Mouse Megakaryocytes Research Articles

Mice Lacking α-Actinin-1 in Megakaryocytes Display the Feature of Thrombocytopenia and Impair Platelet Functions

Cytoskeleton remodeling and mitochondrial bioenergetics play important roles in platelet biogenesis and functions. α-Actinin-1, a filament crosslinker, was widely expressed in nonmuscle cells, including megakaryocytes (MKs) and platelets. The cytoskeleton is essential for normal mitochondrial morphology, motility, and distribution. In recent years, the accumulated evidence suggests that inherited mutations in α-actinin-1 are implicated in congenital macrothrombocytopenia. However, the role and underlying mechanism of α-actinin-1 in platelet biogenesis and platelet functions remain poorly studied. In this study, we generated an MK-specific α-actinin-1 knockout mouse model (referred to herein as PF4- Actn1-/-) to investigate the contribution of α-actinin-1 in platelet biogenesis and functions. Blood count tests showed that PF4- Actn1-/- mice exhibited reduced platelet counts. The decreased platelet number in PF4- Actn1-/- mice was not attributed to accelerated platelet clearance or impaired TPO generation in vivo. Platelet count changes observed in an anti-GPIbα antibody-induced thrombocytopenia model and splenectomy indicated that the diminished platelet counts in PF4- Actn1-/- mice might be due to defects in platelet biogenesis. Compared to control mice, PF4- Actn1-/- mice had normal numbers of MK progenitors, including in LSK, MPP, CMP, GMP, MEP, and PreMegE. H&E staining and flow cytometry revealed a decrease in the number of MKs in BM, but an increase in the number of MKs in the spleen. The absence of α-actinin-1 significantly increased the proportion of 2N-4N MKs and decreased the proportion of 8N-32N MKs. CFU-MK colony formation and MK migration in response to SDF-1 signaling was inhibited in PF4- Actn1-/- mice. Furthermore, in vitro studies showed a reduced ratio of PPF-bearing MKs in fetal liver-derived PF4- Actn1-/- MKs. Collectively, these data suggest that α-actinin-1 deficiency in mouse MKs could impair platelet biogenesis, resulting in thrombocytopenia in PF4- Actn1-/- mice. Platelet adhesion and spreading on immobilized fibrinogen and collagen, thrombin-stimulated clot retraction, and platelet aggregation in response to various concentrations of ADP, thrombin, and collagen was significantly decreased in PF4- Actn1-/- platelets. PF4- Actn1-/- platelets also exhibited decreased integrin αIIbβ3 activation and reduced P-selectin exposure in response to various concentrations of ADP, ADP+EPI, thrombin, collagen, and convulxin. Notably, PF4- Actn1-/- platelets showed inhibited calcium mobilization, reactive oxygen species (ROS) generation, and actin polymerization in response to collagen and thrombin. Furthermore, PF4- Actn1-/- mice demonstrated significantly impaired hemostasis. No differences were observed in the PT, APTT, TT, and fibrinogen concentrations between PF4- Actn1-/- mice and Actn1f/f mice. However, tail, liver, and brain bleeding tests revealed that PF4- Actn1-/- mice had significantly prolonged bleeding time and increased bleeding volume. Kaolin-activated whole thromboelastography (TEG) mapping assay indicated that there were no differences in the R-time, K-time, and α-angle values, but the maximum amplitude was decreased in PF4- Actn1-/- mice. To explore the mechanism of α-actinin-1 in platelet biogenesis and platelet functions. Low- (2-4N) and high-ploidy (≥8N) Actn1f/f and PF4- Actn1-/- MKs were sorted for quantitative proteomics analysis. The results of 4D label-free quantitative proteomics revealed that protein expression was significantly decreased in high-ploidy MKs following α-actinin-1 knockdown. Functional enrichment analysis of differentially expressed proteins indicated that α-actinin-1 deletion reduced platelet activation and mitochondrial functions. PF4- Actn1-/- platelets exhibited reduced mitochondrial membrane potential, mitochondrial ROS generation, mitochondrial respiration and glycolysis, suggesting alterations in mitochondrial function in platelets. In this study, we report that mice with α-actinin-1 deficiency in MKs recapitulate the features of thrombocytopenia and exhibit impaired platelet functions.

