An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.

Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche-related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

Co-stimulatory effect of TLR2 and TLR4 stimulation on megakaryocytic development is mediated through PI3K/NF-ĸB and XBP-1 loop.

Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.

The RNA-Binding Protein ATXN2 is Expressed during Megakaryopoiesis and May Control Timing of Gene Expression.

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.

Coronin 1 Is Required for Integrin ß2 Translocation in Platelets.

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin ß2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin ß2 translocation are specifically or predominantly dependent on coronin 1.

Evaluation of platelet surface glycoproteins in patients with Glanzmann thrombasthenia: Association with bleeding symptoms.

BACKGROUND & OBJECTIVES: Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbß3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies. METHODS: Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed. RESULTS: Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002). INTERPRETATION & CONCLUSIONS: Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.

Bone marrow niche-mimetics modulate HSPC function via integrin signaling.

The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and ß3 (CD61) were found to be induced. Signaling focal contacts via ITGß3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.

Modeling Glanzmann thrombasthenia using patient specific iPSCs and restoring platelet aggregation function by CD41 overexpression.

Glanzmann thrombasthenia (GT) is a rare monogenic hemorrhagic disorder involving aggregation defect of non-nuclear platelets. In this study we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of a GT patient with complex heterogeneous mutations of ITGA2B gene. GT-iPSCs could be successfully differentiated into platelets (GT-iPS-platelets). GT-iPS-platelets were CD41-/CD42b+/CD61- and were platelet activation marker (PAC-1) negative after adenosine diphosphate (ADP) activation. Furthermore, GT-iPS-platelets were defective in platelet aggregation tests in vitro. Moreover, exogenous expression of the wild-type ITGA2B gene in GT-iPS platelets restored CD41 expression and normal platelet aggregation. Our study suggested that patient-specific iPSCs could be a potential target of stem cell based gene therapy for platelet diseases.

Granulocyte colony-stimulating factor mobilizes dormant hematopoietic stem cells without proliferation in mice.

Granulocyte colony-stimulating factor (G-CSF) is used clinically to treat leukopenia and to enforce hematopoietic stem cell (HSC) mobilization to the peripheral blood (PB). However, G-CSF is also produced in response to infection, and excessive exposure reduces HSC repopulation capacity. Previous work has shown that dormant HSCs contain all the long-term repopulation potential in the bone marrow (BM), and that as HSCs accumulate a divisional history, they progressively lose regenerative potential. As G-CSF treatment also induces HSC proliferation, we sought to examine whether G-CSF-mediated repopulation defects are a result of increased proliferative history. To do so, we used an established H2BGFP label retaining system to track HSC divisions in response to G-CSF. Our results show that dormant HSCs are preferentially mobilized to the PB on G-CSF treatment. We find that this mobilization does not result in H2BGFP label dilution of dormant HSCs, suggesting that G-CSF does not stimulate dormant HSC proliferation. Instead, we find that proliferation within the HSC compartment is restricted to CD41-expressing cells that function with short-term, and primarily myeloid, regenerative potential. Finally, we show CD41 expression is up-regulated within the BM HSC compartment in response to G-CSF treatment. This emergent CD41Hi HSC fraction demonstrates no observable engraftment potential, but directly matures into megakaryocytes when placed in culture. Together, our results demonstrate that dormant HSCs mobilize in response to G-CSF treatment without dividing, and that G-CSF-mediated proliferation is restricted to cells with limited regenerative potential found within the HSC compartment.

Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis.

Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.

Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

BACKGROUND: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. STUDY DESIGN AND METHODS: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. RESULTS: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2) = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. CONCLUSION: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts.

Inefficient megakaryopoiesis in mouse hematopoietic stem-progenitor cells lacking T-bet.

