43 research outputs found
Proteomics : A Tool to Study Platelet Function
Acknowledgments: Authors acknowledge Laxmikanth Kollipara for the critical review and Julia Lill for support with figures preparation. The Figure 2 was created in Biorender.Peer reviewedPublisher PD
Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages
Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for âŒ15min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.1
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A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function
Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnAloxP/loxP females with GATA1-Cre males. FlnAloxP/y GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbα because they lack the normal cytoskeletal linkage of GPIbα to underlying actin filaments. This results in âŒ70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-likeâmediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased α-granule secretion, integrin αIIbÎČ3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase CâÎł2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnAâSyk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-likeâcontaining receptor signaling and platelet function
NO/cGMP und ROS Singnalwege in Regulation der PlÀttchen Funktion und Megakaryozyten Entwicklung
BlutplĂ€ttchen spielen unter physiologischen Bedingungen eine wichtige Rolle bei der Erhaltung der HĂ€mostase. So verhindern sie ein andauerndes Bluten von Wunden, indem sie in BlutgefĂ€ssen zwischen normalen Zellen des Endothels und beschĂ€digten Bereichen unterscheiden und sich dort gezielt anheften können. Das Zusammenspiel der PlĂ€ttchenagonisten und den dazugehörigen Rezeptoren wird durch intrazellulĂ€re SignalmolekĂŒle kontrolliert, die die Aktivierung der BlutplĂ€ttchen regulieren. Ăusserst wichtige intrazellulare SignalmolekĂŒle stellen dabei die zyklischen Nukleotide cGMP und cAMP dar, die bei der Hemmung der PlĂ€ttchen beteiligt sind. Die Bildung von cGMP und cAMP in den BlutplĂ€ttchen wird durch die aus dem Endothel freigesetzten MolekĂŒle NO und Prostacyclin (PGI2) stimuliert, die ihrerseits BlutplĂ€ttchen hemmen, indem sie Proteinkinase G (PKG) und Proteinkinase A (PKA) aktivieren. Neuerdings wird vorgeschlagen, dass es sich bei ROS (âreactive oxygen speciesâ) um einen neuen Modulator bei der Signaltransduktion zwischen verschiedenen Zelltypen handelt. Die hier zusammengefasste Arbeit beschreibt die Rolle der ROS-Produktion bei der Aktivierung von BlutplĂ€ttchen, die Beziehung zwischen dem NO/cGMP/PKG I Signalweg und der ROS bzw. MAP-Kinase Signaltransduktion, und die Rolle von zyklischen Nukleotiden bei der Entwicklung von Megakaryozyten und BlutplĂ€ttchen. Werden BlutplĂ€ttchen durch unterschiedliche EinflĂŒsse aktiviert, so produzieren sie ĂŒber die Aktivierung von NAD(P)H-Oxidase nur intrazellulĂ€res aber nicht extrazellulĂ€res ROS. Dabei beinflusst das in den BlutplĂ€ttchen produzierte ROS signifikant die Aktivierung von αIIbβ3 Integrin, nicht jedoch die Sekretion von alpha- bzw. dichten Granula oder die Gestalt der BlutplĂ€ttchen. Die Thrombin-induzierte Integrin αIIbβ3-Aktivierung ist nach Behandlung der BlutplĂ€ttchen mit Hemmstoffen der NAD(P)H-Oxidase oder Superoxid-FĂ€ngern signifikant reduziert. Diese Inhibitoren reduzieren auch die Aggregation der BlutplĂ€ttchen bzw. die Thrombusbildung auf Kollagen, wobei diese Effekte unabhĂ€ngig vom NO/cGMP Signalweg vermittelt werden. Sowohl ADP, das von dichten Granula der BlutplĂ€ttchen sezerniert wird und zur Aktivierung von P2Y12-Rezeptoren fĂŒhrt, als auch die Freigabe von Thromboxan A2 stellen wichtige, vorgeschaltete Vermittler bei der p38 MAP Kinase-Aktivierung durch Thrombin dar. Jedoch spielt die p38 MAP-Kinase-Aktivierung keine signifikante Rolle bei der Thrombin-induzierten Kalzium-Mobilisierung, P-Selektin Exprimierung, αIIbβ3 Integrin Aktivierung oder Aggregation der BlutplĂ€ttchen. Abschliessend kann festgestellt werden, dass sich die Aktivierung der PKG insgesamt klar hemmend auf die p38 and ERK MAP-Kinasen in menschlichen BlutplĂ€ttchen auswirkt. Desweiteren zeigt diese Studie, dass zyklische Nukleotide nicht nur die BlutplĂ€ttchen hemmen, sondern auch einen Einfluss auf die Entwicklung der Megakaryozyten und BlutplĂ€ttchen haben, aber auf unterschiedliche Weise. cAMP ist an der Differenzierung von embryonalen hĂ€matopoietischen Zellen zu Megakaryozyten beteiligt, wobei cGMP keine Rolle bei diesem Prozess spielt. WĂ€hrend PKA in embryonalen Zellen schon vertreten ist, steigt beim Reifungsprozess der Megakaryozyten die Expression von Proteinen, die bei der cGMP Signalverbreitung (âsoluble guanylyl cyclaseâ, sGC; PKG) mitwirken, stetig an. In der letzten Phase der Reifung von Megakaryozyten, die durch die Freisetzung der BlutplĂ€ttchen charakterisiert ist, zeigen cGMP und cAMP leicht divergierende Effekte: cGMP verstĂ€rkt die Bildung von BlutplĂ€ttchen, wĂ€hrend cAMP dieselbe reduziert. Dies deutet auf einen fein abgestimmten Prozess hin, abhĂ€ngig von einem Stimulus, der von den benachbarten Zellen des Sinusoid-Endothels stammen könnte. Die Ergebnisse dieser Dissertation tragen zu einen besseren VerstĂ€ndnis der Regulation von BlutplĂ€ttchen sowie der möglichen molekularen Mechanismen bei, die eine Rolle bei der Reifung von Megakaryozyten im vaskularen Mikroumfeld des Knochenmarks innehaben.In physiological conditions platelets have a major role in maintaining haemostasis. Platelets prevent bleeding from wounds by distinguishing normal endothelial cells in vasculature from areas with lesions to which they adhere. Interaction of platelet agonists and their receptors is controlled by intracellular signaling molecules that regulate the activation state of platelets. Very important intracellular signaling molecules are cyclic nucleotides (cGMP and cAMP), both involved in inhibition of platelet activation. Formation of cGMP and cAMP in platelets is stimulated by endothelial-derived NO and prostacyclin (PGI2), which then mediate inhibition of platelets by activating protein kinase G (PKG) and protein kinase A (PKA). Recently, it has been suggested that reactive oxygen species (ROS) represent new modulators of cell signaling within different cell types. The work summarized here describes the involvement of platelet ROS production in platelet activation, the relation of NO/cGMP/PKG I pathway to ROS and to mitogen-activated protein kinases (MAP kinase) signaling, and the involvement of cyclic nucleotides in megakaryocyte and platelet development. Platelets activated with different agonists produce intracellular but not extracellular ROS by activation of NAD(P)H oxidase. In addition, ROS produced in platelets significantly affects αIIbβ3 integrin activation but not alpha/dense granule secretion and platelet shape change. Thrombin induced integrin αIIbβ3 activation is significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors and superoxide scavengers. These inhibitors also reduce platelet aggregation and thrombus formation on collagen under high shear and achieve their effects independently of the NO/cGMP pathway. ADP secreted from platelet dense granules with subsequent activation of P2Y12 receptors as well as thromboxane A2 release are found to be important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation does not significantly contribute to calcium mobilization, P-selectin expression, αIIbβ3 integrin activation and aggregation of human platelets in response to thrombin. Finally, PKG activation does not stimulate, but rather inhibit, p38 and ERK MAP kinases in human platelets. Further study revealed that cyclic nucleotides not only inhibit platelet activation, but are also involved, albeit differentially, in megakaryocyte and platelet development. cAMP is engaged in haematopoietic stem cell differentiation to megakaryocytes, and cGMP has no impact on this process. While PKA is already present in stem cells, expression of proteins involved in cGMP signaling (soluble guanylyl cyclase, sGC; PKG) increases with maturation of megakaryocytes. In the final step of megakaryocyte maturation that includes release of platelets, cGMP and cAMP have mild but opposing effects: cGMP increases platelet production while cAMP decreases it indicating a finely regulated process that could depend on stimulus coming from adjacent endothelial cells of sinusoids in bone marrow. The results of this thesis contribute to a better understanding of platelet regulation and of the possible molecular mechanisms involved in megakaryocyte maturation in bone marrow vascular microenvironment
Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P<sub>2</sub> and PI4P in Human Platelets
Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P2 and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation
Developmental differences of in vitro cultured murine bone marrow- and fetal liver-derived megakaryocytes
Multiple lines of evidence support differences in the megakaryopoiesis during development. Murine in vitro models to study megakaryopoiesis employ cultured megakaryocytes MKs derived from adult bone marrow (BM) or fetal livers (FL) of mouse embryos. Mouse models allow to study the molecular basis for cellular changes utilizing conditional or knock-out models and permit further in vitro genetic or pharmacological manipulations. Despite being extensively used, MKs cultured from these two sources have not been systematically compared. In the present study, we compared BM- and FL-derived MKs, assessing their size, proplatelet production capacity, expression of common MK markers (αIIb, ÎČ3, GPIb α, ÎČ) and cytoskeletal proteins (filamin A, ÎČ1-tubulin, actin), the subcellular appearance of α-granules (VWF), membranes (GPIbÎČ) and cytoskeleton (F-actin) throughout in vitro development. We demonstrate that FL MKs although smaller in size, spontaneously produce more proplatelets than BM MKs and at earlier stages express more ÎČ1-tubulin. In addition, early FL MKs show increased internal GPIbÎČ staining and present higher GPIbÎČ (early and late) and VWF (late stages) total fluorescence intensity (TFI)/cell size than BM MKs. BM MKs have up-regulated TPO signaling corresponding to their bigger size and ploidy, without changes in c-Mpl. Expressing endogenous ÎČ1-tubulin or the presence of heparin improves BM MKs ability to produce proplatelets. These data suggest that FL MKs undergo cytoplasmic maturation earlier than BM MKs and that this, in addition to higher ÎČ1-tubulin levels and GPIb, supported with an extensive F-actin network, could contribute to more efficient proplatelet formation in vitro