OC 39.3 Platelet Biogenesis from Mouse Megakaryocytes at Variable Shear Stress Levels by using Microfluidic Systems

OC 39.3 Platelet Biogenesis from Mouse Megakaryocytes at Variable Shear Stress Levels by using Microfluidic Systems

Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation

AbstractThe process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage–specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1−/− mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1−/− mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1−/− MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

Erratum: Isolation of Mouse Megakaryocyte Progenitors.

An erratum was issued for:Isolation of Mouse Megakaryocyte Progenitors. A figure was updated. Figure 2 was updated from: Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. Please click here to view a larger version of this figure. to: Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. Please click here to view a larger version of this figure.

Mitochondrial Fission Protein Drp1 Regulates Megakaryocyte and Platelet Mitochondrial Morphology, Platelet Numbers, and Platelet Function

The mitochondrial architecture is dynamic, undergoing constant fusion and fission. Through mitochondrial fusion, mitochondria form elongated inter-connected networks to promote maximal mitochondrial efficiency. Dynamin Like Protein 1 (Drp1) mediated mitochondrial division (“fission”) on the other hand compartmentalizes mitochondria into smaller sized organelles to facilitate cell division and mitochondrial destruction. Recent published pharmacologic studies suggested an intriguing non-mitochondrial role for Drp1 in platelet degranulation. However, the mitochondrial roles of Drp1 in Megakaryocytes (MKs) and platelets have not been addressed. Furthermore, genetic studies are needed to establish a functional role for Drp1 in platelets. Therefore we examined the contribution of Drp1 mediated mitochondrial fission to MK and platelet mitochondrial morphology, platelet numbers, and platelet function.Using mitotracker labeling of live mitochondria and EVOS fluorescent cell imaging, we examined mitochondrial dynamics and mitochondrial architecture in human and mouse MKs and platelets. In MKs, mitochondria were motile, and formed elaborate interconnected structures. On the other hand, mitochondria in platelets were non-motile, small, and punctate suggesting active mitochondrial fission in late stage MKs and/or platelets.To establish a causative role of Drp1 in regulating MK/platelet mitochondrial morphology, and to assess the role of Drp1 in platelet numbers and function, we generated MK/platelet specific Drp1 null mice (KO). We crossed mice harboring flox (Drp1 fl/fl) sites flanking exons 3-5 of the gene that codes for Drp1 (Dnm1l) with mice harboring Cre recombinase driven by the MK Pf4 promoter (Pf4-Cre). Western blot confirmed that Drp1 protein is abundantly present in platelets from wild type (WT) mice but is absent in platelets from KO mice. Deletion of Drp1 in MKs led to altered mitochondrial morphology: mitochondria in KO MKs formed more hyperextended mitochondrial networks compared to WT MKs (Figure 1A). In Drp1 KO platelets, mitochondria also became less punctate, and more inter-connected compared to WT platelets.Deletion of Drp1 significantly reduced platelet counts in mice (830±27 vs 698±31 K/μL, p =.004, n=10-12 per group, Figure 1B). Conversely, MPV was significantly increased (4.18±.08 vs 4.58±.08, p=.002, n=10-12 per group, Figure 1C). To assess mechanisms contributing to the difference in platelet numbers, we injected an antibody fab fragment that labels platelets, and tracked platelet life span by flow cytometry. The mean platelet life span of Drp1 KO platelets was modestly, yet consistently reduced (65.1±1.1 vs 56.2±2.3 hours, p=.01, n=4-5 per group). On the other hand, there was not a significant difference in the rate of platelet recovery following antibody depletion of platelets (n=5-6 per group).Using PAR4 agonist, which activates mouse thrombin receptors on platelets, we found that platelet degranulation is significantly decreased in DRP1 KO platelets compared to WT platelets (Figure 1D, *p<.05, n=4-6 per group). This is consistent with previously published pharmacologic inhibitor studies of DRP1 in human platelets. Unexpectedly, DRP1 KO also reduced activation of the αIIb/β3 fibrinogen binding receptor (Figure 1E, *p<.05, n=4-6 per group), as measured with JonA antibody (binds to the activated heterodimer).In summary, Drp1 mediated mitochondrial fission regulates mitochondrial morphology in MKs, and disruption of Drp1 in MKs leads to hyperextended mitochondrial networks in MKs and platelets. This corresponds with decreased platelet counts, and increased MPV. Coupled with our observation that αIIb/β3 activation is reduced in DRP1 KO platelets, our findings suggest a novel involvement of DRP1 in regulating platelet numbers and function that may be related to mitochondrial roles of Drp1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thiéry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 °C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Isolation of Mouse Megakaryocyte Progenitors.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