Differentiation of hematopoietic stem-progenitor cells (HSPCs) into mature blood lineages results from the translation of extracellular signals into changes in the expression levels of transcription factors controlling cell fate decisions. Multiple transcription factor families are known to be involved in hematopoiesis. Although the T-box transcription factor family is known to be involved in the differentiation of multiple tissues, and expression of T-bet, a T-box family transcription factor, has been observed in HSPCs, T-box family transcription factors do not have a described role in HSPC differentiation. In the current study, we address the functional consequences of T-bet expression in mouse HSPCs. T-bet protein levels differed among HSPC subsets, with highest levels observed in megakaryo-erythroid progenitor cells (MEPs), the common precursor to megakaryocytes and erythrocytes. HSPCs from T-bet-deficient mice exhibited a defect in megakaryocytic differentiation when cultured in the presence of thrombopoietin. In contrast, erythroid differentiation in culture in the presence of erythropoietin was not substantially altered in T-bet-deficient HSPCs. Differences observed with respect to megakaryocyte number and maturity, as assessed by level of expression of CD41 and CD61, and megakaryocyte ploidy, in T-bet-deficient HSPCs were not associated with altered proliferation or survival in culture. Gene expression micro-array analysis of MEPs from T-bet-deficient mice exhibited diminished expression of multiple genes associated with the megakaryocyte lineage. These data advance our understanding of the transcriptional regulation of megakaryopoiesis by supporting a new role for T-bet in the differentiation of MEPs into megakaryocytes.

CD41 and CD45 expression marks the angioformative initiation of neovascularisation in human haemangioblastoma.

The initiation and formation of haemangioblastoma (HB) neovascularisation remain unknown, with concomitant controversy on its cytological origin. We detected HB-derived specific haematopoietic progenitors identified by surface expression of CD41 and CD45, which are similar to human embryonic vasculogenesis. CD41/CD45 cells expressed mesodermal markers, including SCL, Flk1 and c-kit. CD41 also seemed to appear before CD45 on haematopoietic progenitors. In vitro analysis showed that the CD41(+)/CD45 subpopulation gave rise to occasional primitive erythroid activity and endothelial marker expression. Meanwhile, kinetic investigation of the CD41(+)/CD45(+) subpopulation showed that some molecules, including SCL, Flk1 and c-kit, were involved in vascular formation. The CD45(+)/c-kit(+) population that lacked primitive haematopoiesis came from CD41(+) cells. Acquisition of CD45 expression by the haematopoietic progenitors was associated with advanced differentiation towards the vascular cell lineage. Taken together, the present data suggested that CD41 and CD45 expression marked the onset of HB neovascularisation and the stepwise development of the angioformative period. Our findings provide new insights into the mechanisms of HB neovascularisation and the underlying therapeutic targets of anti-vascular treatment.

Novel mutations of integrin αIIb and ß3 genes in Turkish children with Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) is an inherited disorder of platelet aggregation, characterized by qualitative and quantitative defect on platelet αIIbß3 integrin (GpIIb/IIIa), resulting in lifelong bleeding tendency due to defective platelet plug formation. The αIIb gene (ITGA2B) and ß3 gene (ITGB3) are closely located at chromosome 17q21.31-32. ITGA2B consist of 30 exons and encoding α chain, whereas ITGB3 has 15 exons and encoding ß chain. Until now, according to the Human Gene Mutation Database (HGMD), 138 mutations at ITGA2B gene and 101 mutations at ITGB3 gene have been identified. We aimed to determine whether there was any mutation in the ITGA2B and ITGB3 genes, and a correlation between clinical phenotype and genotype in Turkish GT patients. We examined 20 patients with GT followed at the Department of Pediatric Hematology, Meram Faculty of Medicine, for Clinical and Laboratory Findings and Molecular Genetic Analysis. Peripheral blood was collected from patients, and a written informed consent for genetic analysis was obtained from parents. DNA was isolated from by proteinase K and phenol/chloroform extraction. ITGA2B and ITGB3 genes were screened by polymerase chain reaction. There were 12 females and 8 males with a median age of 15.25 years. Major clinical presentations of these patients were mucocutaneous bleedings. The most common bleeding type was epistaxis (85%). Life-threatening bleedings were seen in five patients. Seven (35%) patients showed various mutations in the ITGA2B or ITGB3 genes. We detected four novel mutations in three different regions and two mutations defined previously within the ITGA2B gene. These changes are at exon 4; c.570 T > G alteration, at exon 13 c.1277 T > A, c.1291 T > G alterations, at exon 19 c.1921A > G alterations. And from the start point of exon 14, behind 107 bases, we detected a heterozygous alteration at Thymine to Guanine. According to PolyPhen Database Program and NCBI Multiple Alignment Tool Database, four transitions are conserved at evolutionary process, so we can say that these transitions are novel mutations. c. 468T > G alteration at exon 4 and c. 1378 T > A alteration at exon 13 were reported to HGMD previously. Screening the exons of the ITGB3 gene from the same patient groups, we reported a novel missense mutation at exon 5, at nucleotide 680. No correlation was found between clinical phenotype and genotype. These mutations were described for the first time in Turkish population, and all novel mutations are not defined previously. Furthermore, collaborative studies are needed for the population point of view.

Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies.

Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12(+) thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12(+) thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.

Thrombopoietin/MPL signaling confers growth and survival capacity to CD41-positive cells in a mouse model of Evi1 leukemia.

Ecotropic viral integration site 1 (Evi1) is a transcription factor that is highly expressed in hematopoietic stem cells and is crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in 5% to 10% of de novo acute myeloid leukemia (AML) patients and predicts poor prognosis, reflecting multiple leukemogenic properties of Evi1. Here, we show that thrombopoietin (THPO) signaling is implicated in growth and survival of Evi1-expressing cells using a mouse model of Evi1 leukemia. We first identified that the expression of megakaryocytic surface molecules such as ITGA2B (CD41) and the THPO receptor, MPL, positively correlates with EVI1 expression in AML patients. In agreement with this finding, a subpopulation of bone marrow and spleen cells derived from Evi1 leukemia mice expressed both CD41 and Mpl. CD41(+) Evi1 leukemia cells induced secondary leukemia more efficiently than CD41(-) cells in a serial bone marrow transplantation assay. Importantly, the CD41(+) cells predominantly expressing Mpl effectively proliferated and survived on OP9 stromal cells in the presence of THPO via upregulating BCL-xL expression, suggesting an essential role of the THPO/MPL/BCL-xL cascade in enhancing the progression of Evi1 leukemia. These observations provide a novel aspect of the diverse functions of Evi1 in leukemogenesis.

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.

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 αvß3 and αIIbß3 integrins. Cholesterol inhibitor 6-O-α-maltosyl-ß 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 ß3 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 ß3 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.

CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.

The hematopoietic stem cell (HSC) compartment is heterogeneous, yet our understanding of the identities of different HSC subtypes is limited. Here we show that platelet integrin CD41 (αIIb), currently thought to only transiently mark fetal HSCs, is expressed on an adult HSC subtype that accumulates with age. CD41+ HSCs were largely quiescent and exhibited myeloerythroid and megakaryocyte gene priming, governed by Gata1, whereas CD41- HSCs were more proliferative and exhibited lymphoid gene priming. When isolated without the use of blocking antibodies, CD41+ HSCs possessed long-term repopulation capacity on serial transplantations and showed a marked myeloid bias compared with CD41- HSCs, which yielded a more lymphoid-biased progeny. CD41-knockout (KO) mice displayed multilineage hematopoietic defects coupled with decreased quiescence and survival of HSCs, suggesting that CD41 is functionally relevant for HSC maintenance and hematopoietic homeostasis. Transplantation experiments indicated that CD41-KO-associated defects are long-term transplantable, HSC-derived and, in part, mediated through the loss of platelet mass leading to decreases in HSC exposure to important platelet released cytokines, such as transforming growth factor ß1. In summary, our data provide a novel marker to identify a myeloid-biased HSC subtype that becomes prevalent with age and highlights the dogma of HSC regulation by their progeny.

OP9 bone marrow stroma cells differentiate into megakaryocytes and platelets.

Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells.