Primary Megakaryocytes Research Articles

Platelet RNA Biomarker of Ticagrelor-Responsive Genes Is Associated With Platelet Function and Cardiovascular Events.

Identifying patients with the optimal risk:benefit for ticagrelor is challenging. The aim was to identify ticagrelor-responsive platelet transcripts as biomarkers of platelet function and cardiovascular risk. Healthy volunteers (n=58, discovery; n=49, validation) were exposed to 4 weeks of ticagrelor with platelet RNA data, platelet function, and self-reported bleeding measured pre-/post-ticagrelor. RNA sequencing was used to discover platelet genes affected by ticagrelor, and a subset of the most informative was summarized into a composite score and tested for validation. This score was further analyzed (1) in CD34+ megakaryocytes exposed to an P2Y12 inhibitor in vitro, (2) with baseline platelet function in healthy controls, (3) in peripheral artery disease patients (n=139) versus patient controls (n=30) without atherosclerosis, and (4) in patients with peripheral artery disease for correlation with atherosclerosis severity and risk of incident major adverse cardiovascular and limb events. Ticagrelor exposure differentially expressed 3409 platelet transcripts. Of these, 111 were prioritized to calculate a Ticagrelor Exposure Signature score, which ticagrelor reproducibly increased in discovery and validation cohorts. Ticagrelor's effects on platelets transcripts positively correlated with effects of P2Y12 inhibition in primary megakaryocytes. In healthy controls, higher baseline scores correlated with lower baseline platelet function and with minor bleeding while receiving ticagrelor. In patients, lower scores independently associated with both the presence and extent of atherosclerosis and incident ischemic events. Ticagrelor-responsive platelet transcripts are a biomarker for platelet function and cardiovascular risk and may have clinical utility for selecting patients with optimal risk:benefit for ticagrelor use.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

Megakaryocytes Direct CD4 + T Cell Responses Via Major Histocompatibility Complex Class II Antigen Presentation

Megakaryocytes (MKs) are rare, large, hematopoietic cells that canonically have the role of producing platelets. Advances in single-cell RNA-sequencing (scRNA-seq) have revealed a previously under-appreciated degree of MK heterogeneity suggesting that both murine and human MKs may perform functions beyond platelet production. Among other inflammatory molecules, these studies identified that select MK subsets express major histocompatibility complex (MHC II) antigen presentation-associated transcripts and display an upregulation of antigen processing pathways. Together, these findings suggest a potential immunoregulatory function of select MK subsets. However, functional validation substantiating these transcriptional signatures and the proposed MK immunoregulatory capacities has lagged, and the expression of MHC II in bone marrow MKs remains controversial. In our studies, we aimed to both quantify and functionally assess immunomodulatory bone marrow MKs. Using flow cytometry and high-resolution confocal imaging, we showed that MHC class II is expressed on ~20% of MKs at various stages of differentiation. Of the cell populations identified, MHC class II expression was observed in CD41+ and vWF+ platelet-biased hematopoietic stem cells (HSCs), c-Kit+CD150+CD41+ MK progenitors, immature CD41+ MKs and mature CD41+ CD42d+ MKs. Intriguingly, we also observed that MHC class II expression was dramatically upregulated, (2.5-fold increase in expression) in mature MKs and MK progenitors of aged mice. Additionally, MHCII expression was significantly correlated with vWF and CD41 expression in long-term HSCs, with 60-70% of MHCII+ HSCs co-expressing both of these markers. To further validate MHC class II expression in the MK lineage, we utilized two unique murine MHCII transgenic reporter models. The first was an MHCII-EGFP knock-in mouse in which endogenous class II product is replaced by GFP, and the second was a Cd74tdT reporter mouse in which the invariant chain (CD74) can be visualized via tdTomato upon MHC class II pathway activation. In both models, approximately 20-30% of MKs were MHCII positive, consistent with antibody staining in wild-type mice. To substantiate the immunoregulatory function of primary murine MKs, we assayed antigen presentation capacity and ability to stimulate T-cells in vitro. We observed that bone marrow derived MKs express basal levels of multiple co-stimulatory receptors including CD80, CD83, CD86, and CD40 in addition to MHC class II. We also observed significant upregulated of these receptors upon TLR stimulation (TLR3, TLR4 TLR9) in a dose dependent manner. Notably, co-culture of MKs with CD4+ T cells resulted in naive T cell activation and proliferation. To verify that these results extended to human MKs, we examined HLA class II expression in MKs differentiated from CD34+ cells obtained from human bone marrow. Comparable with the murine studies, we found that human MKs also upregulated class II pathway molecules, suggesting that human MKs may also engage with the adaptive immune system. Together, our results support the hypothesis that there is an immunomodulatory sub-population of MKs that can be distinguished by MHCII surface expression, engages in class II antigen presentation, and can activate CD4+ T cells to prime adaptive immune responses. Future studies will investigate the in vivo role of these immunomodulatory functions of MK and how they may be exploited to alter T cell differentiation in the context of inflammatory, infectious, or age-associated thrombotic diseases.

