biochemical and structural definition of the l-afadin- and actin-binding sites of alpha-catenin.

    Science.gov (United States)

    Pokutta, Sabine; Drees, Frauke; Takai, Yoshimi; Nelson, W James; Weis, William I

    2002-05-24

    alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.

  14. Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

    Directory of Open Access Journals (Sweden)

    Andrew Yee

    Full Text Available von Willebrand factor (VWF tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

  15. Mechanisms of platelet-mediated liver regeneration.

    Science.gov (United States)

    Lisman, Ton; Porte, Robert J

    2016-08-04

    Platelets have multiple functions beyond their roles in thrombosis and hemostasis. Platelets support liver regeneration, which is required after partial hepatectomy and acute or chronic liver injury. Although it is widely assumed that platelets stimulate liver regeneration by local excretion of mitogens stored within platelet granules, definitive evidence for this is lacking, and alternative mechanisms deserve consideration. In-depth knowledge of mechanisms of platelet-mediated liver regeneration may lead to new therapeutic strategies to treat patients with failing regenerative responses.

  16. Platelets as delivery systems for disease treatments

    OpenAIRE

    Shi, Qizhen; Montgomery, Robert R.

    2010-01-01

    Platelets are small, anucleate, discoid shaped blood cells that play a fundamental role in hemostasis. Platelets contain a large number of biologically active molecules within cytoplasmic granules that are critical to normal platelet function. Because platelets circulate in blood through out the body, release biological molecules and mediators on demand, and participate in hemostasis as well as many other pathophysiologic processes, targeting expression of proteins of interest to platelets an...

  17. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Wilbur, Jeremy D., E-mail: jwilbur@msg.ucsf.edu [Graduate Program in Biophysics, University of California, San Francisco, California 94143 (United States); Hwang, Peter K. [Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 (United States); Brodsky, Frances M. [The G. W. Hooper Foundation, Departments of Microbiology and Immunology and of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143 (United States); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 (United States); Fletterick, Robert J. [Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 (United States); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 (United States); Graduate Program in Biophysics, University of California, San Francisco, California 94143 (United States)

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  18. Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

    Energy Technology Data Exchange (ETDEWEB)

    Prochazkova, Katerina; Shuvalova, Ludmilla A.; Minasov, George; Voburka, Zden& #283; k; Anderson, Wayne F.; Satchell, Karla J.F.; (NWU); (Czech Academy)

    2009-10-05

    The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP{sub 6}). In this study, we demonstrated that InsP{sub 6} is not simply an allosteric cofactor, but rather binding of InsP{sub 6} stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-{angstrom} crystal structure of this InsP{sub 6}-bound unprocessed form of CPD was determined and revealed the scissile bond Leu{sup 3428}-Ala{sup 3429} captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP{sub 6}, but was reactivated for high affinity binding of InsP{sub 6} by cooperative binding of both a new substrate and InsP{sub 6}. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.

  19. [Protein kinase C activation induces platelet apoptosis].

    Science.gov (United States)

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  20. Inherited platelet disorders and oral health.

    Science.gov (United States)

    Valera, Marie-Cécile; Kemoun, Philippe; Cousty, Sarah; Sie, Pierre; Payrastre, Bernard

    2013-02-01

    Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention. © 2012 John Wiley & Sons A/S.

  1. Minimally Invasive Treatment with Platelet Rich Plasma in Patients with Knee Osteoarthritis

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    Albu Daniel–Emil

    2016-09-01

    Full Text Available Background: The main target of the structural damage in osteoarthritisis the hyaline cartilage. New options such as PRP (platelet rich plasma may cause structural improvement of the cartilage.

  2. Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

    NARCIS (Netherlands)

    S. Patel-Hett (Sunita); J.L. Richardson (Jennifer); H. Schulze (Harald); K. Drabek (Ksenija); N.A. Isaac (Natasha); K. Hoffmeister (Karin); R.A. Shivdasani (Ramesh); J.C. Bulinski (J. Chloë); N.J. Galjart (Niels); J.H. Hartwig (John); J. Italiano (Joseph)

    2008-01-01

    textabstractThe marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized

  3. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.

    Science.gov (United States)

    Dohan, David M; Choukroun, Joseph; Diss, Antoine; Dohan, Steve L; Dohan, Anthony J J; Mouhyi, Jaafar; Gogly, Bruno

    2006-03-01

    Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this second article, we investigate the platelet-associated features of this biomaterial. During PRF processing by centrifugation, platelets are activated and their massive degranulation implies a very significant cytokine release. Concentrated platelet-rich plasma platelet cytokines have already been quantified in many technologic configurations. To carry out a comparative study, we therefore undertook to quantify PDGF-BB, TGFbeta-1, and IGF-I within PPP (platelet-poor plasma) supernatant and PRF clot exudate serum. These initial analyses revealed that slow fibrin polymerization during PRF processing leads to the intrinsic incorporation of platelet cytokines and glycanic chains in the fibrin meshes. This result would imply that PRF, unlike the other platelet concentrates, would be able to progressively release cytokines during fibrin matrix remodeling; such a mechanism might explain the clinically observed healing properties of PRF.

  4. Platelet-containing tantalum powders

    Energy Technology Data Exchange (ETDEWEB)

    Schiele, E.K.

    1988-04-26

    A method of forming platelet tantalum powders is described comprising the steps of: (a) providing an ingot-derived precursor tantalum powder, and (b) ball-milling the precursor powder for a time sufficient to form a platelet powder having an average FSSS of less than about 2 micrometers, a Scott density not greater than about 30 g/in/sup 3/ and a BET surface area of at least about 0.7 in/sup 2//g.

  5. In vitro function of random donor platelets stored for 7 days in composol platelet additive solution

    Directory of Open Access Journals (Sweden)

    Gupta Ashish

    2011-01-01

    Full Text Available Background and Aim: Platelets are routinely isolated from whole blood and stored in plasma for 5 days. The present study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C. Materials and Methods: The study sample included 30 blood donors of both sex in State Blood Bank, CSM Medical University, Lucknow. Random donor platelets were prepared by platelet rich plasma method. Whole blood (350 ml was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators, with and without additive solution. Results: Platelet swirling was present in all the units at 22°C on day 7, with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution, whereas platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001 on day 7 without additive solution at 22°C. Conclusion: Our study infers that platelet viability and aggregation were best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

  6. Evidence of platelet activation in multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Alexander J Steven

    2008-06-01

    Full Text Available Abstract Objective A fatality in one multiple sclerosis (MS patient due to acute idiopathic thrombocytopenic purpura (ITP and a near fatality in another stimulated our interest in platelet function abnormalities in MS. Previously, we presented evidence of platelet activation in a small cohort of treatment-naive MS patients. Methods In this report, 92 normal controls and 33 stable, untreated MS patients were studied. Platelet counts, measures of platelet activation [plasma platelet microparticles (PMP, P-selectin expression (CD62p, circulating platelet microaggragtes (PAg], as well as platelet-associated IgG/IgM, were carried out. In addition, plasma protein S activity was measured. Results Compared to controls, PMP were significantly elevated in MS (p Conclusion Platelets are significantly activated in MS patients. The mechanisms underlying this activation and its significance to MS are unknown. Additional study of platelet activation and function in MS patients is warranted.

  7. VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

    Science.gov (United States)

    Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L; Aisiku, Omozuanvbo; Danglot, Lydia; Galli, Thierry; Flaumenhaft, Robert

    2015-07-30

    Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

  8. Structural and functional evaluation of C. elegans filamins FLN-1 and FLN-2.

    Directory of Open Access Journals (Sweden)

    Christina R DeMaso

    Full Text Available Filamins are long, flexible, multi-domain proteins composed of an N-terminal actin-binding domain (ABD followed by multiple immunoglobulin-like repeats (IgFLN. They function to organize and maintain the actin cytoskeleton, to provide scaffolds for signaling components, and to act as mechanical force sensors. In this study, we used transcript sequencing and homology modeling to characterize the gene and protein structures of the C. elegans filamin orthologs fln-1 and fln-2. Our results reveal that C. elegans FLN-1 is well conserved at the sequence level to vertebrate filamins, particularly in the ABD and several key IgFLN repeats. Both FLN-1 and the more divergent FLN-2 colocalize with actin in vivo. FLN-2 is poorly conserved, with at least 23 IgFLN repeats interrupted by large regions that appear to be nematode-specific. Our results indicate that many of the key features of vertebrate filamins are preserved in C. elegans FLN-1 and FLN-2, and suggest the nematode may be a very useful model system for further study of filamin function.

  9. Contribution of blood platelets to vascular pathology in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Zhang W

    2013-11-01

    Full Text Available Wei Zhang,1,2 Wei Huang,1 Fang Jing11Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, People's Republic of China; 2Shanghai Engineering Research Center of Molecular Therapy and Pharmaceutical Innovation, Shanghai, People's Republic of ChinaAbstract: Cerebral amyloid angiopathy (CAA is a critical factor in the pathogenesis of Alzheimer's disease (AD. In the clinical setting, nearly 98% AD patients have CAA, and 75% of these patients are rated as severe CAA. It is characterized by the deposition of the β-amyloid peptide (mainly Aβ40 in the walls of cerebral vessels, which induces the degeneration of vessel wall components, reduces cerebral blood flow, and aggravates cognitive decline. Platelets are anuclear cell fragments from bone marrow megakaryocytes and their function in hemostasis and thrombosis has long been recognized. Recently, increasing evidence suggests that platelet activation can also mediate the onset and development of CAA. First, platelet activation and adhesion to a vessel wall is the initial step of vascular injury. Activated platelets contribute to more than 90% circulating Aß (mainly Aβ1-40, which in turn activates platelets and results in the vicious cycle of Aβ overproduction in damaged vessel. Second, the uncontrolled activation of platelets leads to a chronic inflammatory reaction by secretion of chemokines (eg, platelet factor 4 [PF4], regulated upon activation normal T-cell expressed and presumably secreted [RANTES], and macrophage inflammatory protein [MIP-1α], interleukins (IL-1 β, IL-7, and IL-8, prostaglandins, and CD40 ligand (CD40L. The interaction of these biological response modulators with platelets, endothelial cells, and leukocytes establishes a localized inflammatory response that contributes to CAA formation. Finally, activated platelets are the upholder of fibrin clots, which are structurally abnormal and resistant to degradation

  10. 机采血小板志愿无偿献血人群结构分析及招募策略应对%Population structure analysis and recruitment strategy for voluntary apheresis platelet donors

    Institute of Scientific and Technical Information of China (English)

    范小伊; 杨婷婷; 曾芬; 邹海玉; 叶有玩

    2015-01-01

    Objective To plan recruitment strategy suitable for local region,keep the voluntary blood donors stable and ensure the safety of blood supply, by analyzing voluntary apheresis platelet donors population structure form central blood station of Longgang district Shenzhen city from 2011 to 2013. Methods On the basic of information from voluntary blood donors registration, population structure was analyzed for 3 368 voluntary apheresis platelet donors from the station. Results The voluntary apheresis platelet donors population structure from our station in this period remained stability. By gender statistics, the male donors was 3 069 times(91.12%), while the female donors was 299 times(8.87%). The male female ratio was 10.2∶1, most of the donors were male. According to age, 18 to 25 was 259 times(7.69%), 26 to 45 was 2 513 times(74.61%), 46 to 55 was 596 times(17.69%). It concluded that the age of voluntary apheresis platelet donors were mainly distributed in 26 to 45. According to degree of education, the most more was senior high school which occupied 1 134 times and 33.66%, while the secend most was junior high school which owned 873 times and 25.92%. By profession, worker which occupyed 569 times and 16.89% was the secend most, clerk was 534 times and 15.85%, the other was the most which owned 2 156 times and 64.01%. Conclusion The voluntary apheresis platelet donors population from our station was mainly distributed in male, senior high school and junior high school in education, 26 to 45 in age. It needed a powerful targeted publicity to females, high education people, and civil servants who rarely donated blood, and different recruitment strategy and service process must be made to different population.%目的:通过分析深圳市龙岗区中心血站2011—2013年机采血小板志愿无偿献血人群结构情况,制定适合本地区的招募策略,更好地保留献血者,保障供血安全。方法根据《无偿献血登记表献血者基本

  11. Impact of reticulated platelets on antiplatelet response to thienopyridines is independent of platelet turnover.

    Science.gov (United States)

    Stratz, Christian; Nührenberg, Thomas; Amann, Michael; Cederqvist, Marco; Kleiner, Pascal; Valina, Christian M; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald

    2016-10-28

    Reticulated platelets are associated with impaired antiplatelet response to thienopyridines. It is uncertain whether this interaction is caused by a decreased drug exposure due to high platelet turnover reflected by elevated levels of reticulated platelets or by intrinsic properties of reticulated platelets. This study sought to investigate if the impact of reticulated platelets on early antiplatelet response to thienopyridines is mainly caused by platelet turnover as previously suggested. Elective patients undergoing coronary intervention were randomised to loading with clopidogrel 600 mg or prasugrel 60 mg (n=200). Adenosine diphosphate (ADP)-induced platelet reactivity was determined by impedance aggregometry before, at 30, 60, 90, and 120 minutes and at day 1 after loading. Immature platelet count was assessed as marker of reticulated platelets by flow cytometry. Platelet reactivity increased with rising levels of immature platelet count in both groups. This effect was more distinctive in patients on clopidogrel as compared to patients on prasugrel. Overall, immature platelet count correlated well with on-treatment platelet reactivity at all time-points (p < 0.001). These correlations did not change over time in the entire cohort as well as in patients treated with clopidogrel or prasugrel indicating an effect independent of platelet turnover (comparison of correlations 120 minutes/day 1: p = 0.64). In conclusion, the association of immature platelet count with impaired antiplatelet response to thienopyridines is similar early and late after loading. This finding suggests as main underlying mechanism another effect of reticulated platelets on thienopyridines than platelet turnover.

  12. Platelets and infection — an emerging role of platelets in viral infection

    Directory of Open Access Journals (Sweden)

    Alice eAssinger

    2014-12-01

    Full Text Available Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia and several platelet function disorders increase the risk of bleeding. Over the last years more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients.Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favours platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen-antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies.All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count, but also shapes immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation and platelet-mediated modulations of innate and adaptive immune responses.

  13. Platelets and infection - an emerging role of platelets in viral infection.

    Science.gov (United States)

    Assinger, Alice

    2014-01-01

    Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen-antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses.

  14. The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

    Science.gov (United States)

    Jenne, Craig N.; Wong, Connie H. Y.; Petri, Björn; Kubes, Paul

    2011-01-01

    Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation. PMID:21949865

  15. The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.

    Directory of Open Access Journals (Sweden)

    Craig N Jenne

    Full Text Available Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation.

  16. Platelet-type von Willebrand disease: new insights into the molecular pathophysiology of a unique platelet defect.

    Science.gov (United States)

    Othman, Maha; Kaur, Harmanpreet; Emsley, Jonas

    2013-09-01

    Compared with coagulation factor defects, little attention is given to defects of platelet function as causes of rare bleeding disorders. Platelet-type von Willebrand disease (PT-VWD) is an autosomal dominant bleeding disorder and is unique among platelet disorders because it is characterized by platelet hyperresponsiveness rather than decreased function. The disease is caused by gain-of-function mutations in the platelet GP1BA gene, which codes for the platelet von Willebrand factor (VWF) receptor, GPIbα. Only five mutations (four missense and one deletion) have so far been reported. Affected patients suffer from mild to moderate mucocutaneous bleeding, low VWF activity compared with antigen, decreased high-molecular-weight VWF multimers, variable degree of thrombocytopenia and typically platelet aggregation in response to low concentrations of ristocetin. All reported PT-VWD missense mutations occur within the R-loop of GPIbα and it was speculated that the introduction of short branched chain mutations such as Val in PT-VWD stabilized the extended β-hairpin. Examination of this theory by surveying all the available GPIbα structures showed that a distinct conformation predominates for the R-loop when GPIbα is not bound to VWF-A1 and this provides the framework of a new hypothesis for the molecular basis of PT-VWD. Worldwide efforts to improve diagnosis of PT-VWD continue, and international systematic studies are required to further our understanding of the phenotype and the influence of the hyperresponsive GPIbα beyond hemostasis.

