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Supplementary Materials and Methods, Supplementary Figures S1-S12

Supplementary Table 3 HD and MPN samples.

Supplementary Table 1 VEGFAC Top Differentially Expressed Genes by Cluster

MM ALL Donor Details

Supplementary Table 2 Gene sets for GSEA

Supplementary Table 6 qRT PCR Probes

Supplementary Table 5 Antibodies

Supplementary Table 7 NGS Panel

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow—stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor–derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow–like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers.Significance:We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics.See related commentary by Derecka and Crispino, p. 263.This article is highlighted in the In This Issue feature, p. 247

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

Introduction The thrombin generation (TG) test is a global hemostasis assay sensitive to procoagulant conditions. However, some TG assays may underestimate elevated TG when the thrombin fluorogenic substrate is depleted or fluorescence is attenuated by the inner filter effect (IFE). Objectives We sought to elucidate the extent to which procoagulant conditions require correcting for fluorogenic substrate depletion and/or IFE. Methods We analyzed corrections for substrate depletion and IFE and their effect on TG parameters in plasma samples with elevated blood coagulation factors in the presence or absence of thrombomodulin via commercial calibrated automated thrombogram (CAT) platform and in‐house software capable of internal thrombin calibration with or without CAT‐like artifact correction. Results Elevated thrombin peak height (TPH) and endogenous thrombin potential (ETP) were detected with 2× and 4× increases in blood coagulation factors I, V, VIII, IX, X, and XI, or prothrombin in the presence or absence of artifact correction. The effect of the CAT algorithm was evident in TG curves from both low procoagulant (thrombomodulin‐supplemented) and procoagulant (factor‐supplemented) plasma samples. However, in all samples, with the exception of elevated prothrombin, CAT’s correction was small (

The traditional role of platelets is in the formation of blood clots for physiologic (e.g., in hemostasis) or pathologic (e.g., in thrombosis) functions. The cellular and subcellular mechanisms and signaling in platelets involved in these functions have been extensively elucidated and new knowledge continues to emerge, resulting in various therapeutic developments in this area for the management of hemorrhagic or thrombotic events. Nanomedicine, a field involving design of nanoparticles with unique biointeractive surface modifications and payload encapsulation for disease-targeted drug delivery, has become an important component of such therapeutic development. Beyond their traditional role in blood clotting, platelets have been implicated to play crucial mechanistic roles in other diseases including inflammation, immune response, and cancer, via direct cellular interactions, as well as secretion of soluble factors that aid in the disease microenvironment. To date, the development of nanomedicine systems that leverage these broader roles of platelets has been limited. Additionally, another exciting area of research that has emerged in recent years is that of platelet-derived extracellular vesicles (PEVs) that can directly and indirectly influence physiological and pathological processes. This makes PEVs a unique paradigm for platelet-inspired therapeutic design. This review aims to provide mechanistic insight into the involvement of platelets and PEVs beyond hemostasis and thrombosis, and to discuss the current state of the art in the development of platelet-inspired therapeutic technologies in these areas, with an emphasis on future opportunities.

The 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 that originate from the MK cell body. Proplatelet formation is a highly dynamic and asynchronous process which poses unique challenges for researchers to accurately capture and analyze. We have designed an open-source, high-content, high-throughput, label-free analysis platform. Phase-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 then processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphologic parameters. CellProfiler Analyst, provides a final, supervised, machine learning classification to bolster accurate identification of cellular structures. This entire workflow yields the percent of proplatelet production, area, and count of proplatelets and MKs, as well as 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. This 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.

Megakaryocytes (MKs) are relatively rare in bone marrow, comprising

During inflammation, steady-state hematopoiesis switches to emergency hematopoiesis to repopulate myeloid cells, with a bias toward the megakaryocytic lineage. Soluble inflammatory cues are thought to be largely responsible for these alterations. However, how these plasma factors rapidly alter the bone marrow (BM) is not understood. Inflammation also drives platelet activation, causing the release of platelet-derived extracellular vesicles (PEVs), which package diverse cargo and reprogram target cells. We hypothesized that PEVs infiltrate the BM, providing a direct mode of communication between the plasma and BM environments. We transfused fluorescent, wild-type (MPL+) platelets into recipient cMpl−/−mice before triggering systemic inflammation. Twenty hours postinfusion, we observed significant infiltration of donor platelet-derived particles in the BM, which we tracked immunophenotypically (MPL+ immunohistochemistry staining) and quantified by flow cytometry. To determine if this phenomenon relates to humans, we extensively characterized both megakaryocyte-derived and PEVs generated in vitro and in vivo, and found enrichment of extracellular vesicles in bone marrow compared with autologous peripheral blood. Last, BM from cMpl−/− mice was cultured in the presence or absence of wild-type (MPL+) PEVs. After 72 hours, flow cytometry revealed increased megakaryocytes only in cultures with added PEVs. The majority of CD41+ cells were bound to PEVs, suggesting a PEV-mediated rescue of megakaryopoiesis. In conclusion, we report for the first time that plasma-residing PEVs infiltrate the BM. Further, PEVs interact with BM cells in vivo and in vitro, causing functional reprogramming that may represent a novel model of inflammation-induced hematopoiesis.

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from iPSCs committed to mesenchymal, endothelial and hematopoietic lineages. These 3-dimensional structures capture key features of human bone marrow - stroma, lumen-forming sinusoidal vessels and myeloid cells including pro-platelet forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate discovery and prioritization of novel targets for bone marrow disorders and blood cancers.Significance StatementWe present a 3D, vascularised human bone marrow organoid that supports growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment, and provides a much-needed, ex vivo tool for prioritization of new therapeutics.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress. ispartof: RESEARCH AND PRACTICE IN THROMBOSIS AND HAEMOSTASIS vol:5 issue:5 ispartof: location:United States status: published

Megakaryocytes (MKs) are widely known as the progenitor cells of platelets. These large, polyploid cells are a derivative of the hematopoietic stem cell (HSC), and reside in the bone marrow, lining blood vessel walls where they release their platelet progeny into circulation. Although little is known about how MKs differ under various environmental stressors, both chronic and acute inflammation alter the differentiation and molecular content of MKs. Furthermore, evidence suggests that the release of inflammatory cytokines may induce MK rupture and rapid release of platelets as a mechanism to quickly replenish diminished platelet counts in response to inflammation. Similarities between MKs and their close relatives, white blood cells, have introduced the notion that MKs may play a role in combating infection by engulfing and presenting antigens, and passing this information to circulating platelets. In addition, MKs exposed to varying bone marrow environments produce different platelets which enter circulation primed to respond to and combat inflammation, infection, or injury. This review focuses on how inflammation alters MK production, maturation, and platelet production. In addition, it introduces the idea that inflammation reprograms MKs to create different, more pathogenic platelets and leads them to take on different roles as responders to deleterious conditions. In the future, studies determining how platelets are altered in disease states may lead to novel MK- and platelet-based therapeutic targets to mitigate inflammation-related morbidity and mortality.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow–resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term Mk(L), had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an Mk(L)-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that Mk(L) internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, Mk(L) induced CD4(+) T cell activation in an MHC II–dependent manner both in vitro and in vivo. These data indicated that Mk(L) had key immune regulatory roles dictated in part by the tissue environment.

Extracellular vesicles (EVs) are a means of cell-to-cell communication and can facilitate the exchange of a broad array of molecules between adjacent or distant cells. Platelets are anucleate cells derived from megakaryocytes and are primarily known for their role in maintaining hemostasis and vascular integrity. Upon activation by a variety of agonists, platelets readily generate EVs, which were initially identified as procoagulant particles. However, as both platelets and their EVs are abundant in blood, the role of platelet EVs in hemostasis may be redundant. Moreover, findings have challenged the significance of platelet-derived EVs in coagulation. Looking beyond hemostasis, platelet EV cargo is incredibly diverse and can include lipids, proteins, nucleic acids, and organelles involved in numerous other biological processes. Furthermore, while platelets cannot cross tissue barriers, their EVs can enter lymph, bone marrow, and synovial fluid. This allows for the transfer of platelet-derived content to cellular recipients and organs inaccessible to platelets. This review highlights the importance of platelet-derived EVs in physiological and pathological conditions beyond hemostasis.

Background: The thrombin generation (TG) test is useful for characterizing global hemostasis potential, but fluorescence substrate artifacts, such as thrombin-&alpha, 2macroglobulin (T-&alpha, 2MG) signal, inner filter effect (IFE), substrate consumption, and calibration algorithms have been suggested as sources of intra- and inter-laboratory variance, which may limit its clinical utility. Methods: Effects of internal vs. external normalization, IFE and T-&alpha, 2MG on TG curves in normal plasma supplemented with coagulation factors, thrombomodulin, and tissue factor were studied using the Calibrated Automated Thrombinography (CAT, Diagnostica Stago, Parsippany, NJ, USA) and in-house software. Results: The various calibration methods demonstrated no significant difference in producing TG curves, nor increased the robustness of the TG assay. Several TG parameters, including thrombin peak height (TPH), produced from internal linear calibration did not differ significantly from uncalibrated TG parameters. Further, TPH values from internal linear and nonlinear calibration with or without T-&alpha, 2MG correction correlated well with TPH from external calibration. Higher coefficients of variation (CVs) for TPH values were observed in both platelet-free and platelet-rich plasma with added thrombomodulin. Conclusions: Our work suggests minimal differences between distinct computational approaches toward calibrating and correcting fluorescence signals into TG levels, with most samples returning similar or equivalent TPH results.