Panaxadiol saponin ameliorates ferroptosis in iron-overload aplastic anemia mice and Meg-01 cells by activating Nrf2/HO-1 and PI3K/AKT/mTOR signaling pathway

Panaxadiol saponin (PND) is a latent targeted drug for the treatment of aplastic anemia (AA). In this study, we examined the effects of PND on ferroptosis in iron-overload AA and Meg-01 cells. We utilized RNA-seq to analyze differentially expressed genes in iron-induced Meg-01 cells treated with PND. The effects of PND or combined with deferasirox (DFS) on iron deposition, labile iron pool (LIP), several ferroptosis events, apoptosis, mitochondrial structure, as well as ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR pathway-related markers in iron-induced Meg-01 cells were examined by Prussian-blue staining, flow cytometer, ELISA, Hoechst 33342 staining, transmission electron microscope, and Western blot assays, respectively. Additionally, an AA mice model with iron overload was established. Then, the blood routine was assessed, and the number of bone marrow-derived mononuclear cells (BMMNCs) in mice was counted. Also, serum iron, ferroptosis events, apoptosis, histology, T lymphocyte percentage, ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR signaling-related targets in primary megakaryocytes of AA mice with iron overload were assessed by commercial kits, TUNEL staining, hematoxylin and eosin (H&E) staining, Prussian blue staining, flow cytometer, and qRT-PCR analysis, respectively. PND suppressed iron-triggered iron overload, and apoptosis, and ameliorated mitochondrial morphology in Meg-01 cells. Importantly, PND ameliorated ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR signaling-related marker expressions in iron-induced Meg-01 cells or primary megakaryocytes of AA mice with iron overload. Moreover, PND ameliorated body weight, peripheral blood cell counts, the number of BMMNCs, and histological injury in the iron-overload AA mice. Also, PND improved the percentage of T lymphocytes in the iron-overload AA mice. PND attenuates ferroptosis against iron-overload AA mice and Meg-01 cells via activating Nrf2/HO-1 and PI3K/AKT/mTOR pathway and is a promising novel therapeutic candidate for AA.

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes.

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

PIKfyve-Dependent Phosphoinositide Dynamics in Megakaryocyte/Platelet Granule Integrity and Platelet Functions.

Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P2 and the cation channel TRPML (transient receptor potential mucolipin) 1 and TPC (two pore segment channel) 2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P2 control granule integrity both in megakaryocytes and platelets.

Establishment of a platelet production model by bone marrow cavity transplantation of mouse primary megakaryocytes

目的利用小鼠原代巨核细胞骨髓腔移植构建血小板产生模型，为研究巨核细胞功能及血小板产生调控机制提供工具。方法通过磁珠富集绿色荧光蛋白（Green Fluorescent Proteins, GFP）转基因供体小鼠骨髓原代巨核细胞，将其移植入经半致死剂量辐照的受体小鼠骨髓腔，建立原代巨核细胞骨髓腔内血小板生成模型。通过免疫荧光染色、流式细胞术等方法检测受体小鼠中供体来源巨核细胞和血小板形态、大小等指标。结果磁珠富集可将巨核细胞在骨髓细胞悬液中的比例提高40～50倍。供体来源巨核细胞能够在受体小鼠骨髓腔成功定植并正常产生血小板，其产生的子代血小板具有与受体自身来源血小板相似的形态、大小及CD41、CD42d、CD61等表面标志分子表达水平。结论通过磁珠富集与骨髓腔注射可成功构建小鼠原代巨核细胞移植模型，该模型能够客观反映巨核细胞在骨髓内产生血小板的能力。

Manipulation of Fatty Acid Metabolism Impairs Megakaryocyte Differentiation and Platelet Production

Background: Obesity is associated with an altered plasma lipid composition that impacts platelet activation, indicating that lipids directly affect platelet function and reactivity. However, very little is known about which essential lipids are important to enable platelet production from their precursors, megakaryocytes (MKs), and whether obesity influences lipid composition and consequently affects thrombopoiesis.Aim: We aimed to determine which lipids and associated pathways play a significant role in MK differentiation and platelet production in health and upon obesity. Our long-term goal is to determine if we can manipulate MK maturation and platelet production through lipid incorporation or inhibition.Methods: In order to determine the lipid profile of MKs through maturation, we performed an extensive lipidomics screen on primary MK progenitors, mature MKs, and platelets. Based on our findings, we then targeted de novo fatty acid (FA) synthesis in MKs to determine whether manipulation of this pathway attenuates MK differentiation and platelet formation. In parallel, we investigated megakaryo- and thrombopoiesis in an experimental setting of diet-induced obesity (DIO) in mice to examinate how altered lipid compositions within the plasma affect MK maturation.Results: Lipidomic data revealed increased polyunsaturated FA and plasmalogen content in the late stages of MKs maturation, suggesting that lipid composition is undergoing changes that may enable platelet production. To validate and extend these results, we inhibited multiple enzymes in de novo lipogenesis and FA synthesis. Using two different sources of primary murine hematopoietic stem cells (HSCs), we found that MK differentiation and maturation from HSCs was significantly decreased up to 60% after inhibition of acetyl-coA carboxylase (ACC) and 80% after fatty acid synthetase (FASN) inhibition, both enzymes from the de novo lipogenesis pathway. Moreover, we also found that MK differentiation and maturation were highly decreased (up to 70%) after the inhibition of acyl-coA synthetase (ACS). To further examine the role of fatty acids incorporation on platelet production, we next treated mature MKs with an ACS inhibitor to investigate the direct effect of FA incorporation on proplatelet formation; we observed a significant, multifoldreduction in proplatelet area. While dihydroceramides were altered in the lipidomic data, impairment of the de novo ceramide synthesis pathway did not affect either MK maturation or platelet production. Moreover, we observed an increased abundance of HSCs, myeloid cells, and MK progenitors in the bone marrow of DIO mice compared to control mice on a chow diet. This further supports an important role for lipids in megakaryopoiesis and suggests that differences in platelet reactivity during obesity may be caused by altered MK maturation and/or platelet production.Conclusions: Our results elucidate an important and previously unrecognized role for fatty acid synthesis in megakaryopoiesis and platelet production. The findings imply that an altered lipid content, as observed in patients with obesity, may not only impact platelet reactivity but also MK maturation and function. Consequently, platelets with an altered lipid content might originate from pathogenic MKs in obesity, which makes lipid-regulating proteins in MKs novel and viable therapeutic targets. DisclosuresMachlus: KEROS Therapeutics: Consultancy, Honoraria; STRM.BIO: Consultancy, Honoraria.

NKL Homeobox Genes NKX2-3 and NKX2-4 Deregulate Megakaryocytic-Erythroid Cell Differentiation in AML.

NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte–erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes.

SummaryCalreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

Abstract 109: Platelets Amplify Endotheliopathy In Covid-19

In addition to their pivotal role in thrombosis and hemostasis, platelets participate in inflammatory responses and endothelial cell activation - hallmarks in the pathogenesis of coronavirus disease 2019 (COVID-19). Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). To explore this interaction, ECs were treated with platelet releasate from patients with and without COVID-19, and EC mRNA sequencing performed. We demonstrate that platelet released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. Investigation of the COVID-19 platelet transcriptome identified pathways related to organelle/granule release, metabolism, and immune effector function in addition to upregulation of S100A8 and S100A9 mRNA. Incubation of primary megakaryocytes with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also induced upregulation of S100A8 and S100A9 mRNA. Consistent with increased gene expression, the heterodimer protein product of S100A8 / A9 , myeloid-related protein (MRP)8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates microvascular endothelial cells, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to thromboinflammation and poor clinical outcomes in COVID-19 patients. Finally, we present evidence that therapeutic targeting of platelet P2Y 12 represents a promising candidate to reduce proinflammatory and prothrombotic platelet-endothelial interactions. Altogether, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

Synthesis of Inflammatory Cytokines and Expression of Adhesion Molecules in Megakaryocytes

