Thrombosis and Inflammation Are Coupled by a Common Thioredoxin Reductase Pathway Downstream of the P2×7 Receptor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2218-2218
Author(s):  
Andrea S Rothmeier ◽  
Christian Furlan-Freguia ◽  
Brian G. Petrich ◽  
Patrizia Marchese ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Cell Surface ◽  
Conflicts Of Interest ◽  
Thioredoxin Reductase ◽  
Small Gtpase ◽  
Confocal Imaging ◽  
Integrin Β1 ◽  
Disulfide Exchange ◽  
Integrin Α4 ◽  
Purinergic P2x7 Receptor ◽  
Protein Disulfide

Abstract Abstract 2218 The purinergic P2X7 receptor contributes to thrombosis by promoting tissue factor (TF) activation and the release of prothrombotic microparticles (MP). On primed macrophages, P2X7 stimulation induces the procoagulant activity of cell surface TF and the release of MP that carry TF, integrin β1 and protein disulfide isomerase (PDI). The generation of TF+ MP is dependent on extracellular thiol-disulfide exchange, but intermediates leading to MP generation downstream of P2X7 signaling are incompletely defined. Tracking of cell surface TF by confocal microscopy shows that constitutive internalization of TF is prevented by P2X7 activation. In non-stimulated cells, inhibition of dynamin-dependent endocytosis retains TF on the cell surface, rapidly upregulates TF activity, and releases procoagulant MP carrying TF and integrin β1. Integrin recycling is dependent on the small GTPase ARF6 that is found incorporated into MP released when internalization is blocked. In contrast, activation of P2X7 releases ARF6 into the MP-depleted supernatant rather than associated with MP. Decreased ARF6 GTP loading in P2X7-stimulated cells and additional data in integrin α4 S988A mutant mice with reduced ARF6 activity support the conclusion that P2X7 interrupts constitutive ARF6 recycling to increase cell surface availability of TF-integrin complexes for incorporation into MP. Confocal imaging shows that P2X7 stimulation promotes filopodia formation and the peripheral transport of TF to the tips of filipodia. Proteomics identifies γ-actin as a thiol-regulated MP protein that is released by P2X7 signaling. We show that specific inhibition of thioredoxin reductase attenuates P2X7-induced actin remodeling and filopodia formation, the generation of highly procoagulant MP carrying γ-actin and PDI, and the increased availability of extracellular thioredoxin and free thiols. Importantly, inhibition of thioredoxin reductase also prevented the processing and release of the pro-inflammatory cytokine IL1β. Thus, the thioredoxin reductase system is a crucial intermediate downstream of P2X7 activation that may couple coagulation and inflammation in cardiovascular diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 70: P2X7-Induced Thiol-Disulfide Exchange is Critical for the Coupling of Coagulation and Inflammation.

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Andrea S Rothmeier ◽  
Christian Furlan-Freguia ◽  
Patrizia Marchese ◽  
Brian Petrich ◽  
Zaverio M Ruggeri ◽  
...  
Keyword(s):  
Cell Surface ◽  
Thioredoxin Reductase ◽  
Integrin Β1 ◽  
Disulfide Exchange ◽  
Upstream Regulator ◽  
Molecular Events ◽  
Pathway Activation ◽  
Purinergic P2x7 Receptor ◽  
Recycling Pathway ◽  
Key Steps

Macrophages are important players in the maintenance of tissue homeostasis, but promote inflammation through the release of IL1β triggered by danger signals in form of extracellular ATP that activates the purinergic P2X7 receptor. We found that P2X7 signaling also contributes to thrombosis by inducing thiol-dependent tissue factor (TF) activation coupled to procoagulant microparticles (MP) release. In the present study, we identified thiol-regulated proteins released on MP and based on this information delineated key steps in the P2X7-induced generation of prothrombotic TF + MP. We find that TF procoagulant activity of LPS/IFNγ primed macrophages is controlled by internalization through the arf6/integrin-recycling pathway. Activation of P2X7 inactivates arf6 and prevents TF internalization, but additional steps are required to generate highly procoagulant MP carrying TF and integrin β1. Imaging of cell surface TF by confocal microscopy shows translocation of TF onto filopodia that form in response to P2X7 activation. Blocking raft mobility does not inhibit filopodia formation, but rather specifically prevents TF and integrin β1 trafficking and release on MP. We show that filopodia formation is dependent on thioredoxin reductase (TRXR). Remarkably, thioredoxin (TRX), the direct substrate of TRXR, is entirely released from the cytosol. Pharmacological inhibition of TRXR blocks both TRX release and reductive changes on the cell surface and MP, identifying the molecular events that change the extracellular redox environment. TRXR-mediated externalization of TRX was also required for activation of the inflammasome and caspase1 leading to IL1β processing and release. These data elucidate the molecular events required for the generation of highly procoagulant TF + MP and identifies TRXR-TRX dependent thiol-disulfide exchange as common upstream regulator responsible for the induction of inflammation and coagulation in innate immune cells.