  17. Platelet satellitism in infectious disease?

    Science.gov (United States)

    Laskaj, Renata; Sikiric, Dubravka; Skerk, Visnja

    2015-01-01

    Background Platelet satellitism is a phenomenon of unknown etiology of aggregating platelets around polymorphonuclear neutrophils and other blood cells which causes pseudothrombocytopenia, visible by microscopic examination of blood smears. It has been observed so far in about a hundred cases in the world. Case subject and methods Our case involves a 73-year-old female patient with a urinary infection. Biochemical serum analysis (CRP, glucose, AST, ALT, ALP, GGT, bilirubin, sodium, potassium, chloride, urea, creatinine) and blood cell count were performed with standard methods on autoanalyzers. Serum protein fractions were examined by electrophoresis and urinalysis with standard methods on autoanalyzer together with microscopic examination of urine sediment. Erythrocyte sedimentation rate, blood culture and urine culture tests were performed with standard methods. Results Due to typical pathological values for bacterial urinary infection, the patient was admitted to the hospital. Blood smear examination revealed phenomenon, which has persisted for three weeks after the disease has been cured. Blood smears with EDTA as an anticoagulant had platelet satellitism whereas the phenomenon was not observed in tubes with different anticoagulants (Na, Li-heparin) and capillary blood. Discussion We hypothesize that satellitism was induced by some immunological mechanism through formation of antibodies which have mediated platelets binding to neutrophil membranes and vice versa. Unfortunately we were unable to determine the putative trigger for this phenomenon. To our knowledge this is the second case of platelet satellitism ever described in Croatia. PMID:26110042

  18. Evaluation of platelet aggregation in platelet concentrates: storage implications

    Directory of Open Access Journals (Sweden)

    Neiva Teresinha J.C.

    2003-01-01

    Full Text Available The use of hemo-derivatives is nowadays a fundamentally important therapeutic modality in the exercise of medicine. Among the various hemo-components employed, we have the platelet concentrate (PC, indicated in cases of hemorrhagic disturbances. We previously showed that platelet function in blood donors is reduced in their screening phase and after the separation process of PCs. Currently, we are providing evidence for the existence of biochemical and functional changes in PC preparations stored for three days at temperatures of 20 ± 2 ºC. Platelet concentrates from 40 healthy donors, collected in CPD anticoagulant and PL-146 polyvinylchloride containers, were examined in order to determine the pH value, pCO2 ,pO2 and lactate concentrations. In addition, the aggregation of platelets with thrombin and collagen were examined to evaluate platelet function. A pH increase from 7.07 ± 0.04 to 7.36 ± 0.07 (p < 0.01 was observed. The pCO2 concentration decreased progressively from 69.2 ± 7.7 mmHg to 28.8 ± 6.2 mmHg (p < 0.001 during the storage period. In contrast, pO2 value increase from 103.4 ± 30.6 to 152.3 ± 24.6 mmHg (p < 0.001 was evidenced during the 48 hours of storage. The lactate concentration increased from 17.97 ± 5.2 to 57.21 ± 5.7 mg/dl (p < 0.001. Platelet aggregation using 0.25 U/ml-thrombin and 2.0 µg/ml-collagen showed significant hypofunction from 61.8 ± 2.7% to 24.8 ± 9.8% and 62.7±5.0 to 33.4± 6.2 (p < 0.001, respectively. We concluded that the evaluated biochemical parameters and the platelet function changed significantly when the platelets were kept under routine storage conditions.

  19. CD8+ T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia

    Science.gov (United States)

    Qiu, Jihua; Liu, Xuena; Li, Xiaoqing; Zhang, Xu; Han, Panpan; Zhou, Hai; Shao, Linlin; Hou, Yu; Min, Yanan; Kong, Zhangyuan; Wang, Yawen; Wei, Yu; Liu, Xinguang; Ni, Heyu; Peng, Jun; Hou, Ming

    2016-01-01

    In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP. PMID:27321376

  20. An overview of platelet indices and methods for evaluating platelet function in thrombocytopenic patients

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Hvas, Anne-Mette; Nybo, Mads

    2014-01-01

    in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P-selectin and surface coverage by the Cone and Plate[let] analyser™ predict bleeding in selected thrombocytopenic populations...

  1. Effect of photodynamic therapy on mouse platelets

    Science.gov (United States)

    Zhou, Chuannong; Chi, Shunji; Deng, Jinsheng; Zhang, Hua; Liang, Junlin; Ha, Xian-wen

    1993-06-01

    Normal mice received hematoporphyrin derivative (HpD) i.v. prior to red light irradiation and the platelet-rich plasma was prepared and irradiated by red light. The platelets were processed for EM examination and stereological analysis. It was shown the 16 hrs after irradiation almost all platelets were necrotized; 8 hours after irradiation about one fourth of the platelets were necrotized and the remaining were considerably damaged. Immediately after irradiation a small number of platelets became necrotic and most other platelets were swollen and deformated, showing significantly increased mean area, perimeter and short axis, and mean cell volume and cell surface area. The findings indicate that platelets are highly sensitive to PDT action and can be directly and rapidly damaged by PDT even in the absence of vascular endothelial cells. The early platelet photoactivation may play an important role in the initiation of early vascular damage and microcirculatory alterations induced by PDT in vivo.

  2. Platelet Disorders: MedlinePlus Health Topic

    Science.gov (United States)

    ... Article: Erythropoietin and thrombopoietin mimetics: Natural alternatives to erythrocyte and platelet... Article: Detection of CALR Mutation in Clonal and Nonclonal Hematologic Diseases... Platelet Disorders -- see more articles Thrombocytopenias -- see more ...

  3. Effect of platelet age on adhesiveness to collagen and platelet surface charge

    Energy Technology Data Exchange (ETDEWEB)

    Castellan, R.M.; Steiner, M.

    1976-11-30

    Adhesion to collagen was investigated as a function of platelet age in rat platelets. Platelet adherence was measured using EDTA-containing platelet- rich plasma which was added to preparations of collagen fibers clamped between magnetic stirrers by recording changes in light transmission. The plot of light transmission versus logarithm of time was linear and allowed calculation of a slope factor which related to the rate of adherence. Neither the amount of collagen nor the platelet count were limiting in the test. Young platelet populations (less than or equal to 1 day old) were obtained during the recovery phase from immune induced thrombocytopenia. Old platelet populations were prepared by blocking thrombopoiesis with cyclophosphamide. Young platelets did not differ significantly from randomly aged platelets in this function. The electrophoretic mobility of platelets was not affected by their age.

  4. Dengue platelets meet Sir Arthur Conan Doyle.

    Science.gov (United States)

    Bray, Paul F

    2013-11-14

    In this issue of Blood, Hottz et al provide compelling evidence that dengue virus (DV) induces (1) platelet synthesis of interleukin-1b (IL-1b); (2) platelet-derived IL-1b–containing microvesicles (MVs) that increase vascular permeability; and (3) DV-triggered inflammasome activation in platelets.

  5. Molecular Basis Linking Platelet to Inflammation

    Institute of Scientific and Technical Information of China (English)

    马丽萍

    2010-01-01

    @@ Introduction Blood platelets not only play an important role in hemostasis and thrombosis,but increasing evidence show that they participate in the induction of inflammation.Firstly,platelets contain and release cytokines and immune mediators.And platelets are able to modulate and regulate the function of surrounding cells by adhesion molecules or by the release of various factors.

  6. Platelets Inhibit Migration of Canine Osteosarcoma Cells.

    Science.gov (United States)

    Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

    2017-01-01

    The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

  7. Platelet regulating properties of insulin revisited

    NARCIS (Netherlands)

    Andrade Ferreira, I. (Irlando)

    2005-01-01

    Disturbances in platelet responsiveness in diabetes mellitus (DM) lead to platelet-dependent complications in the vasculature. Our studies showed that insulin inhibits platelet activation by inhibiting ADP- and thrombin-induced Ca2+ levels. Ca2+ is under control of cAMP that is a potent endogenous p

  8. Image analysis of blood platelets adhesion.

    Science.gov (United States)

    Krízová, P; Rysavá, J; Vanícková, M; Cieslar, P; Dyr, J E

    2003-01-01

    Adhesion of blood platelets is one of the major events in haemostatic and thrombotic processes. We studied adhesion of blood platelets on fibrinogen and fibrin dimer sorbed on solid support material (glass, polystyrene). Adhesion was carried on under static and dynamic conditions and measured as percentage of the surface covered with platelets. Within a range of platelet counts in normal and in thrombocytopenic blood we observed a very significant decrease in platelet adhesion on fibrin dimer with bounded active thrombin with decreasing platelet count. Our results show the imperative use of platelet poor blood preparations as control samples in experiments with thrombocytopenic blood. Experiments carried on adhesive surfaces sorbed on polystyrene showed lower relative inaccuracy than on glass. Markedly different behaviour of platelets adhered on the same adhesive surface, which differed only in support material (glass or polystyrene) suggest that adhesion and mainly spreading of platelets depends on physical quality of the surface. While on polystyrene there were no significant differences between fibrin dimer and fibrinogen, adhesion measured on glass support material markedly differed between fibrin dimer and fibrinogen. We compared two methods of thresholding in image analysis of adhered platelets. Results obtained by image analysis of spreaded platelets showed higher relative inaccuracy than results obtained by image analysis of platelets centres and aggregates.

  9. Platelet count and platelet indices in women with preeclampsia

    Directory of Open Access Journals (Sweden)

    AlSheeha MA

    2016-11-01

    Full Text Available Muneera A AlSheeha,1 Rafi S Alaboudi,1 Mohammad A Alghasham,1 Javed Iqbal,2 Ishag Adam1 1Department of Obstetrics and Gynaecology, College of Medicine, Qassim University, Buriadah, 2Department of Obstetrics and Gynecology, Maternity and Children’s Hospital, Qassim, Kingdom of Saudi Arabia Background: Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia.Objective: To compare platelet indices, namely platelet count (PC, mean platelet volume (MPV, platelet distribution width (PDW, and PC to MPV ratio in women with preeclampsia compared with healthy controls.Setting: Qassim Hospital, Kingdom of Saudi Arabia.Design: A case–control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls.Results: There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC curves, the PC cutoff was 248.0×103/µL for diagnosis of preeclampsia (P=0.019; the area under the ROC curve was 62.4%. Binary regression suggests that women with PC <248.010×103/µL were at higher risk of preeclampsia (odds ratio =2.2, 95% confidence interval =1.08–4.6, P=0.03. The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia (P=0.035, the area under the ROC curve was 62.2%.Conclusion: PC <248.010×103/µL and PC to MPV ratio 31.2 are valid predictors of preeclampsia. Keywords: preeclampsia, platelets, PDW, mean platelet

  10. Platelet count and platelet indices in women with preeclampsia.

    Science.gov (United States)

    AlSheeha, Muneera A; Alaboudi, Rafi S; Alghasham, Mohammad A; Iqbal, Javed; Adam, Ishag

    2016-01-01

    Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia. To compare platelet indices, namely platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), and PC to MPV ratio in women with preeclampsia compared with healthy controls. Qassim Hospital, Kingdom of Saudi Arabia. A case-control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls. There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC) curves, the PC cutoff was 248.0×10(3)/µL for diagnosis of pre-eclampsia (P=0.019; the area under the ROC curve was 62.4%). Binary regression suggests that women with PC preeclampsia (odds ratio =2.2, 95% confidence interval =1.08-4.6, P=0.03). The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia (P=0.035, the area under the ROC curve was 62.2%). PC preeclampsia.

  11. Pooled platelet concentrates: an alternative to single donor apheresis platelets?

    Science.gov (United States)

    Pietersz, R N I

    2009-10-01

    Three types of platelet concentrates (PC) are compared: PC either processed with the platelet-rich plasma (PRP) or the Buffy coat (BC) method from whole blood units and PC obtained by apheresis. Leuko-reduction (LR) pre-storage is advocated to improve quality of the PC during storage and reduce adverse reactions in recipients. Standardization of methods allow preparation of PC with comparable yields of approximately 400 x 10(9) platelets in pooled non-LR-PRP, approximately 370 x 10(9) in pooled LR-BC-PC and in LR apheresis PC the number of platelets can be targeted on 350 x 10(9) or more with devices of various manufacturers. While viral transmission can be prevented by outstanding laboratory tests, the risk of bacterial contamination should be reduced by improved arm disinfection, deviation of the first 20-30 ml of blood and culture or rapid detection assays of the PC pre-issue. In a large prospective multicenter trial no significant difference was observed between cultures of apheresis PC (n = 15,198): 0.09% confirmed positive units versus 0.06% in pooled BC-PC (n = 37,045), respectively. Though platelet activation as measured by CD62 expression may differ in vitro in PC obtained with various apheresis equipment, and also between PC processed with the two whole blood methods there is scarce literature about the clinical impact of these findings. In conclusion the final products of LR-PC derived from whole blood or obtained by apheresis can be comparable, provided the critical steps of the processing method are identified and covered and the process is in control.

  12. Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, H.; Tamura, S.; Sudo, T.; Suzuki, T. (Kirin Brewery Co., Ltd., Gunma (Japan))

    1990-09-15

    Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused approximately 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of 14C-serotonin from 14C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation.

  13. Effect of ionizing radiation on platelet function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kalovidouris, A.E.; Papayannis, A.G. (Evangelismos Hospital, Athens (Greece))

    1981-01-01

    The effect of ionizing radiation on platelet function was investigated in vitro. Platelet-rich plasma (300x10/sup 9//l) was irradiated with doses of 1, 4, 10, 20 and 50 Gy. Platelet function tests were performed on both irradiated and control (non-irradiated) platelet samples. The platelet function tests were (1) platelet aggregation by ADP (1, 2, 4 ..mu..mol final concentration), adrenaline and collagen, (2) ADP-release from platelets, (3) clot retraction and (4) platelet factor-3 availability. It was found that roentgen irradiation of platelets in vitro did not affect these platelet function tests.

  14. Hemocompatibility of Polyvinyl Alcohol-Gelatin Core-Shell Electrospun Nanofibers: A Novel Scaffold for Modulating Platelet Deposition and Activation

    Science.gov (United States)

    Merkle, Valerie M.; Martin, Daniel; Hutchinson, Marcus; Tran, Phat L.; Behrens, Alana; Hossainy, Samir; Bluestein, Danny; Wu, Xiaoyi; Slepian, Marvin J.

    2015-01-01

    In this study, we evaluate coaxial electrospun nanofibers with gelatin in the shell and polyvinyl (PVA) in the core as a potential vascular material by determining fiber surface roughness, as well as human platelet deposition and activation under varying conditions. PVA scaffolds had the highest surface roughness (Ra = 65.5 ± 6.8 nm) but the lowest platelet deposition (34.2 ± 5.8 platelets) in comparison to gelatin nanofibers (Ra = 36.8 ± 3.0 nm & 168.9 ± 29.8 platelets) and coaxial nanofibers (1 Gel: 1 PVA coaxial – Ra = 24.0 ± 1.5 nm & 150.2 ± 17.4 platelets; 3 Gel: 1 PVA coaxial – Ra = 37.1 ± 2.8 nm & 167.8 ± 15.4 platelets). Therefore, the chemical structure of the gelatin nanofibers dominated surface roughness in platelet deposition. Due to their increased stiffness, the coaxial nanofibers had the highest platelet activation rate – rate of thrombin formation, in comparison to gelatin and PVA fibers. Our studies indicate that mechanical stiffness is a dominating factor for platelet deposition and activation, followed by biochemical moieties, and lastly surface roughness. Overall, these coaxial nanofibers are an appealing material for vascular applications by supporting cellular growth while minimizing platelet deposition and activation. PMID:25815434

  15. Platelets: crossroads of immunity and hemostasis.

    Science.gov (United States)

    Jenne, Craig N

    2014-07-31

    In this issue of Blood, Koupenova and colleagues report that platelets express functional TOLL-like receptor 7 (TLR7) and contribute to host survival during viral infection. Through a series of experiments utilizing mice deficient for TLR7 together with adoptive transfer of wild-type platelets, Koupenova et al demonstrate that platelets specifically respond to viral analogs and intact virus, leading to platelet activation and binding to various leukocyte subsets. Perhaps most importantly, this platelet activation appears absolutely essential for host survival during infection with some viral pathogens such as encephalomyocarditis virus (EMCV).

  16. Evidence that platelet buoyant density, but not size, correlates with platelet age in man.

    Science.gov (United States)

    Mezzano, D; Hwang, K; Catalano, P; Aster, R H

    1981-01-01

    Following infusion of 51Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radio-activity of LD platelets declined rapidly post-infusion (T1/2 = 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3--4-day period and gradually declined for 6--7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P less than 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33) days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P less than 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets = 7.57 mu3, LD platelets = 6.87 mu3, 0.05 less than P less than 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions.

  17. Evidence that platelet buoyant density, but not size, correlates with platelet age in man

    Energy Technology Data Exchange (ETDEWEB)

    Mezzano, D.; Hwang, K.; Catalano, P.; Aster, R.H.

    1981-01-01

    Following infusion of 51Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radio-activity of LD platelets declined rapidly post-infusion (T1/2 . 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3--4-day period and gradually declined for 6--7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P less than 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33) days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P less than 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets . 7.57 mu3, LD platelets . 6.87 mu3, 0.05 less than P less than 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions.