Inflammatory cytokines and platelet adhesion molecules may play a major role in hemostasis and immuno-pathological processes. Hematopoietic cells of myeloid and lymphoid lineages are known to be important sources of a variety of inflammatory cytokines. However, the immunological role of megakaryocytes is not well known. This project studied the involvement of megakaryocytes on immuno-inflammatory processes and the possible mechanism via the adhesion molecules and the synthesis of relevant cytokines. The expression of interleukin (IL)-1 to IL-10, TNF-a, TNF-g and IFN-g in primary megakaryocytes and four megakaryocytic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e were determined by RT-PCR. The effect of IL-1b, IL-3, IL-6 and TPO on megakaryocyte colony formation (CFU-MK) was studied by using a plasma clot culture system. The CFU-MK was confirmed by AchE staining. The expression of adhesion protein CD36 on human platelets, megakaryocytes and megakaryocytic cell lines was also analyzed by using flow cytometry, ELISA and immunocytochemical methods. Our results showed that CD36 was expressed on platelets, megakaryocytes and the four megakaryocytic cell lines. The relative expression level is as follows: platelets > megakaryocytes > Meg-01 > Dami > CHRF-288-11 > M-07e, suggesting that the level of CD36 expression correlates with the degree of maturity of megakaryocytic differentiation. Inflammatory cytokines TNF-a, IL-1b, IL-3 and IL-6 were detected in primary megakaryocytes and all the four megakaryocytic cell lines, suggesting that different stages of megakaryocytes can be as a source of inflammatory cytokines. IL-1b, IL-3 and IL-6, as well as TPO, play a stimulating effect on CFU-MK formation, suggesting that there is an “autocrine” effect on megakaryopoiesis. Megakaryocytes may involve in immuno-inflammatory processes via the synthesis of inflammatory cytokines and platelet adhesion molecules. DisclosuresNo relevant conflicts of interest to declare.

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5′-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.

Lung megakaryocytes display distinct transcriptional and phenotypic properties.

Megakaryocytes (MKs) are responsible for platelet biogenesis, which is believed to occur canonically in adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may contribute significantly to circulating platelet mass. Although a diversity of cells specific to the BM is known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single-cell RNA sequencing, coupled with histological, ploidy, and flow cytometric analyses, to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts, with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors that are known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed toward roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. The immune phenotype displayed by lung MKs also introduces their potential role in microbial surveillance and antigen presentation.

Effect of Indirubin and Sheng-Xue-Xiao-Ban Capsule (SXXBC) on Promoting Peripheral Platelet in ITP Model Mice

To compare the effect of Sheng-Xue-Xiao-Ban Capsule (SXXBC) and indirubin to the peripheral platelets of the Idiopathic thrombocytopenic purpura (ITP) model mouse. The ITP mouse model was established by the method of passive immunization. SXXBC and indirubin were used for intervention treatment. Then the hemorrhagic phenomena of ITP mice were observed and the numbers of peripheral platelets, hemoglobin and white blood cells, bone marrow megakaryocytes and their classification and coagulation function were detected and compared. The improvement rate of hemorrhage in SXXBC group was 40% for small dose, 60% for medium dose and 80% for high dose, while the improvement rate of hemorrhage in indirubin group was 30% for small dose, 50% for medium dose and 60% for high dose. There was no statistically significant difference in the improvement rate of hemorrhage between the two groups (P＞0.05). Compared with the model control group, PLT and Hb increased in different doses of SXXBC and indirubin group 4th-8th day after drug intervention (P＜0.05, 0.01). However, there was no significant difference between the different doses of SXXBC group and indirubin group (P＞0.05). Compared with the model control group, the WBC in each group was significantly lower (P＜0.05, 0.01) on the 4th-8th day after drug intervention; However, there was no statistical significance between the two groups of SXXBC and indirubin (P＞0.05). Compared with the model control group, the total number of megakaryocytes in each treatment group were decreased (P＜0.05, P＜0.01), in which the number of primary megakaryocytes in the large and medium dose groups of SXXBC and indirubin were decreased (P＜0.05, 0.01), and the number of juvenile megakaryocytes in the large dose group of SXXBC and indirubin were also decreased (P＜0.05). The number of granular megakaryocytes were decreased in each intervention groups (P＜0.05, 0.01), and the number of thromocytogenic megakaryocyte was increased in the high and medium dose groups of SXXBC and indirubin (P＜0.01). The time of prothrombin was shortened in the high and medium dose groups of SXXBC and indirubin (P＜0.05), and the fibrinogen (FIB) content in the high and medium dose groups of SXXBC was close to that of the normal control group. Both of the SXXBC and the indirubin standard all show good hemostatic effects. Indirubin shows a positive effect on increasing the peripheral platelet and hemoglobin in ITP model mice, regulating the immune response, reducing the total number of bone marrow megakaryocytes, increasing the thromocytogenic megakaryocyte, and increasing coagulation function.