Isolation of Mouse Megakaryocyte Progenitors

Isolation of Mouse Megakaryocyte Progenitors

Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function

An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.

Transcriptional and Spatial Heterogeneity of Mouse Megakaryocytes at Single-Cell Resolution

Megakaryocytes (MKs) have long been described solely as platelet progenitors. However, recent studies show that MKs also act as an essential component of the bone marrow niche to maintain hematopoietic stem cell function, and combat infection by engulfing and presenting antigens. However, it is not known whether these diverse programs are executed by a single cell population or distinct subsets of cells. We performed single-cell RNA sequencing (scRNA-seq) to dissect the heterogeneity of MKs. To overcome the difficulty in obtaining the rare (0.1% in BM) and oversized (up to 65μm) highly polyploid, fragile MKs, we developed an efficient isolation strategy by combining fluorescence-activated cell sorting (FACS) sorting, manual selection of highly viable cells, and FISH verification of ploidy. We obtained 920 CD41+ highly-purified, bone-marrow derived, murine MKs spanning each ploidy stages (2N-32N) for scRNA-seq with modified Smart-seq2 protocol. On average, we detected over 6800 expressed genes and 250,000 transcripts in each MK. All cells expressed classical markers such as Pf4 and Itga2b (CD41). Four cell clusters were identified using an unsupervised clustering method. Cells in Cluster 1 expressed mature MK markers CD42 and CD61, were enriched for hemostasis and platelet activation expression signatures, and consist of ≥8N cells, suggesting these MKs may represent platelet generating MKs. Cells in cluster 2 had lower CD42 and CD61 expression, were low ploidy (≤8N), and had higher expression of inflammation-related genes, including Ctss and Itgam ("inflammatory response-associated MKs"). Cells in Cluster 3 were enriched for DNA replication and DNA strand elongation (GO terms) and were in all ploidy stages ("MKs in polyploidization stage"). Cluster 4, most of which were high-ploidy, expressed high levels of CD42, CD61, TGF-β, and IGF1: factors regulating HSC behavior ("HSC niche cells"). Furthermore, we identified cell population-specific surface markers and transcription factors (TFs) for each of the 4 clusters. Then, immunostaining using antibodies against corresponding markers were performed to confirm the presence of respective MK subpopulations in the bone marrow. Our analyses suggest that defining MK stages by ploidy and traditional markers CD42 and CD61 alone may result in a genetically and developmentally heterogenous population of MK. Rather, MKs at various stages may be more specifically identified by these gene signatures. MKs with different functions are known to have specific spatial distribution in the bone marrow (BM) niche. To test whether MKs with distinct expression signatures are uniquely localized within BM, we performed immunofluorescence staining on BM sections using antibodies against cell population-specific marker genes. Remarkably, we observed that Cluster 1 cells directly contacted blood vessels while most of Cluster 4 cells resided within one cell diameter of HSCs. The unique spatial distribution of cluster 1 and 4 population are in consistency with their respective transcriptomic signatures, and support that platelet generation and HSC maintenance are carried out by two distinct MK subpopulations. We further investigated the potential intrinsic relationship of these four Clusters during megakaryopoiesis. Developmental time courses were reconstructed using Monocle analysis, demonstrating that polyploidization (Cluster 3) occurs at the early stage of MK development with subsequent differentiation toward three orientations. While MKs with low ploidy appear to have two distinct cell fates (immuno-modulation or polyploidization), MKs with high ploidy (≥8N) differentiate towards populations associated with platelet production or stem cell regulation. In summary, our study provides the first in vivo transcriptomic profile of megakaryopoiesis and a potential map of megakaryocyte heterogeneity at the single-cell resolution. MKs may be classified into different functional subpopulations irrespective to their developmental stage and degree of ploidy. These observations suggest that megakaryopoiesis does not occur merely in a stepwise process, but is dynamic and adaptive to locations in the BM and biological needs. Disclosures No relevant conflicts of interest to declare.