  18. Platelets: bridging hemostasis, inflammation, and immunity.

    Science.gov (United States)

    Jenne, C N; Urrutia, R; Kubes, P

    2013-06-01

    Although the function of platelets in the maintenance of hemostasis has been studied in great detail, more recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Platelets by virtue of their large numbers and their ability to rapidly release a broad spectrum of immunomodulatory cytokines, chemokines, and other mediators act as circulating sentinels. Upon detection of a pathogen, platelets quickly activate and begin to drive the ensuing inflammatory response. Platelets have the ability to directly modulate the activity of neutrophils (phagocytosis, oxidative burst), endothelium (adhesion molecule and chemokine expression), and lymphocytes. Due to their diverse array of adhesion molecules and preformed chemokines, platelets are able to adhere to leukocytes and facilitate their recruitment to sites of tissue damage or infection. Furthermore, platelets directly participate in the capture and sequestration of pathogens within the vasculature. Platelet-neutrophil interactions are known to induce the release of neutrophil extracellular traps (NETs) in response to either bacterial or viral infection, and platelets have been shown to internalize pathogens, sequestering them in engulfment vacuoles. Finally, emerging data indicate that platelets also participate in the host immune response by directly killing infected cells. This review will highlight the central role platelets play in the initiation and modulation of the host inflammatory and immune responses.

  19. Calpain Activator Dibucaine Induces Platelet Apoptosis

    Directory of Open Access Journals (Sweden)

    Jun Liu

    2011-03-01

    Full Text Available Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

  20. Platelet-mediated cytotoxicity and its enhancement by platelet activating factor.

    Science.gov (United States)

    Bykovskaya, S N; Bolvacheva, A V; Kiselevsky, M V; Khaylenko, V A; Bykovsky, A F

    1991-01-01

    Platelet cytotoxicity was assessed in 70 cancer patients with various tumor localizations and in 30 normal donors. The data presented reveal that the ACL cell line displays the highest sensitivity to platelet cytotoxicity. Using the ACL cells, we discovered that platelets from oncological patients and normal donors display comparable cytotoxicity. The level of platelet lytic activity is irrelevant to tumor localisation; however, it appears to be dependent on the stage of tumor growth. Incubation of platelets, both from donors and patients, with PAF (concentration range 10 pM to 10 nM) results in a significant rise of the killing activity of platelets. PAF induces greater cytotoxicity enhancement for platelets with lower initial activity, this pattern appearing to be the specific feature of the PAF mediated effect. Hence, platelets can be considered as effector cells relevant to antitumor immunity; PAF-mediated enhancement of platelet cytotoxicity can appear to be useful in the search for new immunotherapeutic drugs.

  1. Platelet function tests: a comparative review

    Directory of Open Access Journals (Sweden)

    Paniccia R

    2015-02-01

    Full Text Available Rita Paniccia,1,2 Raffaella Priora,1,2 Agatina Alessandrello Liotta,2 Rosanna Abbate1,2 1Department of Experimental and Clinical Medicine, Thrombosis Center, University of Florence, Florence, Italy; 2Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Abstract: In physiological hemostasis a prompt recruitment of platelets on the vessel damage prevents the bleeding by the rapid formation of a platelet plug. Qualitative and/or quantitative platelet defects promote bleeding, whereas the high residual reactivity of platelets in patients on antiplatelet therapies moves forward thromboembolic complications. The biochemical mechanisms of the different phases of platelet activation – adhesion, shape change, release reaction, and aggregation – have been well delineated, whereas their complete translation into laboratory assays has not been so fulfilled. Laboratory tests of platelet function, such as bleeding time, light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, and platelet activation investigated by flow cytometry, are traditionally utilized for diagnosing hemostatic disorders and managing patients with platelet and hemostatic defects, but their use is still limited to specialized laboratories. To date, a point-of-care testing (POCT dedicated to platelet function, using pertinent devices much simpler to use, has now become available (ie, PFA-100, VerifyNow System, Multiplate Electrode Aggregometry [MEA]. POCT includes new methodologies which may be used in critical clinical settings and also in general laboratories because they are rapid and easy to use, employing whole blood without the necessity of sample processing. Actually, these different platelet methodologies for the evaluation of inherited and acquired bleeding disorders and/or for monitoring antiplatelet therapies are spreading and the study of platelet function is strengthening. In this review, well

  2. In vitro function of random donor platelets stored for 7 days in composol platelet additive solution

    Directory of Open Access Journals (Sweden)

    Gupta Ashish

    2011-01-01

    Full Text Available Background and Aim: Platelets are routinely isolated from whole blood and stored in plasma for 5 days. This study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C. Materials and Methods: The study sample included 30 blood donors of both sex in State Blood Bank, C S M Medical University, Lucknow. Random donor platelets were prepared by the platelet-rich plasma method. Whole blood (350 ml was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators with and without additive solution. Results: Platelet swirling was present in all the units at 22°C on day 7 with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution while platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001 on day 7 without additive solution at 22°C. Conclusion: Our study infers that the platelet viability and aggregation were the best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

  3. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    Science.gov (United States)

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

  4. Utrophins compensate for Dp71 absence in mdx3cv in adhered platelets.

    Science.gov (United States)

    Cerecedo, Doris; Mondragón, Ricardo; Candelario, Aurora; García-Sierra, Francisco; Mornet, Dominique; Rendón, Alvaro; Martínez-Rojas, Dalila

    2008-01-01

    Platelet adhesion is a critical step due to its hemostatic role in stopping bleeding after vascular damage. Short dystrophins are the most abundant dmd gene products in nonmuscle tissues, and in association with cytoskeleton proteins contribute to their intrinsic function; while utrophins are dystrophin-homologous related family proteins with structural and functional similarities. We previously demonstrated the presence of Dp71 isoforms, utrophins, and various dystrophin-associated proteins and their participation in cytoskeleton re-organization, filopodia and lamellipodia extension, and in centralizing cytoplasmic granules during the adhesion process of human platelets. To evaluate the morphologic changes and actin-based structures of mdx(3cv) platelets during the adhesion process, we compared the topographic distribution of Dp71d/Dp71Delta110(m) and dystrophin-associated protein in adhered platelets from dystrophic mdx(3cv) mouse. By confocal microscopy, we showed that absence of Dp71 isoforms in platelets from this animal model disrupted dystrophin-associated protein expression and distribution without modifying the platelet morphology displayed during the glass-adhesion process. By immunoprecipitation assays, we proved that up-regulated utrophins were associated with dystrophin-associated proteins to conform the dystrophin-associated protein complex corresponding to utrophins, which might compensate for Dp71 absence in mdx(3cv) platelets.

  5. Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

    Science.gov (United States)

    Clemetson, Kenneth J

    2015-01-01

    Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.

  6. Platelet-derived growth factor B retention is essential for development of normal structure and function of conduit vessels and capillaries

    DEFF Research Database (Denmark)

    Nyström, Henrik C.; Lindblom, Per; Wickman, Anna;

    2006-01-01

    : Passive and active properties of conduit vessels were studied using myograph techniques and histological examination. Capillary structure and function was studied using measurements of capillary density in skeletal muscle and by assessing aerobic physical performance in a treadmill setup. Cardiac function...... was assessed using echocardiography. RESULTS: Myograph experiments revealed an increased diameter and stiffness of the aorta in RetKO. Histological examination showed increased media collagen content and a decreased number of aortic wall layers, however with a similar number of vascular smooth muscle cells....... This outward eutrophic remodelling of the aorta was accompanied by endothelial dysfunction. RetKO showed decreased capillary density in skeletal muscle and signs of a defective delivery of capillary oxygen to skeletal muscle, as shown by a decreased physical performance. In RetKO mice, echocardiography...

  7. Platelet-derived growth factor B retention is essential for development of normal structure and function of conduit vessels and capillaries

    DEFF Research Database (Denmark)

    Nyström, Henrik C.; Lindblom, Per; Wickman, Anna

    2006-01-01

    : Passive and active properties of conduit vessels were studied using myograph techniques and histological examination. Capillary structure and function was studied using measurements of capillary density in skeletal muscle and by assessing aerobic physical performance in a treadmill setup. Cardiac function...... was assessed using echocardiography. RESULTS: Myograph experiments revealed an increased diameter and stiffness of the aorta in RetKO. Histological examination showed increased media collagen content and a decreased number of aortic wall layers, however with a similar number of vascular smooth muscle cells....... This outward eutrophic remodelling of the aorta was accompanied by endothelial dysfunction. RetKO showed decreased capillary density in skeletal muscle and signs of a defective delivery of capillary oxygen to skeletal muscle, as shown by a decreased physical performance. In RetKO mice, echocardiography...

  8. Platelet count and platelet indices in women with preeclampsia

    OpenAIRE

    AlSheeha MA; Alaboudi RS; Alghasham MA; Iqbal J; Adam I

    2016-01-01

    Muneera A AlSheeha,1 Rafi S Alaboudi,1 Mohammad A Alghasham,1 Javed Iqbal,2 Ishag Adam1 1Department of Obstetrics and Gynaecology, College of Medicine, Qassim University, Buriadah, 2Department of Obstetrics and Gynecology, Maternity and Children’s Hospital, Qassim, Kingdom of Saudi Arabia Background: Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia.Obj...

  9. Pathogen-Reduced, Platelet Additive Solution, Extended Stored Platelets (PREPS)

    Science.gov (United States)

    2015-10-01

    associated sepsis remains the principal lethal risk associated with platelet transfusion. Cold storage (4°C) is known to reduce post transfusion...and no residual radiation is detectable . *P-selectin samples will be prepped on end of storage day and batch tested. **Bacterial Culture sample...temperature controlled room until such time as they have no detectable residual radiation. This is generally about 3-4 months. At that point they are

  10. Influence of Oxidative Stress on Stored Platelets

    Directory of Open Access Journals (Sweden)

    K. Manasa

    2016-01-01

    Full Text Available Platelet storage and its availability for transfusion are limited to 5-6 days. Oxidative stress (OS is one of the causes for reduced efficacy and shelf-life of platelets. The studies on platelet storage have focused on improving the storage conditions by altering platelet storage solutions, temperature, and materials. Nevertheless, the role of OS on platelet survival during storage is still unclear. Hence, this study was conducted to investigate the influence of storage on platelets. Platelets were stored for 12 days at 22°C. OS markers such as aggregation, superoxides, reactive oxygen species, glucose, pH, lipid peroxidation, protein oxidation, and antioxidant enzymes were assessed. OS increased during storage as indicated by increments in aggregation, superoxides, pH, conjugate dienes, and superoxide dismutase and decrements in glucose and catalase. Thus, platelets could endure OS till 6 days during storage, due to the antioxidant defense system. An evident increase in OS was observed from day 8 of storage, which can diminish the platelet efficacy. The present study provides an insight into the gradual changes occurring during platelet storage. This lays the foundation towards new possibilities of employing various antioxidants as additives in storage solutions.

  11. Effects of Physical (Inactivity on Platelet Function

    Directory of Open Access Journals (Sweden)

    Stefan Heber

    2015-01-01

    Full Text Available As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (inactivity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects’ cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i acute, strenuous exercise can lead to platelet activation, (ii regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality.

  12. Trehalose lyophilized platelets for wound healing.

    Science.gov (United States)

    Pietramaggiori, Giorgio; Kaipainen, Arja; Ho, David; Orser, Cindy; Pebley, Walter; Rudolph, Alan; Orgill, Dennis P

    2007-01-01

    Fresh platelet preparations are utilized to treat a wide variety of wounds, although storage limitations and mixed results have hampered their clinical use. We hypothesized that concentrated lyophilized and reconstituted platelet preparations, preserved with trehalose, maintain and possibly enhance fresh platelets' ability to improve wound healing. We studied the ability of a single dose of trehalose lyophilized and reconstituted platelets to enhance wound healing when topically applied on full-thickness wounds in the genetically diabetic mouse. We compared these results with the application of multiple doses of fresh platelet preparations and trehalose lyophilized and reconstituted platelets as well as multiple doses of vascular endothelial growth factor (VEGF) and wounds left untreated. Trehalose lyophilized and reconstituted platelets, in single and multiple applications, multiple applications of fresh platelets and multiple applications of VEGF increased granulation tissue deposition, vascularity, and proliferation when compared with untreated wounds, as assessed by histology and immunohistochemistry. Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of nonhealing wounds.

  13. Small RNAs as potential platelet therapeutics.

    Science.gov (United States)

    Edelstein, Leonard C; Bray, Paul F

    2012-01-01

    MicroRNAs (miRNAs) are 21-23 nucleotide RNAs that regulate more than 60% of mammalian protein coding genes. miRNAs play critical roles in hematopoiesis and megakaryocyte function and development. Platelets, in addition to possessing functional miRNA processing machinery, have miRNA levels that have been correlated with platelet reactivity, and these miRNAs have been shown to target mRNAs that encode proteins that alter platelet function. There are potential uses of platelet miRNA as biomarkers and therapeutic agents. Due to the ability of platelets to release miRNA-containing microparticles at sites of activation, including angiogenic regions, tumors, and atherosclerotic plaques, there is the possibility of engineering platelets to deliver miRNA-based therapies to these sites. Cellpreferential expression of miRNAs could be exploited to restrict transgene expression in hematopoietic stem cell gene therapy to the desired lineage, including megakaryocytes and platelets. Finally, manipulation of gene expression in stored platelets may allow more effective platelet storage. Although much work remains to be done, there is great potential in miRNA-based platelet therapies.

  14. Platelet MicroRNAs: An Overview.

    Science.gov (United States)

    Dahiya, Neetu; Sarachana, Tewarit; Vu, Long; Becker, Kevin G; Wood, William H; Zhang, Yongqing; Atreya, Chintamani D

    2015-10-01

    MicroRNAs (miRNAs) are short ~22-nucleotide noncoding RNA that have been found to influence the expression of many genes and cellular processes by either repressing translation or degrading messenger RNA transcripts. Platelet miRNA expression has been shown to be perturbed during ex vivo storage of platelets and in platelet-associated disorders. Although bioinformatics-based miRNA target predictions have been established, direct experimental validation of the role of miRNAs in platelet biology has been rather slow. Target prediction studies are, nonetheless, valuable in directing the design of appropriate experiments to test specific miRNA:messenger RNA interactions relevant to the underlying mechanisms of platelet function in general and in disease as well as in ex vivo storage-associated "storage lesions," a collective term used to include physiologic, biochemical, and morphologic changes that occur in stored platelets. This brief review will focus on emerging human platelet miRNA studies to emphasize their potential role relevant to transfusion medicine field in terms of regulating platelet signaling pathways, markers of platelet associated disorders, and remote impactors of gene expression (intercellular biomodulators) as well as potential platelet quality markers of storage and pathogen reduction treatments.

  15. Understanding platelet function through signal transduction.

    Science.gov (United States)

    Lazarus, Alan H; Song, Seng; Crow, Andrew R

    2003-01-01

    Platelets are activated by a number of stimuli resulting in the expression and/or activation of surface receptors, secretion of vasoactive substances, adhesion, aggregation, and finally thrombus formation. These events are propagated by a process known as transmembrane signaling, which relays the activating signal from the platelet membrane (eg, von Willebrand Factor binding to glycoprotein Ib) to the inside of the platelet which then serves to activate the platelet via a cascade of biochemical interactions. Inhibition of these transmembrane signaling molecules with a variety of available inhibitors or antagonists can in many cases prevent the platelet from becoming activated. An awareness of the mechanisms involved in platelet transmembrane signaling and the recent availability of new reagents to inhibit signaling may provide us with additional means to prevent platelet activation and perhaps even ameliorate the platelet storage lesion. This review will provide an introduction to the field of platelet transmembrane signaling and give an overview of some of the platelet signaling mechanisms that are relevant to transfusion medicine. Copyright 2003, Elsevier Science (USA). All rights reserved.

  16. Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets

    Energy Technology Data Exchange (ETDEWEB)

    Stratton, J.R.; Ballem, P.J.; Gernsheimer, T.; Cerqueira, M.; Slichter, S.J.

    1989-05-01

    Using autologous /sup 111/In-labeled platelets, platelet kinetics and the sites of platelet destruction were assessed in 16 normal subjects (13 with and three without spleens), in 17 studies of patients with primary autoimmune thrombocytopenic purpura (AITP), in six studies of patients with secondary AITP, in ten studies of patients with AITP following splenectomy, and in five thrombocytopenic patients with myelodysplastic syndromes. In normal subjects, the spleen accounted for 24 +/- 4% of platelet destruction and the liver for 15 +/- 2%. Untreated patients with primary AITP had increased splenic destruction (40 +/- 14%, p less than 0.001) but not hepatic destruction (13 +/- 5%). Compared with untreated patients, prednisone treated patients did not have significantly different spleen and liver platelet sequestration. Patients with secondary AITP had similar platelet counts, platelet survivals, and increases in splenic destruction of platelets as did patients with primary AITP. In contrast, patients with myelodysplastic syndromes had a normal pattern of platelet destruction. In AITP patients following splenectomy, the five nonresponders all had a marked increase (greater than 45%) in liver destruction compared to five responders (all less than 40%). Among all patients with primary or secondary AITP, there was an inverse relationship between the percent of platelets destroyed in the liver plus spleen and both the platelet count (r = 0.75, p less than 0.001) and the platelet survival (r = 0.86, p less than 0.001). In a stepwise multiple linear regression analysis, total liver plus spleen platelet destruction, the platelet survival and the platelet turnover were all significant independent predictors of the platelet count. Thus platelet destruction is shifted to the spleen in primary and secondary AITP. Failure of splenectomy is associated with a marked elevation in liver destruction.

  17. Role of dystrophins and utrophins in platelet adhesion process.

    Science.gov (United States)

    Cerecedo, Doris; Mondragón, Ricardo; Cisneros, Bulmaro; Martínez-Pérez, Francisco; Martínez-Rojas, Dalila; Rendón, Alvaro

    2006-07-01

    Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

  18. STABILIZATION OF STANDARD PLATELET CONCENTRATES AND MINIMIZATION OF THE PLATELET STORAGE LESION BY A PROSTACYCLIN ANALOG

    NARCIS (Netherlands)

    ELIAS, M; HEETHUIS, A; BOM, [No Value; BLOM, N; MCSHINE, RL; HALIE, MR; SIBINGA, CTS

    Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss. of platelet sensitivity to aggregating agents. To mimic the situation after

  19. Mean platelet volume in acute rheumatic fever.

    Science.gov (United States)

    Sert, Ahmet; Aypar, Ebru; Odabas, Dursun

    2013-01-01

    Acute rheumatic fever (ARF) is still an endemic disease, especially among school-aged children in developing countries. Mean platelet volume (MPV), which is commonly used as a measure of platelet size, indicates the rate of platelet production and platelet activation. We aimed to investigate MPV in children with ARF. The study population consisted of 40 children with ARF (32 patients with carditis and 8 patients without carditis) and 40 healthy control subjects. White blood cell (WBC) and platelet counts were significantly higher and MPV values were significantly lower in patients with ARF during the acute stage when compared to controls. Erythrocyte sedimentation rate (ESR) and C-reactive protein values significantly decreased in patients with ARF after the treatment when compared to baseline, whereas MPV values increased. MPV values were negatively correlated with ESR and WBC, and platelet counts. In conclusion, during the acute stage of ARF, MPV values were lower when compared to controls.

  20. Therapeutic platelet reduction: Use in postsplenectomy thrombocytosis

    Directory of Open Access Journals (Sweden)

    Gita Negi

    2015-01-01

    Full Text Available Therapeutic platelet reduction is an effective modality for the reduction of platelet count in patients with treatment of extreme thrombocytosis resulting from a variety of primary and secondary causes of thrombocytosis, which may be associated with thrombotic or hemorrhagic complications of varying degrees. These cases when symptomatic fall into the ASFA Category II indication for therapeutic platelet apheresis procedure. Here, we report a case of postsplenectomy secondary thrombocytosis presenting with extremely high platelet counts and subsequent thrombosis in the shunt and successful treatment after therapeutic platelet reduction. The case is being presented to bring forth the fact that therapeutic platelet reduction is an easy procedure that gives quick and good results and also to bring to the attention of transfusion specialists an associated but as yet unreported procedural finding.

  1. Laboratory testing for platelet function disorders.

    Science.gov (United States)

    Israels, S J

    2015-05-01

    Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

  2. Differential effects of platelets and platelet inhibition by ticagrelor on TLR2- and TLR4-mediated inflammatory responses

    NARCIS (Netherlands)

    Tunjungputri, R.N.; Ven, A.J.A.M. van der; Riksen, N.P.; Rongen, G.A.P.J.M.; Tacke, S.; Berg, T.N.A. van den; Fijnheer, R.; Gomes, M.E.; Dinarello, C.A.; Veerdonk, F.L. van de; Gasem, M.H.; Netea, M.G.; Joosten, L.A.B.; Groot, P.G. de; Mast, Q. de

    2015-01-01

    Platelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y12 receptor antagonist

  3. LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

    Science.gov (United States)

    Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

    2014-11-01

    Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. The effects of selective serotonin reuptake inhibitors on platelet function in whole blood and platelet concentrates.

    Science.gov (United States)

    Reikvam, Anne-Grete; Hustad, Steinar; Reikvam, Håkon; Apelseth, Torunn Oveland; Nepstad, Ina; Hervig, Tor Audun

    2012-01-01

    Several studies report that patients who are treated with selective serotonin reuptake inhibitors (SSRIs) for depression may have increased risk of bleeding, particularly from the gastrointestinal tract. This may be related to low intraplatelet serotonin concentrations. Several blood banks do not store platelets from donors using SSRIs for transfusion, although the possible effects of SSRIs on platelet storage are not documented. We conducted a case-control pilot study of apheresis platelet concentrates prepared from donors using SSRIs (n=8) and from donors without medication (n=10). The platelet concentrates were stored for 5 days. Light transmission aggregometry (LTA), thrombelastography (TEG), and flow cytometric analyses were preformed for in vitro measurements of platelet function. Platelet function and platelet serotonin content were investigated in whole blood and in platelet concentrates stored for up to 5 days. LTA, TEG, and flow cytometric analysis of glycoprotein expression did not reveal any significant differences between the two groups. All 18 platelet concentrates performed well according to the standards set for platelet quality in relation to transfusion. Blood donors using SSRIs had significantly lower platelet serotonin compared to blood donors without medication. The results from our pilot study indicate that platelets from donors using SSRIs may be suitable for transfusion after storage for 5 days, but further laboratory and clinical studies are necessary to confirm this.

  5. Platelet aggregation and quality control of platelet concentrates produced in the Amazon Blood Bank

    Directory of Open Access Journals (Sweden)

    Maria José Dantas Coêlho

    2011-01-01

    Full Text Available BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%. The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5 and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5. CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

  6. Ultrastructural studies of the gray platelet syndrome.

    Science.gov (United States)

    White, J G

    1979-05-01

    The gray platelet syndrome (GPS) is a rare inherited disorder in which peripheral blood platelets are relatively large, vacuolated, and almost devoid of cytoplasmic granulation. In the present study we have evaluated the ultrastructure and cytochemistry of platelets from 2 patients with the GPS to determine precisely which organelles are missing from their cells. The findings indicate that gray platelets contain normal numbers of mitochondria, dense bodies, peroxisomes, and lysosomes but specifically lack alpha-granules. Preliminary studies of megakaryocytes from 1 of the 2 patients suggest that the defect in granule formation may lie at the level of the Golgi zone.

  7. Identification of platelet refractoriness in oncohematologic patients

    Directory of Open Access Journals (Sweden)

    Aline Aparecida Ferreira

    2011-01-01

    Full Text Available OBJECTIVES: To identify the occurrence and the causes of platelet refractoriness in oncohematologic patients. INTRODUCTION: Platelet refractoriness (unsatisfactory post-transfusion platelet increment is a severe problem that impairs the treatment of oncohematologic patients and is not routinely investigated in most Brazilian services. METHODS: Forty-four episodes of platelet concentrate transfusion were evaluated in 16 patients according to the following parameters: corrected count increment, clinical conditions and detection of anti-platelet antibodies by the platelet immunofluorescence test (PIFT and panel reactive antibodies against human leukocyte antigen class I (PRA-HLA. RESULTS: Of the 16 patients evaluated (median age: 53 years, nine (56% were women, seven of them with a history of pregnancy. An unsatisfactory increment was observed in 43% of the transfusion events, being more frequent in transfusions of random platelet concentrates (54%. Platelet refractoriness was confirmed in three patients (19%, who presented immunologic and non-immunologic causes. Alloantibodies were identified in eight patients (50% by the PIFT and in three (19% by the PRA-HLA. Among alloimmunized patients, nine (64% had a history of transfusion, and three as a result of pregnancy (43%. Of the former, two were refractory (29%. No significant differences were observed, probably as a result of the small sample size. CONCLUSION: The high rate of unsatisfactory platelet increment, refractoriness and alloimmunization observed support the need to set up protocols for the investigation of this complication in all chronically transfused patients, a fundamental requirement for the guarantee of adequate management.

  8. Does bipolar pacemaker current activate blood platelets?

    DEFF Research Database (Denmark)

    Gjesdal, Grunde; Hansen, Annebirthe Bo; Brandes, Axel

    2009-01-01

    OBJECTIVE: The aim of this study was to investigate whether bipolar pacemaker current lead can activate blood platelets. The null hypothesis was that 1 minute of electrical stimulation of platelets would not influence their subsequent reactivity to adenosine diphosphate (ADP). BACKGROUND: Both...... platelets and muscle cells contain actin and myosin filaments, and both cells are activated following calcium influx. Muscle cells open their calcium channels and contract when exposed to an electric current. Current through a bipolar pacemaker lead will expose a small volume of blood, including platelets...

  9. Platelet cytoskeleton and its hemostatic role.

    Science.gov (United States)

    Cerecedo, Doris

    2013-12-01

    Upon vascular injury, platelets adhere to the exposed extracellular matrix, which triggers the platelet activation and aggregation to form a hemostatic plug to seal the wound. All of these events involve dramatic changes in shape because of the cytoskeleton reorganization. The versatility of the cytoskeleton's main elements depends on the biochemical nature of the elements, as well as on the associated proteins that confer multiple functions within the cell. The list of these associated proteins grows actively, increasing our knowledge concerning the complexity of platelet cytoskeleton machinery. The present review evidences the recently described platelet proteins that promote characteristic modifications in their cytoskeleton organization, with special focus on the dystrophin-glycoprotein complex.

  10. 富血小板纤维蛋白提取液对MC3T3-E1细胞影响的实验研究%Effects of platelet-rich fibrin extract on MC3T3-E1 cell

    Institute of Scientific and Technical Information of China (English)

    董凯; 柳忠豪; 张晓洁; 许丰伟

    2013-01-01

    Objective To evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts.Methods The experimental group used the α-minimum essential medium(α-MEM) containing PRFe(10% fetal bovine serum),and the control group used the α-MEM (10% fetal bovine serum).The number of the osteoblasts at 1st,3rd,5th d was detected by methyl thiazolyl tetrazolium(MTT) assay,and the differentiation of osteoblast at lst,3rd,5th,7 th d detected by the activity of alkaline phosphatase(ALP).The alizarin red dye was used to observe the number of calcium nodus at 14th,21st d.The F-actin cytoskeleton was evaluated by confocal laser scanning microscope (CLSM) at 3rd,6th,9th,12th h.The level of osteogenetic biomarkers osteocalcin(OCN) and core-binding factor α1 (Cbfα1) at 3rd,7th d were quantified by real-time PCR.Results A significant increase of absorbance at 1st,3rd,5th d was showed in experimental group (0.336 ± 0.011,0.571 ± 0.039,0.787 ± 0.050) compared to control group (0.300 ± 0.021,0.387 ±0.040,0.527 ±0.034) (P <0.05).The absorbance of experimental group at lst,3rd,5th,7th d(0.146 ± 0.014,0.199 ±0.017,0.390 ±0.020,0.492 ±0.019) was significantly higher than that of control group(0.115 ± 0.014,0.145 ± 0.015,0.190 ± 0.015,0.230 ± 0.026) (P < 0.05).The integrated absorbance of the calcium nodus in experimental group at 14th,21st d (22.119 ± 3.694,31.528 ± 3.162) was significantly higher than in control group(8.498 ±2.041,15.162 ±2.526) (P <0.05).The Cbfα1 and OCN gene expression in experimental group was higher than in control group (P < 0.05).Conclusions PRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin cytoskeleton.%目的 探讨富血小板纤维蛋白提取液(platelet-rich fibrin extract,PRFe)对成骨细胞增殖、分化及细胞骨架的影响,以期为富血小板纤维蛋白在临床的应用提供理论基础.方法

  11. Platelet function alterations in dengue are associated with plasma leakage

    NARCIS (Netherlands)

    Michels, M.; Alisjahbana, B.; Groot, P.G. de; Indrati, A.R.; Fijnheer, R.; Puspita, M.; Dewi, I.M.; Wijer, L. van de; Boer, E.M. de; Roest, M.; Ven, A.J. van der; Mast, Q. de

    2014-01-01

    Severe dengue is characterised by thrombocytopenia, plasma leakage and bleeding. Platelets are important for preservation of endothelial integrity. We hypothesised that platelet activation with secondary platelet dysfunction contribute to plasma leakage. In adult Indonesian patients with acute dengu

  12. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and

  13. Structural basis for the interaction of the adaptor protein grb14 with activated ras.

    Directory of Open Access Journals (Sweden)

    Rohini Qamra

    Full Text Available Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA domain, a pleckstrin-homology (PH domain, a family-specific BPS (between PH and SH2 region, and a C-terminal Src-homology-2 (SH2 domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V. The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.

  14. Platelet-rich plasma preparation using three devices : Implications for platelet activation and platelet growth factor release

    NARCIS (Netherlands)

    Everts, Peter A. M.; Mahoney, Christine Brown; Hoffmann, Johannes J. M. L.; Schonberger, Jacques P. A. M.; Box, Henk A. M.; Van Zundert, Andre; Knape, Johannes T. A.

    2006-01-01

    Background: In this study, three commercial systems for the preparation of platelet-rich plasma (PRP) were compared and platelet growth factors release was measured. Methods: Ten healthy volunteers donated whole blood that was fractionated by a blood cell separator, and a table-top centrifuge to pre

  15. Hereditary sideroblastic anemia with associated platelet abnormalities.

    Science.gov (United States)

    Soslau, G; Brodsky, I

    1989-12-01

    A 62 year old male (R.H.) presented with a mild anemia (Hb 11-12 gm%) and a history of multiple hemorrhagic episodes. The marrow had 40-50% sideroblasts. Marrow chromosomes were normal. His wife was hematologically normal, while one daughter, age 30 years, had a sideroblastic anemia (Hb 11-12 gm%) with 40-50% sideroblasts in the marrow. Her anemia was first noted at age 15 years. Administration of vitamin B6 did not correct the anemia in either the father or daughter. Platelet abnormalities inherited jointly with this disorder are described for the first time. Both R.H. and his daughter had prolonged bleeding times, with normal PTT, PT times, fVIII:C, fVIII:Ag levels, and vWF multimers, which may rule out a von Willebrand's disease. They have normal platelet numbers but abnormally low platelet adhesiveness and greatly depressed ADP, collagen, and epinephrine responsiveness. Response to ristocetin was in the low normal range, and aggregation with thrombin was normal. While desmopressin completely normalized R.H.'s bleeding time, none of these platelet parameters were improved. No differences in the SDS PAGE protein patterns of RH platelets could be detected in comparison to normal samples. His platelets took up and released serotonin (5HT) normally, and electron micrographs defined no morphological abnormalities. However, no ATP was released from platelets activated with collagen, and when followed by thrombin about fourfold greater ATP was released by control platelets as compared to RH platelets. The dense granule fraction derived from RH platelets contained about 20% the level of ATP, 40% the level of ADP, and 50% the level of 5HT detected in a normal sample. The results indicate that the bleeding disorder is related to a non-classical heritable storage pool defect. The connection between the inherited sideroblastic anemia and platelet defects is obscure.

  16. Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

    DEFF Research Database (Denmark)

    Pleines, Irina; Eckly, Anita; Elvers, Margitta;

    2010-01-01

    formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form...... reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling....

  17. Platelet-rich fibrin: Evolution of a second-generation platelet concentrate

    Directory of Open Access Journals (Sweden)

    Sunitha Raja V

    2008-01-01

    Full Text Available Platelet-rich plasma (PRP is a platelet concentrate that has been used widely to accelerate soft-tissue and hard-tissue healing. The preparation of PRP has been described by several authors. Platelet-rich fibrin (PRF was first described by Choukroun et al. in France. It has been referred to as a second-generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Its chief advantages include ease of preparation and lack of biochemical handling of blood, which makes this preparation strictly autologous. This article describes the evolution of this novel platelet concentrate, referred to as PRF.

  18. Human platelets antigens influence the viral load of platelets after the interaction of the platelets with HCV and HIV in vitro

    Directory of Open Access Journals (Sweden)

    Rejane Maria Tommasini Grotto

    Full Text Available Abstract: INTRODUCTION: In this study, we evaluated hepatitis C virus (HCV and human immunodeficiency virus (HIV - platelet interactions in vitro as well as human platelets antigen (HPA polymorphisms. METHODS: Platelets were obtained from 100 healthy HPA-genotyped volunteer donors and incubated with HIV or HCV. The viral load after in vitro exposure was detected. RESULTS: The viral load in the platelets after exposure to the virus was higher in the HIV exposure than in the HCV exposure. CONCLUSIONS: HIV-platelet ligation could be more efficient than HCV-platelet interaction. Further, the HPA-1b allele seems to influence the interaction of platelets with HCV.

  19. Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan.

    Science.gov (United States)

    Liu, Zhi-Jian; Hoffmeister, Karin M; Hu, Zhongbo; Mager, Donald E; Ait-Oudhia, Sihem; Debrincat, Marlyse A; Pleines, Irina; Josefsson, Emma C; Kile, Benjamin T; Italiano, Joseph; Ramsey, Haley; Grozovsky, Renata; Veng-Pedersen, Peter; Chavda, Chaitanya; Sola-Visner, Martha

    2014-05-29

    The fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased sevenfold during the first 2 weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in vivo biotinylation and mathematical modeling showed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived 1 day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins showed that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets. © 2014 by The American Society of Hematology.

  20. [Platelet allo-antibodies identification strategies for preventing and managing platelet refractoriness].

    Science.gov (United States)

    Basire, A; Picard, C

    2014-11-01

    Platelet refractoriness is a serious complication for patients receiving recurrent platelet transfusions, which can be explained by non-immune and immune causes. Human Leukocyte Antigens (HLA) allo-immunization, especially against HLA class I, is the major cause for immune platelet refractoriness. To a lesser extent, allo-antibodies against specific Human Platelet Antigen (HPA) are also involved. Pregnancy, transplantation and previous transfusions can lead to allo-immune reaction against platelet antigens. After transfusion, platelet count is decreased by accelerated platelet destruction related to antibodies fixation on incompatible platelet antigens. New laboratory tests for allo-antibodies identification were developed to improve sensibility and specificity, especially with the LUMINEX(®) technology. The good use and interpretation of these antibodies assays can improve strategies for platelet refractoriness prevention and management with a patient adapted response. Compatible platelets units can be selected according to their identity with recipient typing or immune compatibility regarding HLA or HPA antibodies or HLA epitope compatibility. Prospective studies are needed to further confirm the clinical benefit of new allo-antibodies identification methods and consensus strategies for immune platelet refractoriness management.

  1. Platelet antibodies, activated platelets and serum leptin in childhood immune thrombocytopenic purpura.

    Science.gov (United States)

    Badrawy, Hosny; Elsayh, Khalid I; Zahran, Asmaa M; El-Ghazali, Mohamad Hamdy

    2013-01-01

    The aim of this study was to evaluate the levels of platelet-associated antibodies (PAIgG and PAIgM), activated platelets and serum leptin in children with acute immune thrombocytopenic purpura (ITP). The study included 40 patients with ITP and 40 healthy age- and sex-matched controls. PAIgG, PAIgM and activated platelet levels were estimated by flow cytometry, and serum leptin levels were estimated by ELISA. Activated platelets and serum leptin were significantly higher in the ITP patients than in the controls. The percentage and mean fluorescence intensity of PAIgG and PAIgM staining were significantly higher in the patients than in the controls. Serum leptin and activated platelet levels in patients with thrombocytopenia of brief duration were significantly lower than those in patients with thrombocytopenia of prolonged duration. The levels of activated platelets, serum leptin and PAIgG were positively correlated, and the levels of serum leptin, activated platelets and platelet counts were negatively correlated. The increased levels of activated platelets, serum leptin and platelet-associated antibodies in children with acute ITP suggest that these factors could play a role in ITP pathogenesis. Additionally, activated platelets and serum leptin could have prognostic significance in paediatric acute ITP. Copyright © 2013 S. Karger AG, Basel.

  2. Chloride channels of platelets%血小板氯通道

    Institute of Scientific and Technical Information of China (English)

    陈晓琳; 尹松梅

    2004-01-01

    Chloride channels distribute widely in the body, and participate in many physiological actions and regulatory processes. Based on their physiological roles and molecular structures, six kinds of chloride channels have been identified: (1) The chloride channels family; (2) Cystic fibrosis transmembrane conductance regulator; (3) Swelling-activated chloride channels; (4) Calcium-activated chloride channels; (5) The p64 (CLIC) gene family; (6) γ-aminobutyric acid and glycine receptors. The chloride channels do exist in platelets, and their appearances are dependent on the presence of intracellular calcium. Blocking agents of chloride channels inhibit the thrombin-activated platelet aggregation and the elevation of the intracellular calcium concentration in a dose-dependent manner. It is suggested that chloride channels play a role in the activation of platelets. In addition, chloride channels act on both the cell volume regulation and the intracellular pH regulation in platelets.

  3. Bulk fluid phase behaviour of colloidal platelet-sphere and platelet-polymer mixtures.

    Science.gov (United States)

    de las Heras, Daniel; Schmidt, Matthias

    2013-04-13

    Using a geometry-based fundamental measure density functional theory, we calculate bulk fluid phase diagrams of colloidal mixtures of vanishingly thin hard circular platelets and hard spheres. We find isotropic-nematic phase separation, with strong broadening of the biphasic region, upon increasing the pressure. In mixtures with large size ratio of platelet and sphere diameters, there is also demixing between two nematic phases with differing platelet concentrations. We formulate a fundamental measure density functional for mixtures of colloidal platelets and freely overlapping spheres, which represent ideal polymers, and use it to obtain phase diagrams. We find that, for low platelet-polymer size ratio, in addition to isotropic-nematic and nematic-nematic phase coexistence, platelet-polymer mixtures also display isotropic-isotropic demixing. By contrast, we do not find isotropic-isotropic demixing in hard-core platelet-sphere mixtures for the size ratios considered.

  4. IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb.

    Science.gov (United States)

    Fiorin, F; Steffan, A; Pradella, P; Bizzaro, N; Potenza, R; De Angelis, V

    1998-08-01

    EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.

  5. Clinica use of platelet additive solutions.

    Science.gov (United States)

    van Rhenen, Dick J

    2007-12-01

    Randomised clinical trial (RCT) to study the clinical efficacy and safety of new platelet products using platelet additive solutions are scarce. In this paper a number of recent RCT's is discussed. It can be the start of a development where new transfusion products enter a RCT before the product is applied in clinical practice.

  6. Platelets in liver transplantation : Friend or foe?

    NARCIS (Netherlands)

    Pereboom, Ilona T. A.; Lisman, Ton; Porte, Robert J.

    2008-01-01

    Apart from the well-known role of blood platelets in hemostasis, there is emerging evidence that platelets have various nonhemostatic properties that play a critical role in inflammation, angiogenesis, tissue repair and regeneration, and ischemia/reperfusion (I/R) injury. All these processes may be

  7. Studies on megakaryopoiesis and platelet function

    NARCIS (Netherlands)

    Meinders, M.

    2015-01-01

    Platelets are blood circulating specialized subcellular fragments, which are produced by megakaryocytes. Platelets are essential for hemostasis and wound healing but also play a role in non-hemostatic processes such as the immune response or cancer metastasis. Considering the immediate precursors of

  8. BETA-ADRENOBLOCKERS AND PLATELET AGGREGATION. CARVEDILOL

    Directory of Open Access Journals (Sweden)

    A. N. Zakirova

    2008-01-01

    Full Text Available Approaches evolution to studying of beta-blockers influence on platelet aggregation is reviewed. The current view on of beta-blocker antiplatelet effects is presented on the basis of physical and chemical drug properties (water repellency, dipole moment, molecular mass. Trail results on carvedilol influence on platelet aggregation are focused.

  9. Novel agents for anti-platelet therapy

    Directory of Open Access Journals (Sweden)

    Ji Xuebin

    2011-11-01

    Full Text Available Abstract Anti-platelet therapy plays an important role in the treatment of patients with thrombotic diseases. The most commonly used anti-platelet drugs, namely, aspirin, ticlopidine, and clopidogrel, are effective in the prevention and treatment of cardio-cerebrovascular diseases. Glycoprotein IIb/IIIa antagonists (e.g., abciximab, eptifibatide and tirofiban have demonstrated good clinical benefits and safety profiles in decreasing ischemic events in acute coronary syndrome. However, adverse events related to thrombosis or bleeding have been reported in cases of therapy with glycoprotein IIb/IIIa antagonists. Cilostazol is an anti-platelet agent used in the treatment of patients with peripheral ischemia, such as intermittent claudication. Presently, platelet adenosine diphosphate P2Y(12 receptor antagonists (e.g., clopidogrel, prasugrel, cangrelor, and ticagrelor are being used in clinical settings for their pronounced protective effects. The new protease-activated receptor antagonists, vorapaxar and atopaxar, potentially decrease the risk of ischemic events without significantly increasing the rate of bleeding. Some other new anti-platelet drugs undergoing clinical trials have also been introduced. Indeed, the number of new anti-platelet drugs is increasing. Consequently, the efficacy of these anti-platelet agents in actual patients warrants scrutiny, especially in terms of the hemorrhagic risks. Hopefully, new selective platelet inhibitors with high anti-thrombotic efficiencies and low hemorrhagic side effects can be developed.

  10. Performance evaluation of PL-11 platelet analyzer

    Institute of Scientific and Technical Information of China (English)

    张有涛

    2013-01-01

    Objective To evaluate and report the performance of PL-11 platelet analyzer. Methods Intravenous blood sam-ples anticoagulated with EDTA-K2 and sodium citrate were tested by the PL-11 platelet analyzer to evaluate the intra-assay and interassay coefficient of variation(CV),

  11. Platelet affinity for burro aorta collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.D.

    1977-10-01

    Despite ingenious concepts, there are no unequivocal clues as to what, when, and how some undefined biochemical factor(s) or constituent(s) that localizes in the arterial wall can precipitate a thromboatheromatous lesion or arterial disease. The present study focused on the extraction, partial purification, and characterization of a collagen-active platelet stimulator from the aortas of aged burros. The aggregator moiety in the aorta extracts invariably had a higher affinity for platelets in citrated platelet-rich plasma of human beings than for platelets of homologous burros. The platelet-aggregating factor(s) in the aorta extract was retained by incubation with ..cap alpha..-chymotrypsin. Platelet-aggregating activity was rapidly abolished after incubation with collagenase, as determined by platelet-aggregometry tests. Evidence based on light microscope and polysaccharide histochemical reactions indicates a probability that the intracellular amorphous matrix (PAS-positive) and filamentous components (PTAH-positive) expelled from smooth muscle cells disrupted during homogenization of the aorta may be a principal source of a precursor collagen species which is a potent inducer of platelet aggregation.

  12. The origin and function of platelet glycosyltransferases

    DEFF Research Database (Denmark)

    Wandall, Hans H; Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll;

    2012-01-01

    Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and anti-angiogenic factors throughout the vascular system. Platelets are anucleated cells and lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartme...

  13. Abacavir induces platelet-endothelium interactions by interfering with purinergic signalling: A step from inflammation to thrombosis.

    Science.gov (United States)

    Alvarez, Angeles; Rios-Navarro, Cesar; Blanch-Ruiz, Maria Amparo; Collado-Diaz, Victor; Andujar, Isabel; Martinez-Cuesta, Maria Angeles; Orden, Samuel; Esplugues, Juan V

    2017-03-02

    The controversy connecting Abacavir (ABC) with cardiovascular disease has been fuelled by the lack of a credible mechanism of action. ABC shares structural similarities with endogenous purines, signalling molecules capable of triggering prothrombotic/proinflammatory programmes. Platelets are leading actors in the process of thrombosis. Our study addresses the effects of ABC on interactions between platelets and other vascular cells, while exploring the adhesion molecules implicated and the potential interference with the purinergic signalling pathway. The effects of ABC on platelet aggregation and platelet-endothelium interactions were evaluated, respectively, with an aggregometer and a flow chamber system that reproduced conditions in vivo. The role of adhesion molecules and purinergic receptors in endothelial and platelet populations was assessed by selective pre-incubation with specific antagonists and antibodies. ABC and carbovir triphosphate (CBT) levels were evaluated by HPLC. The results showed that ABC promoted the adherence of platelets to endothelial cells, a crucial step for the formation of thrombi. This was not a consequence of a direct effect of ABC on platelets, but resulted from activation of the endothelium via purinergic ATP-P2X7 receptors, which subsequently triggered an interplay between P-selectin and ICAM-1 on endothelial cells with constitutively expressed GPIIb/IIIa and GPIbα on platelets. ABC did not induce platelet activation (P-selectin expression or Ca(2+) mobilization) or aggregation, even at high concentrations. CBT levels in endothelial cells were lower than those required to induce platelet-endothelium interactions. Thus, ABC interference with endothelial purinergic signalling leads to platelet recruitment. This highlights the endothelium as the main cell target of ABC, which is in line with previous experimental evidence that ABC induces manifestations of vascular inflammation.

  14. Function of eltrombopag-induced platelets compared to platelets from control patients with immune thrombocytopenia.

    Science.gov (United States)

    Haselboeck, Johanna; Kaider, Alexandra; Pabinger, Ingrid; Panzer, Simon

    2013-04-01

    Data on the in vivo function of platelets induced by the thrombopoietin receptor agonist eltrombopag are scarce. To assess a possible influence of eltrombopag we compared platelet function of eltrombopag-treated immune thrombocytopenia (ITP) patients (group 1; n=10) after treatment response to that from control ITP patients (group 2; n=12). We further analysed platelet function at baseline and after one, three, and four weeks of eltrombopag treatment and estimated daily changes of platelet function during the eltrombopag-induced platelet rise. The formation of platelet-monocyte aggregates (PMA), P-selectin expression [MFI], and platelet adhesion under high shear conditions (surface coverage, SC) in vivo and after in vitro addition of agonists (ADP, TRAP-6, Collagen) were similar between both groups after response to eltrombopag treatment. Only TRAP-6 induced a lower SC in the eltrombopag group (p=0.03). All platelet function parameters except for Collagen-induced P-selectin expression changed significantly during treatment with eltrombopag. PMA, naïve and after addition of ADP or TRAP-6 increased with increasing platelet counts. P-selectin expression decreased, when measured without and upon addition of ADP, increased in the presence of TRAP-6, and remained unchanged after addition of Collagen. SC increased during the eltrombopag-induced platelet rise. All significant changes of platelet function correlated to changes in platelet counts. Two patients developed venous thromboses during eltrombopag treatment, but no association with any distinct single platelet function parameter or combinations thereof was identifiable. Thus, eltrombopag-induced platelets function similar to those from control ITP patients without discernible increased hyper-reactivity.

  15. A platelet monoclonal antibody inhibition assay for detection of glycoprotein IIb/IIIa-related platelet alloantibodies.

    Science.gov (United States)

    Reiner, A P; Teramura, G; Nelson, K A; Slichter, S J

    1995-08-18

    Post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAT) result from formation of alloantibodies to platelet membrane glycoprotein-associated antigens. The detection and identification of platelet-specific alloantibodies in patient sera is often complicated by the presence of co-existing HLA antibodies and/or more than one platelet specificity in the same serum. We describe a solid phase assay that specifically detects antibodies to platelet membrane associated alloantigens by measuring the ability of patient antisera to inhibit the binding of glycoprotein GPIIb or GPIIIa monoclonal antibodies to intact platelets. When tested in the GPIIIa assay against a panel of random platelet donors, the reactivities of two known PLAI antisera that also contained different HLA antibodies were highly correlated (r = 0.99) and allowed PLA phenotyping of the population. A standard direct binding platelet ELISA, on the other hand, was unable to accurately PLA phenotype the same population. The reactivities of two known Baka antisera (one containing additional anti-PLA2 and the other anti-Brb specificities) were highly correlated (r = 0.95) in the GPIIb assay, and Bak phenotype determination was similarly accomplished for a random platelet panel. Furthermore, a comparison of platelet phenotype results (using the monoclonal inhibition assay) and genotype results (using DNA analysis) for the PLA and Bak systems showed a concordance of 98% for 146 alleles tested. In conclusion, the platelet monoclonal antibody inhibition assay: (1) allows determination of platelet-specific alloantibodies in the presence of contaminating HLA antibodies and/or in sera containing multiple platelet alloantibodies; (2) allows accurate platelet phenotyping for the GPIIIa-associated PLA and GPIIb-associated Bak antigen systems; and (3) may be applicable to the detection of other known or even novel platelet glycoprotein-associated antigens.

  16. Modulatory effect of coffee on platelet function.

    Science.gov (United States)

    Bhaskar, Shobha; Rauf, Arun A

    2010-01-01

    Blood platelets play a major role in cardiovascular disease (CVD) and thrombosis. Conflicting information exists regarding the effect of coffee consumption on the cardiovascular system. We have investigated whether the consumption of moderate amount of coffee affect platelet functions and primary hemostasis in vivo in normal and high fat diet fed rats. Coffee fed group showed significant (P production from membrane arachidonic acid and it was decreased in coffee treated group. Platelet aggregation studies with ADP, collagen, arachidonic acid and epinephrine showed significant (P coffee fed group. Scanning electron microscopic studies revealed that platelet aggregation tendency increased in HFD group and was reduced in coffee fed group. These results indicate that coffee is active in inhibiting platelet aggregation, a critical step involved in thrombosis.

  17. Platelet mitochondrial function and dysfunction: physiological consequences

    Energy Technology Data Exchange (ETDEWEB)

    Popov, D.

    2015-07-01

    There is a general trend in revisiting mitochondria using the up-to-date technologies that uncovered novel attributes of this organelle, such as the intracellular displacement to locations where an energy supply is needed, the dynamic shape changes and turnover, the initiation of signaling to the rest of the cell, and the ability to crosstalk with other cellular organelles. The in-depth scrutiny of platelet mitochondria role in health and pathology is included within this ongoing revisiting trend. The current article puts into a nutshell the most recent data on platelet mitochondria function and disease-related ion, focusing on generation of stress- and apoptosis-related signaling molecules, overproduction of reactive oxygen species during activation and disease, on the biomarker potential of platelets mitochondria, and their prospective exploitation in translational applications. These novel findings complete the physiological profile of platelets and could have potential therapeutic effectiveness in platelet-associated disorders.

  18. Platelets as immune cells in infectious diseases.

    Science.gov (United States)

    Speth, Cornelia; Löffler, Jürgen; Krappmann, Sven; Lass-Flörl, Cornelia; Rambach, Günter

    2013-11-01

    Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.

  19. Platelet bioreactor-on-a-chip

    Science.gov (United States)

    Mazutis, Linas; Wu, Stephen; Sylman, Joanna L.; Ehrlicher, Allen; Machlus, Kellie R.; Feng, Qiang; Lu, Shijiang; Lanza, Robert; Neeves, Keith B.; Weitz, David A.; Italiano, Joseph E.

    2014-01-01

    Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. PMID:25606631

  20. Platelet-rich plasma in otolaryngology.

    Science.gov (United States)

    Stavrakas, M; Karkos, P D; Markou, K; Grigoriadis, N

    2016-12-01

    Platelet-rich plasma is a novel material that is being used more frequently in many surgical specialties. A literature review on the current and potential uses of platelet-rich plasma in otolaryngology was performed. There is limited evidence on the use of platelet-rich plasma in otolaryngology compared with other specialties: only 11 studies on various subspecialties (otology, rhinology and laryngology) were included in the final review. Based on the limited number of studies, we cannot draw safe conclusions about the value of platelet-rich plasma in otolaryngology. Nevertheless, the available literature suggests that platelet-rich plasma holds promise for future research and may have a number of clinical applications.

  1. Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica*

    Science.gov (United States)

    Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie; Burridge, Keith; Siderovski, David P.

    2011-01-01

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases. PMID:21930699

  2. Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica.

    Science.gov (United States)

    Bosch, Dustin E; Wittchen, Erika S; Qiu, Connie; Burridge, Keith; Siderovski, David P

    2011-11-11

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

  3. Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie; Burridge, Keith; Siderovski, David P. (UNC)

    2012-08-10

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

  4. Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.

    Science.gov (United States)

    Phang, Juanita M; Harrop, Stephen J; Duff, Anthony P; Sokolova, Anna V; Crossett, Ben; Walsh, James C; Beckham, Simone A; Nguyen, Cuong D; Davies, Roberta B; Glöckner, Carina; Bromley, Elizabeth H C; Wilk, Krystyna E; Curmi, Paul M G

    2016-09-15

    Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

  5. Depressed patients have decreased binding of tritiated imipramine to platelet serotonin ''transporter''

    Energy Technology Data Exchange (ETDEWEB)

    Paul, S.M.; Rehavi, M.; Skolnick, P.; Ballenger, J.C.; Goodwin, F.K.

    1981-12-01

    The high-affinity tritiated (3H) imipramine binding sites are functionally (and perhaps structurally) associated with the presynaptic neuronal and platelet uptake sites for serotonin. Since there is an excellent correlation between the relative potencies of a series of antidepressants in displacing 3H-imipramine from binding sites in human brain and platelet, we have examined the binding of 3H-imipramine to platelets from 14 depressed patients and 28 age- and sex-matched controls. A highly significant decrease in the number of 3H-imipramine binding sites, with no significant change in the apparent affinity constants, was observed in platelets from the depressed patients compared with the controls. These results, coupled with previous studies showing a significant decrease in the maximal uptake of serotonin in platelets from depressed patients, suggest that an inherited or acquired deficiency of the serotonin transport protein or proteins may be involved in the pathogenesis of depression.

  6. Decreased TGF-β1 and VEGF Release in Cystic Fibrosis Platelets: Further Evidence for Platelet Defects in Cystic Fibrosis

    Science.gov (United States)

    Maloney, James P.; Narasimhan, Jayashree; Biller, Julie

    2016-01-01

    Purpose Cystic fibrosis (CF) patients suffer from chronic lung inflammation. This inflammation may activate platelets. There are limited data on the role of platelet-secreted cytokines in CF. Platelet cytokines with inflammatory effects include vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1). As levels of these cytokines are tenfold greater in serum than plasma due to platelet release, serum levels may be one index of platelet content; but a more specific index is release during the aggregation of isolated platelets. We postulated that altered release of these platelet cytokines occurs in CF. Methods We obtained sera and plasma from CF outpatients (n=21) and from healthy controls (n=20), measured VEGF and TGF-β1, assessed for correlations with platelet number, analyzed cytokine release during platelet aggregation to collagen, and analyzed differences in maximal platelet aggregation. Results Platelet number and maximal aggregation levels were higher in CF. Plasma and serum levels of TGF-β1 and VEGF were higher in CF, but these levels were similar after adjusting for platelet number (serum cytokines correlated with platelet count). The release of VEGF and TGF-β1 during aggregation was decreased in CF platelets (by 52% and 29%, respectively). Conclusion Platelet release is not a source of the elevated blood proinflammatory cytokines TGF-β1 and VEGF in CF, as platelets from CF patients actually release less of these cytokines. These data provide further evidence for platelet defects in CF. PMID:27423781

  7. Spatial and temporal dynamics of receptor for advanced glycation endproducts, integrins, and actin cytoskeleton as probed with fluorescence-based imaging techniques

    Energy Technology Data Exchange (ETDEWEB)

    Syed, Aleem [Iowa State Univ., Ames, IA (United States)

    2016-01-01

    Systematic spatial and temporal fluctuations are a fundamental part of any biological process. For example, lateral diffusion of membrane proteins is one of the key mechanisms in their cellular function. Lateral diffusion governs how membrane proteins interact with intracellular, transmembrane, and extracellular components to achieve their function. Herein, fluorescence-based techniques are used to elucidate the dynamics of receptor for advanced glycation end-products (RAGE) and integrin membrane proteins. RAGE is a transmembrane protein that is being used as a biomarker for various diseases. RAGE dependent signaling in numerous pathological conditions is well studied. However, RAGE lateral diffusion in the cell membrane is poorly understood. For this purpose, effect of cholesterol, cytoskeleton dynamics, and presence of ligand on RAGE lateral diffusion is investigated.

  8. Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge.

    Science.gov (United States)

    Biersmith, Bridget; Wang, Zong-Heng; Geisbrecht, Erika R

    2015-06-01

    The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to engulfment and cell motility (ELMO) proteins, Dock-ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes, including cell motility, apoptotic cell clearance, and axon guidance. Overlapping expression patterns and functional redundancy among the 11 vertebrate Dock family members, which are subdivided into four families (Dock A, B, C, and D), complicate genetic analysis. In both vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism for understanding Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock A) and Sponge (Spg; Dock B). Here we show that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel. Moreover, we confirm the in vivo role of Mbc upstream of Rac and provide evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. Our findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis.

  9. The AGC Ser/Thr kinase Aga1 is essential for appressorium formation and maintenance of the actin cytoskeleton in the smut fungus Ustilago maydis.

    Science.gov (United States)

    Berndt, Patrick; Lanver, Daniel; Kahmann, Regine

    2010-12-01

    On the plant surface the dimorphic fungus Ustilago maydis switches from budding to hyphal growth and differentiates appressoria. To get more insight into these highly regulated processes we report on the role of a conserved Ser/Thr kinase of the AGC kinase family, Aga1. U. maydis Aga1 could functionally replace Ypk1p in Saccharomyces cerevisiae. aga1 deletion mutants were affected in growth, cell wall integrity, mating as well as the ability to form appressoria and showed defects in actin organization and actin-dependent endocytosis. With respect to appressorium formation and endocytosis, the aga1 deletion phenotype could be mimicked by inhibiting the formation of actin filaments with Latrunculin A. These data suggest a critical role of Aga1 in F-actin organization during the morphological changes accompanying the development of appressoria.

  10. Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton

    Science.gov (United States)

    Cameron, Paul U.; Saleh, Suha; Sallmann, Georgina; Solomon, Ajantha; Wightman, Fiona; Evans, Vanessa A.; Boucher, Genevieve; Haddad, Elias K.; Sekaly, Rafick-Pierre; Harman, Andrew N.; Anderson, Jenny L.; Jones, Kate L.; Mak, Johnson; Cunningham, Anthony L.; Jaworowski, Anthony; Lewin, Sharon R.

    2010-01-01

    Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4+ T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4+ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4+ T cells during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue. PMID:20837531

  11. Omega-3 fatty acids modulate Weibel-Palade body degranulation and actin cytoskeleton rearrangement in PMA-stimulated human umbilical vein endothelial cells.

    Science.gov (United States)

    Bürgin-Maunder, Corinna S; Brooks, Peter R; Russell, Fraser D

    2013-11-08

    Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  12. Omega-3 Fatty Acids Modulate Weibel-Palade Body Degranulation and Actin Cytoskeleton Rearrangement in PMA-Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Corinna S. Bürgin-Maunder

    2013-11-01

    Full Text Available Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF in cytoplasmic Weibel-Palade bodies (WPBs. We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs. HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA, and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  13. Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles

    Directory of Open Access Journals (Sweden)

    Katie L Lannan

    2015-02-01

    Full Text Available Platelets are small anucleate blood cells derived from megakaryocytes. In addition to their pivotal roles in hemostasis, platelets are the smallest, yet most abundant, immune cell and regulate inflammation, immunity, and disease progression. Although platelets lack DNA, and thus no functional transcriptional activities, they are nonetheless rich sources of RNAs, possess an intact spliceosome, and are thus capable of synthesizing proteins. Previously, it was thought that platelet RNAs and translational machinery were remnants from the megakaryocyte. We now know that the initial description of platelets as cellular fragments is an antiquated notion, as mounting evidence suggests otherwise. Therefore, it is reasonable to hypothesize that platelet transcription factors are not vestigial remnants from megakaryoctes, but have important, if only partly understood functions. Proteins play multiple cellular roles to minimize energy expenditure for maximum cellular function; thus, the same can be expected for transcription factors. In fact, numerous transcription factors have non-genomic roles, both in platelets and in nucleated cells. Our lab and others have discovered the presence and nongenomic roles of transcription factors in platelets, such as the nuclear factor kappa β (NFκB family of proteins and peroxisome proliferator activated receptor gamma (PPARγ. In addition to numerous roles in regulating platelet activation, functional transcription factors can be transferred to vascular and immune cells through platelet microparticles. This method of transcellular delivery of key immune molecules may be a vital mechanism by which platelet transcription factors regulate inflammation and immunity. At the very least, platelets are an ideal model cell to dissect out the nongenomic roles of transcription factors in nucleated cells. There is abundant evidence to suggest that transcription factors in platelets play key roles in regulating inflammatory and

  14. Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Sandra Mjoll Jonsdottir-Buch

    Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

  15. T lymphocyte migration: an action movie starring the actin and associated actors

    Directory of Open Access Journals (Sweden)

    Loïc eDupré

    2015-11-01

    Full Text Available The actin cytoskeleton is composed of a dynamic filament meshwork that builds the architecture of the cell to sustain its fundamental properties. This physical structure is characterized by a continuous remodeling, which allows cells to accomplish complex motility steps such as directed migration, crossing of biological barriers and interaction with other cells. T lymphocytes excel in these motility steps to ensure their immune surveillance duties. In particular, actin cytoskeleton remodeling is key to facilitate the journey of T lymphocytes through distinct tissue environments and to tune their stop and go behavior during the scanning of antigen-presenting cells. The molecular mechanisms controlling actin cytoskeleton remodeling during T lymphocyte motility have been only partially unraveled, since the function of many actin regulators has not yet been assessed in these cells. Our review aims to integrate the current knowledge into a comprehensive picture of how the actin cytoskeleton drives T lymphocyte migration. We will present the molecular actors that control actin cytoskeleton remodeling, as well as their role in the different T lymphocyte motile steps. We will also highlight which challenges remain to be addressed experimentally and which approaches appear promising to tackle them.

  16. Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis.

    Science.gov (United States)

    Koupenova, Milka; Vitseva, Olga; MacKay, Christopher R; Beaulieu, Lea M; Benjamin, Emelia J; Mick, Eric; Kurt-Jones, Evelyn A; Ravid, Katya; Freedman, Jane E

    2014-07-31

    Viral infections have been associated with reduced platelet counts, the biological significance of which has remained elusive. Here, we show that infection with encephalomyocarditis virus (EMCV) rapidly reduces platelet count, and this response is attributed to platelet Toll-like receptor 7 (TLR7). Platelet-TLR7 stimulation mediates formation of large platelet-neutrophil aggregates, both in mouse and human blood. Intriguingly, this process results in internalization of platelet CD41-fragments by neutrophils, as assessed biochemically and visualized by microscopy, with no influence on platelet prothrombotic properties. The mechanism includes TLR7-mediated platelet granule release, translocation of P-selectin to the cell surface, and a consequent increase in platelet-neutrophil adhesion. Viral infection of platelet-depleted mice also led to increased mortality. Transfusion of wild-type, TLR7-expressing platelets into TLR7-deficient mice caused a drop in platelet count and increased survival post EMCV infection. Thus, this study identifies a new link between platelets and their response to single-stranded RNA viruses that involves activation of TLR7. Finally, platelet-TLR7 stimulation is independent of thrombosis and has implications to the host immune response and survival.

  17. Brief Report: Platelet-Poor Plasma Serotonin in Autism

    Science.gov (United States)

    Anderson, George M.; Hertzig, Margaret E.; McBride, P. A.

    2012-01-01

    Possible explanations for the well-replicated platelet hyperserotonemia of autism include an alteration in the platelet's handling of serotonin (5-hydroxyserotonin, 5-HT) or an increased exposure of the platelet to 5-HT. Measurement of platelet-poor plasma (PPP) levels of 5-HT appears to provide the best available index of in vivo exposure of the…

  18. 21 CFR 864.5700 - Automated platelet aggregation system.

    Science.gov (United States)

    2010-04-01

    ... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet...

  19. Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

    Directory of Open Access Journals (Sweden)

    Singh Ravindra

    2009-01-01

    Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

  20. Platelet thrombosis in cardiac-valve prostheses

    Energy Technology Data Exchange (ETDEWEB)

    Dewanjee, M.K.

    1989-01-01

    The contribution of platelets and clotting factors in thrombosis on cardiovascular prostheses had been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and patients with indium-111, Technetium-99m labeled platelets, iodine-123, iodine-131 labeled fibrinogen, and In-111 and Tc-99m labeled antibody to the fibrinogen-receptor on the platelet- membrane, or fibrin. The early studies demonstrated that certain platelet-inhibitors, e.g. sulfinpyrazone, aspirin or aspirin- persantine increased platelet survival time with mechanical valves implanted in the baboon model and patients. Thrombus localization by imaging is possible for large thrombus on thrombogenic surface of prosthesis in the acute phase. The majority of thrombus was found in the sewing ring (Dacron) in the acute phase in both the mechanical and tissue valves. The amount of retained thrombus in both mechanical and tissue valves in our one-day study in the dog model was similar (< 1% if injected In-111 platelets = 5 billion platelets). As the fibrous ingrowth covered the sewing ring, the thrombus formation decreased significantly. Only a small amount of thrombus was found on the leaflets at one month in both the dog and calf models. 38 refs., 9 figs., 5 tabs.

  1. Changes in platelet parameters in leukocytosis.

    Science.gov (United States)

    Ozturk, Nurinnisa; Baygutalp, Nurcan Kilic; Bakan, Ebubekir; Altas, Gulsum Feyza; Polat, Harun; Dorman, Emrullah

    2016-01-01

    In recent years, platelets are known to have a large variety of functions in many pathophysiological processes and their interaction with endothelial cells and leukocytes is known to play an important role in the pathophysiology of vascular inflammation. The aim of this study was to investigate the relationship between white blood cell count in conditions resulting in leukocytosis and platelet count and platelet parameters including mean platelet volume, platelet distribution width, and plateletcrit. White blood cell counts count and all platelet parameters were evaluated in 341 results of normal complete blood count (of which the white blood cell counts were within reference range, group 1) and 327 results of elevated white blood cell counts count (group 2). There was a significant difference between these two groups in PLT counts and PCT values, being higher in Group 2. However, there was no statistically significant difference between two groups in MPV and PDW values. On the other hand, there were statistically significant, but weak, correlations between the WBC and platelet counts in both groups (p<0.01, r=0.235 for group 1, p<0.05, r=0.116 for group 2). As a conclusion PLT count and PCT values increase in infectious conditions. This study and previous studies show that PLTs are employed in infectious conditions but the exact mechanism and the exact clinical importance of this response remains to be cleared by further studies.

  2. Human thromboxane A2 receptor genetic variants: in silico, in vitro and "in platelet" analysis.

    Directory of Open Access Journals (Sweden)

    Scott Gleim

    Full Text Available Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity or promote (hyperactivity vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C(68S, V(80E, E(94V, A(160T, V(176E, and V(217I for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C(68S to normal (V(217I, with most variants demonstrating functional alteration in binding, expression or activation (V(80E, E(94V, A(160T, and V(176E. In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and "in platelet" evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.