High‐content, label‐free analysis of proplatelet production from megakaryocytes

BackgroundThe mechanisms that regulate platelet biogenesis remain unclear; factors that trigger megakaryocytes (MKs) to initiate platelet production are poorly understood. Platelet formation begins with proplatelets, which are cellular extensions originating from the MK cell body. ObjectivesProplatelet formation is an asynchronous and dynamic process that poses unique challenges for researchers to accurately capture and analyze. We have designed an open‐source, high‐content, high‐throughput, label‐free analysis platform. MethodsPhase‐contrast images of live, primary MKs are captured over a 24‐hour period. Pixel‐based machine‐learning classification done by ilastik generates probability maps of key cellular features (circular MKs and branching proplatelets), which are processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphology. A subsequent reinforcement classification, by CellProfiler Analyst, improves the detection of cellular structures. ResultsThis workflow yields the percent of proplatelet production, area, count of proplatelets and MKs, and other statistics including skeletonization information for measuring proplatelet branching and length. We propose using a combination of these analyzed metrics, in particular the area measurements of MKs and proplatelets, when assessing in vitro proplatelet production. Accuracy was validated against manually counted images and an existing algorithm. We then used the new platform to test compounds known to cause thrombocytopenia, including bromodomain inhibitors, and uncovered previously unrecognized effects of drugs on proplatelet formation, thus demonstrating the utility of our analysis platform. ConclusionThis advance in creating unbiased data analysis will increase the scale and scope of proplatelet production studies and potentially serve as a valuable resource for investigating molecular mechanisms of thrombocytopenia.

In vitro megakaryocyte culture from human bone marrow aspirates as a research and diagnostic tool

Megakaryocytes (MKs) are relatively rare in bone marrow, comprising <0.05% of the nucleated cells, which makes direct isolation from human bone marrow impractical. As such, in vitro expansion of primary MKs from patient samples offers exciting fundamental and clinical opportunities. As most of the developed ex vivo methods require a substantial volume of biomaterial, they are not widely performed on young patients. Here we propose a simple, robust, and adapted method of primary human MK culture from 1 mL of bone marrow aspirate. Our technique uses a small volume of bone marrow per culture, uses straightforward isolation methods, and generates approximately 6 × 105 mature MKs per culture. The relative high cell purity and yield achieved by this technique, combined with efficient use of low volumes of bone marrow, make this approach suitable for diagnostic and basic research of human megakaryopoiesis.

Megakaryopoiesis and Platelet-Innate Immune Cell Interactions Are Developmentally Regulated