Megakaryocytes package contents into separate α-granules that are differentially distributed in platelets

AbstractIn addition to their primary roles in hemostasis and thrombosis, platelets participate in many other physiological and pathological processes, including, but not limited to inflammation, wound healing, tumor metastasis, and angiogenesis. Among their most interesting properties is the large number of bioactive proteins stored in their α-granules, the major storage granule of platelets. We previously showed that platelets differentially package pro- and antiangiogenic proteins in distinct α-granules that undergo differential release upon platelet activation. Nevertheless, how megakaryocytes achieve differential packaging is not fully understood. In this study, we use a mouse megakaryocyte culture system and endocytosis assay to establish when and where differential packaging occurs during platelet production. Live cell microscopy of primary mouse megakaryocytes incubated with fluorescently conjugated fibrinogen and endostatin showed differential endocytosis and packaging of the labeled proteins into distinct α-granule subpopulations. Super-resolution microscopy of mouse proplatelets and human whole-blood platelet α-granules simultaneously probed for 2 different membrane proteins (VAMP-3 and VAMP-8), and multiple granular content proteins (bFGF, ENDO, TSP, VEGF) confirmed differential packaging of protein contents into α-granules. These data suggest that megakaryocytes differentially sort and package α-granule contents, which are preserved as α-granule subpopulations during proplatelet extension and platelet production.

TNF-α–driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging

AbstractAging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

Increased levels of the megakaryocyte and platelet expressed cysteine proteases stefin A and cystatin A prevent thrombosis

Increased platelet activity occurs in type 2 diabetes mellitus (T2DM) and such platelet dysregulation likely originates from altered megakaryopoiesis. We initiated identification of dysregulated pathways in megakaryocytes in the setting of T2DM. We evaluated through transcriptomic analysis, differential gene expressions in megakaryocytes from leptin receptor-deficient mice (db/db), exhibiting features of human T2DM, and control mice (db/+). Functional gene analysis revealed an upregulation of transcripts related to calcium signaling, coagulation cascade and platelet receptors in diabetic mouse megakaryocytes. We also evidenced an upregulation (7- to 9.7-fold) of genes encoding stefin A (StfA), the human ortholog of Cystatin A (CSTA), inhibitor of cathepsin B, H and L. StfA/CSTA was present in megakaryocytes and platelets and its expression increased during obesity and diabetes in rats and humans. StfA/CSTA was primarily localized at platelet membranes and granules and was released upon agonist stimulation and clot formation through a metalloprotease-dependent mechanism. StfA/CSTA did not affect platelet aggregation, but reduced platelet accumulation on immobilized collagen from flowing whole blood (1200 s−1). In-vivo, upon laser-induced vascular injury, platelet recruitment and thrombus formation were markedly reduced in StfA1-overexpressing mice without affecting bleeding time. The presence of CA-074Me, a cathepsin B specific inhibitor significantly reduced thrombus formation in-vitro and in-vivo in human and mouse, respectively. Our study identifies StfA/CSTA as a key contributor of platelet-dependent thrombus formation in both rodents and humans.

A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01

TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions.We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic “leak” hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.

Site-specific phosphorylation regulates the functions of kindlin-3 in a variety of cells.