  3. Platelet labelling with indium-hydroxypyridinone and indium-hydroxypyranone complexes

    Energy Technology Data Exchange (ETDEWEB)

    Abeysinghe, R.D. (Dept. of Clinical Haematology, University Coll. Medical School, London (United Kingdom)); Ellis, B.L. (Dept. of Pharmacy, Kings Coll., London (United Kingdom)); Hider, R.C. (Dept. of Pharmacy, Kings Coll., London (United Kingdom)); Porter, J.B. (Dept. of Clinical Haematology, University Coll. Medical School, London (United Kingdom))

    1994-10-01

    In order to identify new compounds which label platelets without affecting their function, three classes of metal chelating agents have been compared with oxine for their efficiency of indium-113m platelet labelling and for their short- and long-term effects on platelet function. The 3-hydroxypyridinones (both 2-ones and 4-ones) and 3-hydroxypyranones are bidentate chelators of trivalent metal ions that are neutrally charged in the metal-complexed form and hence gain access to cells readily. The hydroxypyranone ethylmaltol has been compared with the 3-hydroxypyridin-4-one CP94 and to its structurally related lipophilic analogue CP25 as well as with the 3-hydroxypyridin-2-one, CP02. The platelet labelling efficiencies with these ligands were between 75% and 95% of that obtained with oxine, following a 12-min incubation in saline. The optimal concentration for the hydroxypyridin-2-ones and hydroxypyridin-4-ones was approximately 10 [mu]M compared with 100 [mu]M for the hydroxypyranone ethylmaltol and 60 [mu]M for oxine. Oxine and tropolone were found to produce significant inhibition of platelet aggregation to collagen in short-term experiments (10 min) or in longer term (18 and 42 h) ex vivo platelet cultures respectively. By contrast, ethylmaltol had no such inhibitory effects at either time interval. The relatively hydrophilic hydroxypyridin-4-one CP94 showed no inhibitory effects on collagen-induced aggregation in short-term studies, unlike the more lipid-soluble derivative CP25. These results suggest that ethylmaltol and related pyranones may have advantages over oxine and tropolone as indium platelet labelling agents where it is important not to damage platelets by the labelling process itself. (orig.)

  4. Analysis of aggregation of platelets in thrombosis

    Science.gov (United States)

    Ahuja, Suresh

    Platelets are key players in thrombus formation by first rolling over collagen bound von Willebrand factor followed by formation of a stable interaction with collagen. The first adhered platelets bind additional platelets until the whole injury is sealed off by a platelet aggregate. The coagulation system stabilizes the formed platelet plug by creating a tight fibrin network, and then wound contraction takes place because of morphological changes in platelets. Coagulation takes place by platelet activation and aggregation mainly through fibrinogen polymerization into fibrin fibers. The process includes multiple factors, such as thrombin, plasmin, and local shear-rate which regulate and control the process. Coagulation can be divided into two pathways: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated by the exposure of a negatively charged. It is able to activate factor XII, using a complex reaction that includes prekallikrein and high-molecular-weight kininogen as cofactors.. Thrombin is the final enzyme that is needed to convert fibrinogen into fibrin. The extrinsic pathway starts with the exposure of tissue factor to the circulating blood, which is the major initiator of coagulation. There are several feedback loops that reinforce the coagulation cascade, resulting in large amounts of thrombin. It is dependent on the presence of pro-coagulant surfaces of cells expressing negatively charged phospholipids--which include phosphatidylserine (PS)--on their outer membrane. PS-bearing surfaces are able to increase the efficiency of the reactions by concentrating and co-localizing coagulation factors.. Aggregation of platelets are analyzed and compared to adhesion of platelet to erythrocyte and to endothelial cells. This abstract is replacing MAR16-2015-020003.

  5. Evaluation of platelet function using multiple electrode platelet aggregometry in dogs with septic peritonitis.

    Science.gov (United States)

    Li, Ronald H L; Chan, Daniel L

    2016-09-01

    To assess platelet function via multiple electrode platelet aggregometry (MEPA) in dogs with septic peritonitis and in healthy dogs. The secondary aim was to determine if there is prognostic significance to changes in platelet function observed in septic dogs. Prospective, observational cohort study conducted from January 2012 to March 2014. University teaching hospital. Twenty dogs with septic peritonitis and 23 healthy dogs. None. MEPA using arachidonic acid, adenosine diphosphate, and collagen (COL) as agonists was measured within 24 hours of diagnosis of sepsis. Compared to healthy dogs, platelet aggregation was reduced in dogs with septic peritonitis for all agonists (P peritonitis. Circulating platelets from dogs with septic peritonitis have diminished aggregation in response to multiple platelet agonists. MEPA may serve as an assessment tool for illness severity in this patient population. © Veterinary Emergency and Critical Care Society 2016.

  6. [Glycoproteins, inherited diseases of platelets, and the role of platelets in wound healing].

    Science.gov (United States)

    Nurden, Alan T; Nurden, Paquita

    2013-02-01

    Recognition that platelets have a glycocalyx rich in membrane glycoproteins prompted the discovery in France that inherited bleeding syndromes due to defects of platelet adhesion and aggregation were caused by deficiencies in major receptors at the platelet surface. Identification of the alpha IIb beta3 integrin prompted the development of powerful anti-thrombotic drugs that have gained worldwide use. Since these discoveries, the genetic causes of many other defects of platelet function and production have been elucidated, with the identification of an ADP receptor, P2 Y12, another widespread target for anti-thrombotic drugs. Discovery of the molecular basis of a rare disease of storage of biologically active proteins in platelet alpha-granules has been accompanied by the recognition of the roles of platelets in inflammation, the innate immune system and tissue repair, opening new avenues for therapeutic advances.

  7. Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

    Energy Technology Data Exchange (ETDEWEB)

    Carter, M.G.; Shukla, S.D. (Univ. of Missouri School of Medicine, Columbia (USA))

    1987-05-01

    Human platelet concentrate from the American Red Cross Blood Center was stored at 24{degree}C in a shaker and aliquots were taken out at time intervals aseptically. Platelet activating factor (PAF) stimulated turnover of phosphoinositide (PPI) was monitored by assaying {sup 32}P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 {times} 10{sup {minus}7} M PAF at 37{degree}C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for {sup 32}P-phosphoinositides. The percent stimulation of {sup 32}P incorporation by PAF (over control) into PPI was approximately 250, 100, 60, 25 and 20 on days 1, 2, 3, 5 and 6, respectively. This indicated a dramatic decrease in PAF responsive turnover of platelet PPI during storage. These findings have important implications in relation to PAF receptor activity and viability of platelets at different periods of storage.

  8. Platelets: at the nexus of antimicrobial defence.

    Science.gov (United States)

    Yeaman, Michael R

    2014-06-01

    Platelets have traditionally been viewed as fragmentary mediators of coagulation. However, recent molecular and cellular evidence suggests that they have multiple roles in host defence against infection. From first-responders that detect pathogens and rapidly deploy host-defence peptides, to beacons that recruit and enhance leukocyte functions in the context of infection, to liaisons that facilitate the T cell-B cell crosstalk that is required in adaptive immunity, platelets represent a nexus at the intersection of haemostasis and antimicrobial host defence. In this Review, I consider recent insights into the antimicrobial roles of platelets, which are mediated both directly and indirectly to integrate innate and adaptive immune responses to pathogens.

  9. Transcellular lipoxygenase metabolism between monocytes and platelets

    Energy Technology Data Exchange (ETDEWEB)

    Bigby, T.D.; Meslier, N. (Univ. of California, San Francisco (USA))

    1989-09-15

    We have examined the effects of co-culture and in vitro co-stimulation on lipoxygenase metabolism in monocytes and platelets. Monocytes were obtained from the peripheral blood of normal volunteers by discontinuous gradient centrifugation and adherence to tissue culture plastic. Platelets were obtained from the platelet-rich plasma of the same donor. When 10(9) platelets and 2.5 x 10(6) monocytes were co-stimulated with 1 microM A23187, these preparations released greater quantities of 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid, 5(S),12-(S)dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, and leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic (LTC4) when compared with monocytes alone. Release of arachidonic acid, 5-HETE, delta 6-trans-LTB4, and delta 6-trans-12-epi-LTB4 from monocytes was decreased in the presence of platelets. A dose-response curve was constructed and revealed that the above changes became evident when the platelet number exceeded 10(7). Dual radiolabeling experiments with 3H- and 14C-arachidonic acid revealed that monocytes provided arachidonic acid, 5-HETE, and LTA4 for further metabolism by the platelet. Monocytes did not metabolize platelet intermediates detectably. In addition, as much as 1.2 microM 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid and 12(S)-hydroperoxy-10-trans-5,8,14-cis-eicosatetraenoic acid had no effect on monocyte lipoxygenase metabolism. Platelets were capable of converting LTA4 to LTC4, but conversion of LTA4 to LTB4 was not detected. We conclude that the monocyte and platelet lipoxygenase pathways undergo a transcellular lipoxygenase interaction that differs from the interaction of the neutrophil and platelet lipoxygenase pathways. In this interaction monocytes provide intermediate substrates for further metabolic conversion by platelets in an unidirectional manner.

  10. Influence of 26-hydroxycholesterol on the composition and function of gel-filtered platelets

    Energy Technology Data Exchange (ETDEWEB)

    Kou, I.L.; Pikul, J.; Kummerow, F.A. (Univ. of Southern California, Los Angeles (USA))

    1991-04-01

    The influence of 26-hydroxycholesterol (26-OH-CHOL) on the structure and function of gel-filtered rat platelets, as a model membrane, was studied in vitro. Its influence on structure was determined by a fatty acid and a phospholipid analysis of the platelet lipids and on function by the cytoplasmic calcium concentration of the platelets exposed to increasing concentrations of 26-OH-CHOL for various periods of time. The intracellular free calcium (Ca{sup 2}{sup +})i of the gel-filtered rat platelets was monitored by a fluorescent probe (quin 2) after incubation in a 37{degree}C water bath with 1 mM Ca{sup 2}{sup +} and 20 microM quin 2/AM. The presence of 26-OH-CHOL in the incubation media changed both the phospholipid composition and the mixed fatty acid composition in the membrane and increased the intracellular free Ca{sup 2}{sup +} level of the platelets. As the incubation of platelets with cholesterol (CHOL) or esterified 26-OH-CHOL did not increase intracellular Ca{sup 2}{sup +} levels, these results indicate that the hydrophilic free 26-hydroxy group in 26-OH-CHOL may have influenced the enzymes catalyzing the synthesis of the phospholipid in the platelet membrane so as to allow it to become more and more 'leaky' to Ca{sup 2}{sup +}. Such a fundamental change in membrane structure and function may be responsible for the development of atherosclerosis in the intimal layer of the coronary arteries.

  11. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

    Science.gov (United States)

    Żmigrodzka, M; Guzera, M; Winnicka, A

    2016-01-01

    Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

  12. Pathogen-Reduced, Extended Platelet Storage in Platelet Additive Solution (PAS)

    Science.gov (United States)

    2016-10-01

    concentrations will be performed to ensure the desired concentration was achieved. Platelet Additive Solutions are isotonic solutions used to replace a...Sherrill J. Slichter, MD CONTRACTING ORGANIZATION: Bloodworks Northwest Seattle, WA 98104 REPORT DATE: October 2016 TYPE OF REPORT: Annual...TITLE AND SUBTITLE Pathogen-Reduced, Extended Platelet Storage in Platelet Additive Solution (PAS) 5a. CONTRACT NUMBER W81XWH-12-1-0441 5b. GRANT

  13. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets

    OpenAIRE

    Ye-Ming Lee; Kuo-Hsien Hsieh; Wan-Jung Lu; Hsiu-Chu Chou; Duen-Suey Chou; Li-Ming Lien; Joen-Rong Sheu; Kuan-Hung Lin

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation...

  14. Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

    OpenAIRE

    Sandra Mjoll Jonsdottir-Buch; Ramona Lieder; Olafur Eysteinn Sigurjonsson

    2013-01-01

    BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose ...

  15. Revascularization of Immature Necrotic Teeth: Platelet rich Fibrin an Edge over Platelet rich Plasma

    OpenAIRE

    Neelam Mittal; Isha Narang

    2012-01-01

    Introduction: Revascularization is one such entity that has found its clinical application in the field of endodontics for the manage-ment of immature permanent necrotic teeth. The protocols for revascularization of such teeth focus especially on delivery of stem cells and scaffolds in a nonsurgical manner rather than concentrated growth micro molecules.The hypothesis: This article proposes the role of platelet concentrates such as platelet rich fibrin (PRF) and platelet rich plasma (PRP) in ...

  16. Platelet-rich fibrin matrix for facial plastic surgery.

    Science.gov (United States)

    Sclafani, Anthony P; Saman, Masoud

    2012-05-01

    Platelets are known primarily for their role in hemostasis, but there is increasing interest in the effect of platelets on wound healing. Platelet isolates such as platelet-rich plasma have been advocated to enhance and accelerate wound healing. This article describes the use of a novel preparation, platelet-rich fibrin matrix (PRFM), for facial plastic surgery applications such as volume augmentation, fat transfer supplementation, and as an adjunct to open surgical procedures.

  17. Platelet interaction with bacterial toxins and secreted products.

    Science.gov (United States)

    Shannon, Oonagh

    2015-01-01

    Bacteria that enter the bloodstream will encounter components of the cellular and soluble immune response. Platelets contribute to this response and have emerged as an important target for bacterial pathogens. Bacteria produce diverse extracellular proteins and toxins that have been reported to modulate platelet function. These interactions can result in complete or incomplete platelet activation or inhibition of platelet activation, depending on the bacteria and bacterial product. The nature of the platelet response may be highly relevant to disease pathogenesis.

  18. Evaluation of platelet thromboxane radioimmunoassay method to measure platelet life-span: Comparison with /sup 111/indium-platelet method

    Energy Technology Data Exchange (ETDEWEB)

    Vallabhajosula, S.; Machac, J.; Badimon, L.; Lipszyc, H.; Goldsmith, S.J.; Fuster, V.

    1985-05-01

    The platelet activation during radiolabeling in vitro with Cr-51 and In-111 may affect the platelet life-span (PLS) in vivo. A new RIA method to measure PLS is being evaluated. Aspirin inhibits platelet thromboxane (TxA/sub 2/) by acetylating cyclooxygenase. The time required for the TxA/sub 2/ levels to return towards control values depends on the rate of new platelets entering circulation and is a measure of PLS. A single dose of aspirin (150mg) was given to 5 normal human subjects. Blood samples were collected for 2 days before aspirin and daily for 10 days. TxA/sub 2/ production in response to endogenous thrombin was studied by allowing 1 ml blood sample to clot at 37/sup 0/C for 90 min. Serum TxB/sub 2/ (stable breakdown product of Tx-A/sub 2/) levels determined by RIA technique. The plot of TxB/sub 2/ levels (% control) against time showed a gradual increase. The PLS calculated by linear regression analysis assuming a 2-day lag period before cyclooxygenase recovery is 9.7 +- 2.37. In the same 5 subjects, platelets from a 50ml blood sample were labeled with /sup 111/In-tropolone in 2 ml autologous plasma. Starting at 1 hr after injection of labeled platelets, 10 blood samples were obtained over a 8 day period. The PLS calculated based on a linear regression analysis is 10.2 +. 1.4. The PLS measured from the rate of platelet disappearance from circulation and the rate of platelet regeneration into circulation are quite comparable in normal subjects. TxA/sub 2/ regeneration RIA may provide a method to measure PLS without administering radioactivity to patient.

  19. Single-step separation of platelets from whole blood coupled with digital quantification by interfacial platelet cytometry (iPC).

    Science.gov (United States)

    Basabe-Desmonts, L; Ramstrom, S; Meade, G; O'Neill, S; Riaz, A; Lee, L P; Ricco, A J; Kenny, D

    2010-09-21

    We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip. iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates. By separating platelets from whole blood using specific binding to protein spots of a defined size, iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (1) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet-platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopulations from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated. To date, we have demonstrated 1-4 of the above list. Platelets were separated from whole blood using iPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 μm). The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state. Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets. For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (

  20. Thrombopoietin induces p-selectin expression on platelets and subsequent platelet/leukocyte interactions.

    Science.gov (United States)

    Tibbles, Heather E; Navara, Christopher S; Hupke, Michael A; Vassilev, Alexei O; Uckun, Fatih M

    2002-04-12

    Ligation of thrombopoietin (TPO) to the platelet c-Mpl receptor induces numerous biochemical pathways in the absence of aggregation. Two forms of recombinant TPO are currently in clinical trials for the treatment of thrombocytopenia. This study focuses on the effects of the full-length recombinant human TPO (rhTPO) on platelets in a whole blood system. Platelet-leukocyte associations (PLAs) were visualized following rhTPO stimulation as CD42b/CD 45 double positive clusters by FACS analysis. Treatment of washed platelets with rhTPO induced granule release and expression of the leukocyte adhesion receptor P-selectin (CD 62P) in the absence of aggregation and calcium mobilization. RhTPO also induced platelet-leukocyte interactions in whole blood. Following stimulation, leukocytes were recruited by platelets through P-selectin in a calcium-dependent manner. rhTPO stimulates platelet-leukocyte associations in whole blood through expression of platelet P-selectin. To our knowledge, this is the first report that identifies TPO as a promoter of platelet-leukocyte interactions.