Assembly of Prothrombinase on Endothelial Cells: Receptor-Mediated or Phospholipid-Driven?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2267-2267
Author(s):  
Mikhail Galkin ◽  
Harlan Bradford ◽  
Sriram Krishnaswamy
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Conflicts Of Interest ◽  
Large Body ◽  
Confocal Imaging ◽  
Binding Studies ◽  
Binding Behavior ◽  
Background Levels ◽  
Membrane Bound

Abstract Membrane binding by factors Xa and Va plays an essential role in facilitating their interaction to yield membrane-bound prothrombinase. This concept is backed by a large body of biochemical work using synthetic phospholipid vesicles typically composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC). However, it remains uncertain whether kinetic and thermodynamic findings with these membranes can be extrapolated to explain the assembly of prothrombinase on cell surfaces relevant to coagulation in the vasculature. We examined the binding of fluorescent derivatives of Xa and Va to endothelial cells cultured in flow chambers using confocal imaging of fluorescence intensity. Cell-bound factor Xa was imaged using Alexa488 or Alexa532 covalently linked to the active site with a peptidyl chloromethyl ketone. Minimal fluorescence was observed on the endothelial surface without intentional activation of the cells by thrombin. Following thrombin activation, total fluorescence of bound Xa in the absence of added Va was marginally different from the unactivated cells but increased 16-fold when excess Va was present. Heterogeneity was evident in the distribution of fluorescence over the surface of the cells that supported Xa binding. Thus, the binding of Xa requires the presence of Va and is distributed in a heterogeneous fashion on the activated endothelial cell surface. Factor Va bound to the endothelial cell was assessed using a derivative singly labeled at Cys539 with Alexa488 or Alexa532. Detection of bound Va also required prior activation by thrombin. The same robust signal for bound but heterogeneously distributed fluorescence was observed following activation both in the presence or absence of added Xa. Together the data indicate that Xa binding to the activated endothelial cell requires bound Va whereas Va binding is unaffected by the presence of Xa, implying a receptor-like role for Va. This is in marked contrast to the behavior on membranes containing 25% PS to which either Xa or Va can bind singly with good affinity. More definitive studies of prothrombinase assembly were conducted using donor fluorescence lifetime imaging on the cells using XaAlexa488 as donor and VaAlexa532 as acceptor. XaAlexa488 bound in the presence of unlabeled Va exhibited an average lifetime of 3.1 ns which was decreased to 2.1 ns in the presence of VaAlexa532. Equivalent lifetimes and energy transfer efficiency were measured using 25% PS containing membranes. Although Xa binding to the cells shows a near-absolute requirement for Va, the resulting cell-bound prothrombinase is comparable to that assembled on synthetic vesicles. An explanation could lie in the binding constraints associated with the exposure of limiting amounts of PS on the activated cell surface expected to disproportionately affect the binding of Xa and Va to membranes. These constraints were replicated using membrane bilayers supported on glass microspheres (lipospheres) containing either 0% or 5% PS balanced by PC and analysis of binding by flow cytometry. Background levels of binding were observed for all approaches in the absence of PS. Va binding was studied using a constant number of lipospheres and increasing concentrations of VaAlexa488 in the presence of different concentrations of unlabeled Xa. Mean fluorescence per 5% PS containing liposphere increased saturably with increasing concentrations of VaAlexa488 and was unaffected by Xa. The binding curves were consistent with a nM dissociation constant. In contrast, background levels of Xa were bound when monitored using varying concentrations of XaAlexa488 in the absence of Va. A family of saturable curves was obtained at different fixed concentrations of unlabeled Va, signifying binding with nM affinity, with amplitude proportional to the concentration of Va. Thus, 5% PS containing lipospheres can replicate the paradoxical receptor-like role for Va in prothrombinase assembly on activated endothelial cells. The binding behavior on lipospheres could be fully accounted for by the markedly decreased affinity of Xa for 5% PS membranes and the major contribution of linked interactions in the stabilization of membrane-bound prothrombinase. Despite appearances to the contrary, a model developed from binding studies with pure phospholipids can be generalized to provide a thermodynamic accounting for the peculiarities of prothrombinase assembly on the endothelial cell. Disclosures No relevant conflicts of interest to declare.

scholarly journals Integrin Activity Reduces Immunogenic Cell Death By Inhibiting Cell Surface Presentation of ERp57 and Calreticulin

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3927-3927
Author(s):  
Chi-Chao Liu ◽  
Pascal Leclair ◽  
Foujan Pedari ◽  
Monajemi Mahdis ◽  
Laura Sly ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Surface ◽  
Conflicts Of Interest ◽  
Inhibitory Effect ◽  
Immunogenic Cell Death ◽  
Integrin Expression ◽  
Negative Regulators ◽  
Integrin Β1 ◽  
T Lymphoblasts ◽  
Enhanced Surface

Abstract Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the 'eat me' engulfment signal for professional phagocytes. ERp57 is an interacting and co-translocating protein with calreticulin, but unlike surface calreticulin, surface ERp57 is not known to be immunogenic. Since calreticulin interacts with α-integrins via the conserved GFFKR motif within the integrin cytoplasmic domain, we assessed whether integrin function can modulate cell surface calreticulin and ERp57 levels in ICD. When stimulated to engage integrin substrates, leukemic T-lymphoblasts treated with an ICD-inducer exhibited decreased surface calreticulin and ERp57 compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin and ERp57 was recapitulated for cells in suspension and treated with various agents that stimulate integrin activation, including Mn2+ and the LIBS antibodies 9EG7 and HUTS21. Similarly, cells expressing a mutant truncated α-integrin bearing only GFFKR as the cytosolic tail, also exhibited low surface calreticulin and ERp57 when treated with ICD-inducers under non-adherent conditions. Furthermore, integrin β1 null cells with overall reduced α-integrin expression exhibited enhanced surface calreticulin and ERp57, consistent with an inhibitory role for integrins in ICD. We generated calreticulin or ERp57 null strains by CRISPR-Cas9, and show that ICD-induced surface calreticulin is ERp57-dependent, while surface ERp57 is not calreticulin-dependent. Using permeabilization techniques that distinguish between cytosolic and ER staining, we found that ICD-inducers promoted the accumulation of cytosolic calreticulin and ERp57 that originated from the ER. In similar fashion, ER to cytosolic trafficking for calreticulin is ERp57-dependent, while for ERp57, it is not calreticulin-dependent. We also showed that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation coupled with normal ER-cytosolic release, suggesting that activated integrins acts to sequester calreticulin within the cytosol. T-lymphoblasts co-treated with an ICD-inducer and integrin activators exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD-inducer. Our study reveals a previously uncharacterized function of integrins as negative regulators of immunogenic cell death by suppressing the presentation of cell surface calreticulin. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tissue factor activation: is disulfide bond switching a regulatory mechanism?

Blood ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 3900-3908 ◽  
Author(s):  
Usha R. Pendurthi ◽  
Samit Ghosh ◽  
Samir K. Mandal ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Cell Signaling ◽  
Tissue Factor ◽  
Disulfide Bond ◽  
Annexin V ◽  
Disulfide Exchange ◽  
Anionic Phospholipids ◽  
Coagulant Activity ◽  
Novel Concept ◽  
Cysteine Thiols

AbstractA majority of tissue factor (TF) on cell surfaces exists in a cryptic form (ie, coagulation function inactive) but retains its functionality in cell signaling. Recent studies have suggested that cryptic TF contains unpaired cysteine thiols and that activation involves the formation of the disulfide bond Cys186-Cys 209 and that protein disulfide isomerase (PDI) regulates TF coagulant and signaling activities by targeting this disulfide bond. This study was carried out to investigate the validity of this novel concept. Although treatment of MDA 231 tumor cells, fibroblasts, and stimulated endothelial cells with the oxidizing agent HgCl2 markedly increased the cell-surface TF coagulant activity, the increase is associated with increased anionic phospholipids at the cell surface. Annexin V, which binds to anionic phospholipids, attenuated the increased TF coagulant activity. It is noteworthy that treatment of cells with reducing agents also increased the cell surface TF activity. No evidence was found for either detectable expression of PDI at the cell surface or association of TF with PDI. Furthermore, reduction of PDI with the gene silencing had no effect on either TF coagulant or cell signaling functions. Overall, the present data undermine the recently proposed hypothesis that PDI-mediated disulfide exchange plays a role in regulating TF procoagulant and cell signaling functions.

scholarly journals Thioredoxin Reductase 1 Is a Highly Immunogenic Cell Surface Antigen in Paracoccidioides spp., Candida albicans, and Cryptococcus neoformans

2020 ◽  
Vol 10 ◽  
Author(s):  
Fabiana Freire Mendes de Oliveira ◽  
Verenice Paredes ◽  
Herdson Renney de Sousa ◽  
Ágata Nogueira D’Áurea Moura ◽  
Juan Riasco-Palacios ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Cryptococcus Neoformans ◽  
Surface Antigen ◽  
Thioredoxin Reductase ◽  
Cell Surface Antigen ◽  
Thioredoxin Reductase 1 ◽  
Paracoccidioides Spp

scholarly journals ENOX2 Target for the Anticancer Isoflavone ME-143

2014 ◽  
Vol 22 (1) ◽  
pp. 1-12 ◽  
Author(s):  
D. James Morr ◽  
Theodore Korty ◽  
Christiaan Meadows ◽  
Laura M. C. Ades ◽  
Dorothy M. Morr
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Cancer Cells ◽  
Human Cancer ◽  
Coenzyme Q ◽  
Cell Enlargement ◽  
Activity Inhibition ◽  
Related Cell ◽  
Ca 50 ◽  
Protein Disulfide

ME-143 (NV-143), a synthetic isoflavone under clinical evaluation for efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated ENOX2 (tNOX) and inhibited the growth of cultured cancer cells with EC50s in the range of 2050 nM. Purified recombinant ENOX2 also bound ME-143 with a K d of 43 (4050) nM. Both the oxidative and protein disulfide-thiol interchange activities of ENOX proteins that alternate to generate a complex set of oscillations with a period length of 22 min compared to 24 min for the constitutive counterpart ENOX1 (CNOX) that characterizes ENOX proteins responded to ME-143. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked. In contrast, the protein disulfide-thiol interchange activity measured from the cleavage of dithiodipyridine (EC50 of ca. 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ENOX1 (CNOX) forms of either cancer or noncancer cells were unaffected by ME-143 over the range of concentrations inhibiting ENOX2. Taken together, the findings show that ME-143 binds to ENOX2 with an affinity 4 to 10 times greater than that reported previously for the related anticancer isoflavone, phenoxodiol.

scholarly journals Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling

Blood ◽  
2018 ◽  
Vol 131 (6) ◽  
pp. 674-685 ◽  
Author(s):  
Andrea S. Rothmeier ◽  
Enbo Liu ◽  
Sagarika Chakrabarty ◽  
Jennifer Disse ◽  
Barbara M. Mueller ◽  
...  
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Binding Site ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Binding Motif ◽  
Integrin Β1 ◽  
Key Points ◽  
Integrin Binding Site ◽  
Recycling Pathway

Key Points The FVIIa integrin-binding motif is required for TF-FVIIa complex formation with integrin β1 and proangiogenic signaling. The arf6 integrin recycling pathway controls TF-FVIIa signaling and cell surface availability for procoagulant activity.

scholarly journals Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

2013 ◽  
Vol 305 (8) ◽  
pp. C829-C845 ◽  
Author(s):  
Hanaa K. B. Motawea ◽  
Selvi C. Jeyaraj ◽  
Ali H. Eid ◽  
Srabani Mitra ◽  
Nicholas T. Unger ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Smooth Muscle Cells ◽  
Blood Vessels ◽  
Muscle Cells ◽  
Small Gtpase ◽  
Actin Binding ◽  
Surface Translocation

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase

2004 ◽  
Vol 67 (9) ◽  
pp. 1721-1731 ◽  
Author(s):  
Giuseppe Bellisola ◽  
Giulio Fracasso ◽  
Rodolfo Ippoliti ◽  
Gianfranco Menestrina ◽  
Anders Rosén ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Thioredoxin Reductase ◽  
Reductive Activation ◽  
Ricin A Chain ◽  
Disulfide Isomerase ◽  
A Chain ◽  
Protein Disulfide ◽  
Ricin A

βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3640-3640
Author(s):  
Sofieke E Klamer ◽  
Paula B van Hennik ◽  
Daphne C Thijssen-Timmer ◽  
C. Ellen Van der Schoot ◽  
Carlijn Voermans
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Cell Surface Expression ◽  
Type I ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3640 Poster Board III-576 Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population. Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation. First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources. Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34+ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34+ cells to βig-h3 is mediated by both β1- and β2- integrins. The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction. In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures: No relevant conflicts of interest to declare.

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