Studies of isolated cells, mice, and humans have demonstrated the vital role of the FERM domain protein kindlin-3 in integrin activation in certain hematopoietic and non-hematopoietic cells, consequent to binding to integrin β-subunits. To explore regulatory mechanisms, we developed a monoclonal antibody that selectively recognizes the phosphorylated form of Ser484 (pS484) in kindlin-3. Activation of platelets, HEL megakaryocytic-like cells and BT549 breast cancer cells led to enhanced expression of pS484 as assessed by immunofluorescence or Western blotting. In platelets, pS484 rose rapidly and transiently upon stimulation. When a mutant form of kindlin-3, T482S484/AA kindlin-3, was transduced into mouse megakaryocytes, it failed to support activation of integrin αIIbβ3, whereas wild-type kindlin-3 did. In MDA-MB231 breast cancer cells, expression of T482S484/AA kindlin-3 suppressed cell spreading, migration, invasion, and VEGF production. Wild-type kindlin-3 expressing cells markedly increased tumor growth in vivo, whereas T482S484/AA kindlin-3 significantly blunted tumor progression. Thus, our data establish that a unique phosphorylation event in kindlin-3 regulates its cellular functions.

Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation

BackgroundUpon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation.ResultsHere, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bβ3integrin signaling in D723H cells is sufficient to induce β1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and β1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of β1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation.ConclusionsWe characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.

NBEAL2 (Neurobeachin-Like 2) Is Required for Retention of Cargo Proteins by α-Granules During Their Production by Megakaryocytes.

Objective- Human and mouse megakaryocytes lacking NBEAL2 (neurobeachin-like 2) produce platelets where α-granules lack protein cargo. This cargo is mostly megakaryocyte-synthesized, but some proteins, including FGN (fibrinogen), are endocytosed. In this study, we examined the trafficking of both types of cargo within primary megakaryocytes cultured from normal and NBEAL2-null mice, to determine the role of NBEAL2 in α-granule maturation. We also examined the interaction of NBEAL2 with the granule-associated protein P-selectin in human megakaryocytes and platelets. Approach and Results- Fluorescence microscopy was used to compare uptake of labeled FGN by normal and NBEAL2-null mouse megakaryocytes, which was similar in both. NBEAL2-null cells, however, showed decreased FGN retention, and studies with biotinylated protein showed rapid loss rather than increased degradation. Intracellular tracking via fluorescence microscopy revealed that in normal megakaryocytes, endocytosed FGN sequentially associated with compartments expressing RAB5 (Ras-related protein in brain 5), RAB7 (Ras-related protein in brain 7), and P-selectin, where it was retained. A similar initial pattern was observed in NBEAL2-null megakaryocytes, but then FGN passed from the P-selectin compartment to RAB11 (Ras-related protein in brain 11)-associated endosomes before release. Megakaryocyte-synthesized VWF (Von Willebrand factor) was observed to follow the same route out of NBEAL2-null cells. Immunofluorescence microscopy revealed intracellular colocalization of NBEAL2 with P-selectin in human megakaryocytes, proplatelets, and platelets. Native NBEAL2 and P-selectin were coimmunoprecipitated from platelets and megakaryocytes. Conclusions- NBEAL2 is not required for FGN uptake by megakaryocytes. NBEAL2 is required for the retention of both endocytosed and megakaryocyte-synthesized proteins by maturing α-granules, and possibly by platelet-borne granules. This function may involve interaction of NBEAL2 with P-selectin.

Identification of eNOS‐based Megakaryocyte Subpopulations and Their Pharmacological Characterization by IFNγ and IL‐10

PurposeRecently, our laboratory identified in human blood two platelet subpopulations based on the presence or absence of a functional endothelial nitric oxide synthase (eNOS)‐signalling pathway and the ability to produce nitric oxide (NO). We have found that eNOS‐negative (eNOSneg) platelets, although less abundant are more reactive than eNOS‐positive (eNOSpos) platelets and initiate aggregate/thrombus formation, while eNOS‐positive platelets through their ability to generate NO limit aggregate size. As platelets derive from bone marrow megakaryocytes, we hypothesized that eNOSneg and eNOSpos subpopulations of megakaryocytes exist and give rise to their respective eNOS‐based platelet subpopulations. Additionally, we hypothesized that interferon‐γ (IFNγ) and interleukin‐10 (IL‐10), which are known to counter‐regulate eNOS expression and megakaryocyte differentiation, would determine in a concentration manner the ratio of eNOSneg to eNOSpos megakaryocytes.MethodsThe human megakaryoblastic cell line (Meg‐01) and megakaryocytes isolated from eNOS‐GFP transgenic mice were studied. RT‐PCR and DNA sequencing were performed to validate eNOS presence within Meg‐01. Flow cytometry was used to identify eNOSneg and eNOSpos megakaryocytes, and NO production was measured using DAF‐FM, a cell‐permeable fluorescent probe. Further, Meg‐01 cells were treated for 48 hours with IFNγ (0 – 100 ng/ml) and IL‐10 (0 – 100 ng/ml), and eNOS as well as inducible NOS (iNOS) were measured by flow cytometry and immunoblot, respectively.ResultsThe presence of eNOS was confirmed in Meg‐01 and eNOS‐GFP transgenic mice. Similar to human platelets, the majority of Meg‐01 cells were eNOSpos (91.5%±1.2 vs. 8.5%±1.2 eNOSneg, P&lt;0.05) and NO‐producing (92.0%±3.34 vs. 8.0%±6.68 non‐producing, P &lt; 0.05). Conversely, similar to mouse platelets, the majority of mouse megakaryocytes were eNOSneg (95.8%±0.7 vs. 4.2%±0.7 eNOSpos, P &lt; 0.05). The proinflammatory cytokine IFNγ decreased the percent eNOSpos Meg‐01 in a concentration‐dependent manner and also increased the level of iNOS within Meg‐01. IL‐10 in a concentration‐dependent manner (10–100ng/ml) reversed IFNγ‐induced iNOS expression, but not the decrease in percent eNOSpos Meg‐01. The changes in eNOS expression within Meg‐01 were further confirmed by qPCR within the total Meg‐01 population and demonstrated that IFNγ (10 ng/ml) caused a 6.4 fold decrease of eNOS mRNA but only 2.2 fold decrease in the presence IL‐10 (100ng/ml).ConclusionseNOS‐based megakaryocyte subpopulations exist within the Meg‐01 cell line and in eNOS‐GFP transgenic mice. Similar to previously shown for platelets, major species differences exist in eNOSpos to eNOSneg ratios between human and mouse megakaryocytes. IFNγ decreases eNOS expression and percent eNOSpos Meg‐01; while increasing iNOS expression. However, high concentrations of IL‐10 attenuated this effect. Our data show that pro‐inflammatory IFN‐γ may promote growth/differentiation of megakaryocytes with decreased levels/absence of eNOS, which may lead to formation of higher numbers of more reactive eNOSneg platelets potentially leading to higher risk of a pro‐thrombotic state. Further experiments are required to confirm whether eNOSpos and eNOSneg megakaryocytes give rise to their respective platelet subtypes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Unlabelled Box BackgroundN‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts. ObjectivesThe aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. MethodsMeg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls. ResultsThe most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. ConclusionsNMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

Three-Dimensional Culture in a Methylcellulose-Based Hydrogel to Study the Impact of Stiffness on Megakaryocyte Differentiation.

The differentiation and maturation of megakaryocytes (MKs) occurs in a 3D environment where the cells must constantly adapt to the external physical and mechanical constraints during their development and migration to sinusoid vessels. In this chapter, we present a method for culture of mouse MKs from bone marrow hematopoietic progenitor cells in a methylcellulose 3D medium with a stiffness mimicking that of bone marrow. In addition, we describe how the MKs can be recovered to allow for analysis of their differentiation and maturation state by transmission electron microscopy, immunofluorescence or flow cytometry techniques and to evaluate their ability to form proplatelets. This approach allows (1) generation of MKs with a morphology that more closely resembles the MKs that differentiate in vivo, (2) recovery of megakaryocyte phenotypes sometimes observed in vivo but not found in classical liquid cultures, and (3) study of mechanotransduction pathways induced by the stiffness of the medium.