Megakaryocyte endoreplication, maturation and platelet production have all been reported to be affected by developmental stage (Elagib, Exp Hematol 2018). Additionally, we have recently found that P-selectin expression and function are altered in fetal and neonatal murine platelets (Stolla, Blood Adv 2019), suggesting that both megakaryopoiesis and platelet function are developmentally regulated. The developmental regulation of megakaryopoiesis can have important implications regarding platelet production in premature and full-term neonates, platelet regeneration following cord blood transplantation, and the in vitro generation of platelets from iPS cells (Elagib, Exp Hematol 2018). To further explore differences between fetal and adult megakaryocytes, we analyzed primary murine megakaryocytes in the fetal liver and adult bone marrow, and found that fetal megakaryocytes have lower average ploidy as well as increased cell size and initial accumulation of surface GP1bbeta expression at 2N and 4N stages. These latter findings indicate that cytoplasmic maturation begins at lower ploidy stages in fetal versus adult murine megakaryocytes. Consistent with findings in cultured human cord blood versus adult blood-derived progenitors (Liu, Blood 2015), we found lower ploidy and earlier cytoplasmic maturation in cultures of fetal versus adult megakaryocytes. These findings suggest the existence of a cell intrinsic developmental program of megakaryocyte maturation. Global gene expression studies of cultured megakaryocytes derived from fetal liver versus adult bone marrow progenitors revealed differential expression of Gata1, Cyclin D1, Cyclin D2, and Igf2bp3, supporting their proposed roles in fetal versus adult megakaryopoiesis (Klusmann, Genes Dev 2010; Liu, Blood 2011; Elagib, J Clin Invest 2017). Igf1R transcripts were increased in fetal megakaryocytes consistent with their sensitivity to IGF signaling (Klusmann, Genes Dev 2010). In addition, we found increased expression of Lin28b, a negative regulator of Let7 miRNAs, as well as multiple downstream targets of Let7 miRNAs in fetal liver-derived megakaryocytes. Induction of Lin28b expression in adult mice (Zhu, Cell 2011) led to increased expression of Gata1, Cyclin D1, Igf2bp3 and IGF1R in bone marrow-derived megakaryocytes. Interestingly, adult megakaryocyte average ploidy was decreased and the sensitivity to IGF1R inhibition was increased by Lin28b induction, suggesting that the Lin28b axis regulates key aspects of fetal versus adult megakaryopoiesis. The largest category of upregulated genes in adult versus fetal megakaryocyte transcriptomes was immune-related genes, including PPBP, a neutrophil chemotactic cytokine, and beta2M, which we have recently shown to mediate platelet-monocyte interactions (Hilt, JCI Insight 2019). Consistent with the transcriptomic data, neonatal platelets express less MHCI on their surface (an indirect indication of less platelet beta2M protein expression) and release less PPBP post-activation when compared to adult platelets. Activated neonatal platelets induce less neutrophil migration and activation when compared to activated adult platelets. In addition, the releasates from activated neonatal versus adult platelets differentially altered the balance of proinflammatory versus proreparative signaling in monocytes. Finally, the induction of Lin28b expression in adult mice resulted in platelets that express less MHCI, release less PPBP, and induce less neutrophil chemotaxis. Taken together, our studies support the hypothesis that megakaryocyte maturation and resulting platelet-immune cell interactions are developmentally regulated, in part, through a Lin28b-mediated program. Disclosures Palis: Rubius Therapeutics: Consultancy.

Lung-Resident Megakaryocytes Display Distinct Transcriptional and Functional Properties

Megakaryocytes (MKs) are responsible for producing anucleate platelets which are indispensable due to their roles in hemostasis, growth and development. Recent studies have also demonstrated the influence of platelets in regeneration and immune protection, thus highlighting the functionality of platelets beyond their classically defined roles. Standard models of thrombopoiesis emphasize that MK development occurs initially in the fetal liver during development, followed by long-term residency in sites of adult hematopoiesis such as the bone marrow sinusoids. Outside of the hematopoietic system, MKs have been shown to reside in the interstitium of the lung, with emerging evidence pointing to the lung microvasculature as a specialized environment that may support a significant bulk of platelet production. However, little is known about how MKs traffic to the lung and whether they are influenced by the lung microenvironment to become functionally distinct from their hematopoietic counterparts. To characterize the transcriptional signature of lung-resident MKs, we performed single cell RNA sequencing (scRNA-Seq) on primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Samples were processed to obtain single cell suspensions, followed by flow cytometry to enrich for CD41 positive cells and prepared for scRNA-Seq using the 10x genomics platform. scRNA-Seq analysis revealed a developmental trajectory starting from megakaryocyte erythroid progenitors (MEPs) and ending in two distinct clusters of mature MKs which were made up of mostly lung derived MKs (Fig. 1). One of these mature clusters was made up of 91% lung MKs and possessed a unique gene enrichment profile, including marked upregulation of platelet factor 4 (PF4) and enrichment for cellular processes related to ATP production and translation. To further profile lung-resident MKs, we performed flow cytometric assessment of ploidy and maturation markers. In comparison to bone marrow derived MKs, lung derived MKs displayed a distinct ploidy profile with a significantly increased number of cells expressing CD42, a marker of MK maturation. Collectively, these findings suggest that the subset of MKs found within the lung may be in a more mature and metabolically active state to potentially facilitate increased platelet output. These studies may shed new light on the process of MK maturation, including what triggers MK endoreplication, and may advance methodologies for in-vitro platelet production. Based on the symbiotic relationship between the lung and the hematopoietic system, our ongoing studies are focused upon the interplay between the lung and resident MKs in the developmental and functional patterning of both tissue types. Disclosures No relevant conflicts of interest to declare.

Defective Zn2+ homeostasis in mouse and human platelets with \u03b1- and \u03b4-storage pool diseases

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.