Mice Lacking the ITIM-Containing Receptor G6b-B Exhibit Macrothrombocytopenia and Aberrant Platelet Function (original) (raw)
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Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
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ABSTRACTThe immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation, loss of which results in severe macrothrombocytopenia and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequentin vitrobiochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insight...
Regulator of G-Protein Signaling 18 Controls Both Platelet Generation and Function
PLoS ONE, 2014
RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/-and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/-mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/-platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/-mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/-mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18.
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Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, and the proliferation, maturation and terminal differentiation of the megakaryocytic progenitors. Circulating levels of thrombopoietin (TPO), the primary growth-factor for the megakaryocyte (MK) lineage, induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signalling induction. Decreased platelet turnover rates results in increased concentration of free TPO, enabling the compensatory response of marrow MKs to increased platelet production. C-Mpl activity is orchestrated by a complex cascade of signalling molecules that induces the action of specific transcription factors to drive MK proliferation and maturation. Mature MKs form proplatelet projections that are fragmented into circulating particles. Newly developed thrombopoietic agents operating via c-Mpl receptor may prove useful in supporting platelet production in thrombocytopenic state. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease state, and the new approaches to thrombopoietic therapy.
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Each day in every human, approximately 1 • 10 11 platelets are produced by the cytoplasmic fragmentation of megakaryocytes (MK), their marrow precursor cells. Platelets are the predominating factor in the process of hemostasis and thrombosis. Recent studies have shown that platelets also play a hitherto unsuspected role in several other processes such as inflammation, innate immunity, neoangiogenesis and tumor metastasis. The late phases of MK differentiation identified by polyploidization, maturation and organized fragmentation of the cytoplasm leading to the release of platelets in the blood stream represent a unique model of differentiation. The molecular and cellular mechanisms regulating platelet biogenesis are better understood and may explain several platelet disorders. This review focuses on MK polyploidization, and platelet formation, and discusses their alteration in some platelet disorders.
NO/cGMP and ROS pathways in regulation of platelet function and megakaryocyte maturation
abhängig von einem Stimulus, der von den benachbarten Zellen des Sinusoid-Endothels stammen könnte. Die Ergebnisse dieser Dissertation tragen zu einen besseren Verständnis der Regulation von Blutplättchen sowie der möglichen molekularen Mechanismen bei, die eine Rolle bei der Reifung von Megakaryozyten im vaskularen Mikroumfeld des Knochenmarks innehaben. Glycocalix is a thin layer of glycoproteins, glycolipids, adsorbed plasma proteins, and mucoploysaccharides that coats the plasma membrane on the extracellular side, making a negatively charged net-surface mainly due to sialic acid residues attached to proteins and lipids (Coller 1983). This electrostatic repulsion is likely to prevent resting platelets from attaching to each other as well as to negatively charged endothelium. Zone of Organelles Cytoplasm consists of mitochondria, glycogen stores and three different storage granules: dense granules, α-granules, and lysosomes. These secretory granules posses molecules that affect platelet function, coagulation, and fibrinolysis, vascular tone, inflammation, and wound healing. Some of the components are synthesized by megakaryocytes, other are taken up from plasma and incorporated into the granules (Reed, Fitzgerald et al. 2000). The main components of platelet storage granules are listed in Table 1. Upon activation, granules fuse with platelet 1.2. Adhesive Platelet Receptors Platelets possess a number of adhesive receptors coupled to intracellular signaling systems leading to platelet activation. Most important of these include glycoprotein Ib/IX/V (for vWF), collagen receptors glycoprotein VI and integrin α2β1, and fibrinogen receptor integrin αIIbβ3 that are in details described below. Other adhesive receptors expressed on platelets specific for intracellular matrix components are receptors for fibronectin (α5β1), laminin (α6β1), and vitronectin (αvβ3).
Platelets and Megakaryocytes Contain Functional Nuclear Factor- B
Arteriosclerosis, Thrombosis, and Vascular Biology, 2010
The Nuclear Factor (NF)-κB transcription factor family is well-known for their role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-κB family members including the regulatory Inhibitor (I)-κB and Inhibitor Kappa Kinase (IKK) molecules.
Molecular & Cellular Proteomics, 2006
The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing impor-tance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domaincontaining phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation. Molecular & Cellular Proteomics 6:548 -564, 2007.
Conditional Knockout of Integrin a2b1 in Murine Megakaryocytes Leads to Reduced Mean Platelet Volume
We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK a2b1 develop normally, are fertile, and like their systemic a2b1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet a2b1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of a2b1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of a2 integrin in selected cell types.
Megakaryocyte growth and development factor (MGDF) moderately enhances in-vitro platelet aggregation
Thrombosis research, 1995
Megakaryocyte growth and development factor (MGDF) is a novel cytokine which promotes the development of immature megakaryocytes into platelets. We tested the hypothesis that MGDF would alter the sensitivity of platelets to aggregating agents as assessed by in-vitro platelet aggregometry. Platelet aggregation in the presence or absence of MGDF was tested with single doses of clinically relevant aggregating agents. A dose-dependent enhancement of the aggregation response to epinephrine was noted in MGDF treated platelets. When a range of concentrations of ADP were used to generate an aggregation dose-response curve, the addition of MGDF to platelet rich plasma shifted the dose response curve to the left. The effect of MGDF on platelet aggregation was partially prevented by the coincubation of platelets with a soluble form of the receptor for MGDF, the extracellular domain of c-mpl. In addition, we demonstrate that exogenous MGDF is able to induce tyrosine phosphorylation of platelet proteins with apparent molecular weights of 85 kDa and 130 kDa. From these data we conclude that exogenously added MGDF moderately increases the sensitivity of platelets to aggregating agents through a mechanism which appears to involve tyrosine phosphorylation of platelet proteins. The endogenous modulator of platelet production, long referred to as thrombopoietin, has until recently remained an elusive factor despite an exhaustive search which began with the observation that serum from thrombocytopenic patients produces a thrombopoietic effect. Our knowledge of this long suspected thrombopoietic factor has been dramatically enhanced by a recent wave of reports (l-5) which have ended the mystery. The resulting hematopoietic factor, named