  1. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    Science.gov (United States)

    Beck, Zoltan; Jagodzinski, Linda L; Eller, Michael A; Thelian, Doris; Matyas, Gary R; Kunz, Anjali N; Alving, Carl R

    2013-01-01

    Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC) either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC), platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture) from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a) platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b) platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV-1; (c) DC

  2. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    Directory of Open Access Journals (Sweden)

    Zoltan Beck

    Full Text Available Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC, platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV

  3. The expression levels of platelet adhesive receptors in PRP derived platelet concentrates during storage

    Directory of Open Access Journals (Sweden)

    Fatemeh Nassaji

    2016-04-01

    Full Text Available Background: Major platelet adhesive receptors that contribute significantly to thrombus formation include platelet receptor glycoprotein Ibα (GPIbα of the GPIb-IX-V complex and platelet glycoprotein VI (GPVI. GPIbα plays a crucial role in platelet tethering to sub-endothelial matrix, which initiates thrombus formation at arterial shear rates, whereas GPVI is critically involved in platelets firm adhesion to the site of injury regardless of shear condition. During storage, platelets experience some changes that deleteriously affect the expression levels of platelet receptors, which in turn can alter platelet functional behaviors. Considering the important roles of GPIbα and GPVI in platelet adhesion, it seems that any dramatic changes in the expression levels of these receptors can influence adhesive function of transfused platelets. Thereby examining GPIbα and GPVI expression during the storage of platelet concentrates may provide some useful information about the functional quality of these products after transfusion. Methods: In our experimental study, 5 PRP-platelet concentrates were randomly obtained from Iranian Blood Transfusion Organization (IBTO. All the platelet products met the standard quality assessment based on AABB (American Association of Blood Banks guidelines. Washed platelets were subjected to flowcytometry analysis for the evaluation of GPIbα and GPVI receptor expression in day 1, 3 and 5 after storage. Data were presented as mean fluorescence intensity (MFI and analyzed by Kruskal-Wallis test with Dunn’s multiple comparison test. Results: The GPIbα expression on first day (MFI=86±5.9 was reduced three days after storage (MFI= 69±6.9. The expression levels continued to reduce until day 5 in which GPIbα expression was markedly decreased to (MFI= 61±7.7 (P= 0.0094. GPVI expression on the days 1, 3 and 5 after storage were 20.6±3.3, 24±2.5 and 14±4.9, respectively. The results showed a significant decrease of

  4. Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets.

    Science.gov (United States)

    Sim, Xiuli; Poncz, Mortimer; Gadue, Paul; French, Deborah L

    2016-03-10

    Platelets are anucleate cytoplasmic discs derived from megakaryocytes that circulate in the blood and have major roles in hemostasis, thrombosis, inflammation, and vascular biology. Platelet transfusions are required to prevent the potentially life-threatening complications of severe thrombocytopenia seen in a variety of medical settings including cancer therapy, trauma, and sepsis. Platelets used in the clinic are currently donor-derived which is associated with concerns over sufficient availability, quality, and complications due to immunologic and/or infectious issues. To overcome our dependence on donor-derived platelets for transfusion, efforts have been made to generate in vitro-based platelets. Work in this area has advanced our understanding of the complex processes that megakaryocytes must undergo to generate platelets both in vivo and in vitro. This knowledge has also defined the challenges that must be overcome to bring in vitro-based platelet manufacturing to a clinical reality. This review will focus on our understanding of committed megakaryocytes and platelet release in vivo and in vitro, and how this knowledge can guide the development of in vitro-derived platelets for clinical application.

  5. Donor demographic and laboratory predictors of single donor platelet yield

    Directory of Open Access Journals (Sweden)

    R. Arun

    2013-10-01

    Full Text Available Background: Platelet transfusions are essential to prevent morbidity and mortality in patients who are severely thrombocytopenic and are at risk of spontaneous bleeding. Platelets are currently obtained either by fractionation of whole blood or by platelet apheresis. The quality of single donor platelets (SDP in terms of yield influences platelet recovery in the recipient and allows prolonging intervals between transfusions. Material and Methods: Donor demographic and laboratory data were analyzed prior to performing plateletpheresis to identify donor factors that influence platelet yield. The study was conducted on 130 healthy, first-time plateletpheresis donors over a period of 4 years. The plateletpheresis procedures were performed using Fresenius Kabi COM.TEC and Hemonetics MCS plus separator. A relationship between pre-donation donor variables and yield of platelets was studied using the Pearson correlation. Results: The mean platelet yield was 3.160.62x1011 per unit. A positive correlation was observed between platelet yield and pre-donation platelet count, body mass index (BMI; Kg/m2 of the donor, while a negative correlation was observed between age and the platelet yield. Conclusion: Donor pre-donation platelet count, BMI and donor age influence platelet yield. Young healthy donors with a high platelet count and better BMI can give a better platelet yield in the SDP.

  6. Propranolol modifies platelet serotonergic mechanisms in rats.

    Science.gov (United States)

    Zółtowski, R; Pawlak, R; Matys, T; Pietraszek, M; Buczko, W

    2002-06-01

    Though the mechanisms for the vascular actions of vasodilatory beta-blockers are mostly determined, some of their interactions with monoaminergic systems are not elucidated. Because there are evidences supporting a possible involvement of serotonin (5-HT) in the actions of beta-blockers, we studied the effect of propranolol on peripheral serotonergic mechanisms in normotensive and Goldblatt two-kidney - one clip (2K1C) hypertensive rats. In both groups of animals propranolol decreased systolic blood pressure, significantly increased whole blood serotonin concentration and at the same time it decreased platelet serotonin level. The uptake of the amine by platelets from hypertensive animals was lower than that of normotensive animals and it was decreased by propranolol only in the latter. In both groups propranolol inhibited potentiation of ADP-induced platelet aggregation by serotonin. In conclusion, this study provides evidence that propranolol modifies platelet serotonergic mechanisms in normotensive and renal hypertensive rats.

  7. Activation of human platelets by misfolded proteins

    NARCIS (Netherlands)

    Herczenik, E.; Bouma, B.; Korporaal, J.A.; Strangi, R.; Zeng, Q.; Gros, P.; van Eck, M.; van Berkel, T.J.C.; Gebbink, M.F.B.G.; Akkerman, J.W.N.

    2007-01-01

    Objective: Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis,which are diseases where platelet activation might be

  8. Mapuche herbal medicine inhibits blood platelet aggregation.

    Science.gov (United States)

    Falkenberg, Susan Skanderup; Tarnow, Inge; Guzman, Alfonso; Mølgaard, Per; Simonsen, Henrik Toft

    2012-01-01

    12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM) and collagen- (2.0 μg/mL) induced aggregations in human blood. These four species in respective extracts (in brackets) were Blechnum chilense (MeOH), Luma apiculata (H(2)O), Amomyrtus luma (DCM : MeOH 1 : 1) and Cestrum parqui (DCM : MeOH 1 : 1). The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1), and L. apiculata (H(2)O) were substantial and confirmed by inhibition of platelet surface activation markers.

  9. Mapuche Herbal Medicine Inhibits Blood Platelet Aggregation

    Directory of Open Access Journals (Sweden)

    Susan Skanderup Falkenberg

    2012-01-01

    Full Text Available 12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM and collagen- (2.0 μg/mL induced aggregations in human blood. These four species in respective extracts (in brackets were Blechnum chilense (MeOH, Luma apiculata (H2O, Amomyrtus luma (DCM : MeOH 1 : 1 and Cestrum parqui (DCM : MeOH 1 : 1. The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1, and L. apiculata (H2O were substantial and confirmed by inhibition of platelet surface activation markers.

  10. Relationship between platelet parameters and sudden ...

    African Journals Online (AJOL)

    Relationship between platelet parameters and sudden sensorineural hearing loss: a ... Data source: A PubMed, Science Direct, Scopus, OVID, EMBASE and ... relationship of PDW and SSNHL but due to the limited studies on this subject more ...

  11. Effects of drugs on platelet function.

    Science.gov (United States)

    Morse, E E

    1977-01-01

    Numerous drugs and chemicals affect the function of human blood platelets. The mechanism of action of some medications is partly understood. Aspirin is the most frequently involved drug. It appears to interfere with the platelet release reaction by acetylation of a platelet membrane protein which may be involved in the synthesis of prostaglandins. Other anti-inflammatory drugs, including indomethacin, phenylbutazone, ibuprophen (Motrin) and clonixin, also interfere with the release reaction but have a shorter acting course than aspirin. Some drugs stimulate adenylcyclase (gliclazide) or block phosphodiesterase, (dipyridamole, caffeine) both of which actions lead to an increase in adenosine cyclic 3':5' monophosphate (cAMP) and decrease aggregation by adenosine diphosphate (ADP). These interactions should be known to clinical scientists since patients using these medicaments may manifest abnormal platelet function tests in the laboratory and mild hemorrhagic syndromes in the clinic.

  12. Physiopathology of blood platelets and development of platelet substitutes. Progress report, August 1, 1975--July 31, 1976

    Energy Technology Data Exchange (ETDEWEB)

    Baldini, M G

    1976-04-28

    Progress is reported on studies on the physiology of blood platelets in thrombocytopenic patients and rabbits. Methods for the detection of platelet antibodies and the preservation of platelets in vitro were investigated. Studies on the effect of low doses of x irradiation (up to 1000 R) on platelet function indicate that platelets exposed to ionizing radiation have increased functional activity. A list is included of publications that report the results of the studies in detail.

  13. Platelet Glycoprotein lb-1X and Malignancy

    Science.gov (United States)

    2011-09-01

    patient with systemic lupus erythematosus . Am J Hematol 2001; 67:262-67. 20. Arthur JF, Dunkley S and Andrews RK. Platelet glycoprotein VI-related...Moroi M. Antibody against platelet membrane glyco- protein VI in a patient with systemic lupus erythematosus . Am J Hematol 2001; 67: 262–7. 9 Arthur JF...Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the

  14. Dengue virus binding and replication by platelets.

    Science.gov (United States)

    Simon, Ayo Y; Sutherland, Michael R; Pryzdial, Edward L G

    2015-07-16

    Dengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome in ∼500,000. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. As a probable initiating event, direct purified DENV-platelet binding was followed in the current study by quantitative reverse transcription-polymerase chain reaction and confirmed antigenically. Approximately 800 viruses specifically bound per platelet at 37°C. Fewer sites were observed at 25°C, the blood bank storage temperature (∼350 sites), or 4°C, known to attenuate virus cell entry (∼200 sites). Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and heparan sulfate proteoglycan were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Interestingly, at 37°C and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days. Further time course experiments demonstrated production of viral NS1 protein, which is known to be highly antigenic in patient serum. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. This was shown using a transcription inhibitor and confirmed by freeze-denatured platelets being incapable of replicating the DENV genome. For the first time, these data demonstrate that platelets directly bind DENV saturably and produce infectious virus. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism. These results furthermore draw attention to the possibility that platelets may produce permissive RNA viruses in addition to DENV.

  15. Platelet dynamics in three-dimensional simulation of whole blood.

    Science.gov (United States)

    Vahidkhah, Koohyar; Diamond, Scott L; Bagchi, Prosenjit

    2014-06-03

    A high-fidelity computational model using a 3D immersed boundary method is used to study platelet dynamics in whole blood. We focus on the 3D effects of the platelet-red blood cell (RBC) interaction on platelet margination and near-wall dynamics in a shear flow. We find that the RBC distribution in whole blood becomes naturally anisotropic and creates local clusters and cavities. A platelet can enter a cavity and use it as an express lane for a fast margination toward the wall. Once near the wall, the 3D nature of the platelet-RBC interaction results in a significant platelet movement in the transverse (vorticity) direction and leads to anisotropic platelet diffusion within the RBC-depleted zone or cell-free layer (CFL). We find that the anisotropy in platelet motion further leads to the formation of platelet clusters, even in the absence of any platelet-platelet adhesion. The transverse motion, and the size and number of the platelet clusters are observed to increase with decreasing CFL thickness. The 3D nature of the platelet-RBC collision also induces fluctuations in off-shear plane orientation and, hence, a rotational diffusion of the platelets. Although most marginated platelets are observed to tumble just outside the RBC-rich zone, platelets further inside the CFL are observed to flow with an intermittent dynamics that alters between sliding and tumbling, as a result of the off-shear plane rotational diffusion, bringing them even closer to the wall. To our knowledge, these new findings are based on the fundamentally 3D nature of the platelet-RBC interaction, and they underscore the importance of using cellular-scale 3D models of whole blood to understand platelet margination and near-wall platelet dynamics.

  16. [The role of blood platelets in infections].

    Science.gov (United States)

    Micota, Bartłomiej; Sadowska, Beata; Różalska, Barbara

    2015-05-17

    Platelets are primarily associated with their main function, hemostasis, although it is known that these cells also exhibit biological activity in cancer progression, inflammation and infectious processes. During infection platelets, due to the expression of specific receptors - Toll-like receptors (TLRs) - which recognize molecular patterns associated with pathogens - pathogen-associated molecular patterns (PAMPs) - are activated by the presence of microorganism components and/or substances released from damaged cells/tissue. Further antimicrobial activity of platelets is based on their capacity for phagocytosis, generation of reactive oxygen species (ROS), and the synthesis, storage and release of proteins/peptides with antimicrobial activity. Another mechanism of platelet action is their immunomodulatory activity. It is based mainly on the ability to secrete chemotactic factors allowing the accumulation of professional immunocompetent cells at the site of infection, thus enhancing the effective eradication of an infectious agent. In chronic infections, platelets, due to release of numerous growth factors and various cytokines, support mechanisms of acquired immunity. They accelerate the maturation of dendritic cells, stimulate B cells to be immunoglobulin-producing plasma cells and potentiate the activity of T cells. Unfortunately, in certain situations (the existence of specific risk factors) the interaction of microorganisms with activated platelets may also be the cause of pathology within the cardiovascular system.

  17. Effects of irradiation on platelet function

    Energy Technology Data Exchange (ETDEWEB)

    Rock, G.; Adams, G.A.; Labow, R.S.

    1988-09-01

    Current medical practice involves the irradiation of blood components, including platelet concentrates, before their administration to patients with severe immunosuppression. The authors studied the effect of irradiation on in vitro platelet function and the leaching of plasticizers from the bag, both immediately and after 5 days of storage. The platelet count, white cell count, pH, glucose, lactate, platelet aggregation and release reaction, and serotonin uptake were not altered by the irradiation of random-donor or apheresis units with 2000 rads carried out at 0 and 24 hours and 5 days after collection. The leaching of di(2-ethylhexyl)phthalate from the plastic bags followed by the conversion to mono(2-ethylhexyl)phthalate was not increased by irradiation. Therefore, it is possible to irradiate platelet concentrates on the day of collection and subsequently store them for at least 5 days while maintaining in vitro function. This procedure could have considerable benefit for blood banks involved in the provision of many platelet products.

  18. Treatment of osteochondral injuries with platelet gel

    Directory of Open Access Journals (Sweden)

    Marcus Vinicius Danieli

    2014-12-01

    Full Text Available OBJECTIVES: Treatments for injured articular cartilage have not advanced to the point that efficient regeneration is possible. However, there has been an increase in the use of platelet-rich plasma for the treatment of several orthopedic disorders, including chondral injuries. Our hypothesis is that the treatment of chondral injuries with platelet gel results in higher-quality repair tissue after 180 days compared with chondral injuries not treated with gel. METHODS: A controlled experimental laboratory study was performed on 30 male rabbits to evaluate osteochondral injury repair after treatment with or without platelet gel. Osteochondral injuries were surgically induced in both knees of each rabbit at the medial femoral condyle. The left knee injury was filled with the platelet gel, and the right knee was not treated. Microscopic analysis of both knee samples was performed after 180 days using a histological grading scale. RESULTS: The only histological evaluation criterion that was not significantly different between treatments was metachromasia. The group that was treated with platelet gel exhibited superior results in all other criteria (cell morphology, surface regularity, chondral thickness and repair tissue integration and in the total score. CONCLUSION: The repair tissue was histologically superior after 180 days in the study group treated with platelet gel compared with the group of untreated injuries.

  19. Lymphocyte-platelet crosstalk in Graves' disease.

    Science.gov (United States)

    Kuznik, Boris I; Vitkovsky, Yuri A; Gvozdeva, Olga V; Solpov, Alexey V; Magen, Eli

    2014-03-01

    Platelets can modulate lymphocytes' role in the pathophysiology of thyroid autoimmune diseases. The present study was performed to clarify the status of platelet-lymphocyte subpopulations aggregation in circulating blood in patients with Graves' disease (GD). One hundred