Essentials Identifying genetic variants in platelet disorders is challenging due to its heterogenous nature. We combine WES, RNAseq, and python-based bioinformatics to identify novel gene variants. We find novel candidates in patient data by cross-referencing against a murine RNAseq model of thrombopoiesis. This innovative combined bioinformatic approach provides novel data for future research in the field. ABSTRACT: Background The UK Genotyping and Phenotyping of Platelets study has recruited and analyzed 129 patients with suspected heritable bleeding. Previously, 55 individuals had a definitive genetic diagnosis based on whole exome sequencing (WES) and platelet morphological and functional testing. A significant challenge in this field is defining filtering criteria to identify the most likely candidate mutations for diagnosis and further study. Objective Identify candidate gene mutations for the remaining 74 patients with platelet-based bleeding with unknown genetic cause, forming the basis of future re-recruitment and further functional testing and assessment. Methods Using python-based data frame indexing, we first identify and filter all novel and rare variants using a panel of 116 genes known to cause bleeding across the full cohort of WES data. This identified new variants not previously reported in this cohort. We then index the remaining patients, with rare or novel variants in known bleeding genes against a murine RNA sequencing dataset that models proplatelet-forming megakaryocytes. Results Filtering against known genes identified candidate variants in 59 individuals, including novel variants in several known genes. In the remaining cohort of "unknown" patients, indexing against differentially expressed genes revealed candidate gene variants in several novel unreported genes, focusing on 14 patients with a severe clinical presentation. Conclusions We identified candidate mutations in a cohort of patients with no previous genetic diagnosis. This work involves innovative coupling of RNA sequencing and WES to identify candidate variants forming the basis of future study in a significant number of undiagnosed patients.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.

Essentials Factor VIIa binds activated platelets to promote hemostasis in hemophilia patients with inhibitors. The interactions and sites responsible for platelet-FVIIa binding are not fully understood. Endothelial cell protein C receptor (EPCR) is expressed on activated human platelets. EPCR binding enhances the efficacy of a FVIIa variant and could impact design of new therapeutics. Summary Background High-dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa-DVQ is an analog of FVIIa with enhanced tissue factor (TF)-independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa-DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet-FVIIa interactions. Objectives Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet-FVIIa binding. Methods/results In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa-DVQ is partially inhibited in the presence of excess protein C or an anti-EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa-DVQ compared with wild-type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry. Conclusions This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

Significance Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs in the first step of protein synthesis in the cytoplasm. However, in higher eukaryotes, they acquired additional functions beyond translation. In the present study, we show that an activated form of tyrosyl-tRNA synthetase (YRSACT) functions to enhance megakaryopoiesis and platelet production in vitro and in vivo. These findings were confirmed with human megakaryocytes differentiated from peripheral blood CD34+ hematopoietic stem cells and with human induced pluripotent stem (iPS) cells. The activity of YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from iPS cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. These findings demonstrate a previously unrecognized function of an aaRS which may have implications for therapeutic interventions., New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.

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. Disclosures Machlus: KEROS Therapeutics: Consultancy, Honoraria; STRM.BIO: Consultancy, Honoraria.

Megakaryocytes are hematopoietic cells, which are responsible for the production of blood platelets. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells, through a hierarchical series of progenitor cells, ultimately to a mature megakaryocyte. Once mature, the megakaryocyte then undergoes a terminal maturation process involving multiple rounds of endomitosis and cytoplasmic restructuring to allow platelet formation. However, recent studies have begun to redefine this hierarchy and shed new light on alternative routes by which hematopoietic stem cells are differentiated into megakaryocytes. In particular, the origin of megakaryocytes, including the existence and hierarchy of megakaryocyte progenitors, has been redefined, as new studies are suggesting that hematopoietic stem cells originate as megakaryocyte-primed and can bypass traditional lineage checkpoints. Overall, it is becoming evident that megakaryopoiesis does not only occur as a stepwise process, but is dynamic and adaptive to biological needs. In this review, we will reexamine the canonical dogmas of megakaryopoiesis and provide an updated framework for interpreting the roles of traditional pathways in the context of new megakaryocyte biology. Visual Overview— An online visual overview is available for this article.

SummaryBone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the β2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated marrowin vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of inflammation associated with platelet overproduction, contributing to platelet productionin vitroandin vivo.These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the β2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated murine marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of murine inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

It is now recognized that compounds released from tumor cells can activate platelets, causing the release of platelet-derived factors into the tumor microenvironment. Several of these factors have been shown to directly promote neovascularization and metastasis, yet how the feedback between platelet releasate and the tumor cell affects metastatic phenotype remains largely unstudied. Here, we identify that breast tumor cells secrete high levels of interleukin 8 (IL-8, CXCL8) in response to platelet releasate, which promotes their invasive capacity. Furthermore, we found that platelets activate the Akt pathway in breast tumor cells, and inhibition of this pathway eliminated IL-8 production. We therefore hypothesized inhibiting platelets with aspirin could reverse the prometastatic effects of platelets on tumor cell signaling. Platelets treated with aspirin did not activate the Akt pathway, resulting in reduced IL-8 secretion and impaired tumor cell invasion. Of note, patients with breast cancer receiving aspirin had lower circulating IL-8, and their platelets did not increase tumor cell invasion compared with patients not receiving aspirin. Our data suggest platelets support breast tumor metastasis by inducing tumor cells to secrete IL-8. Our data further support that aspirin acts as an anticancer agent by disrupting the communication between platelets and breast tumor cells.

Megakaryocytes (MKs) are specialized precursor cells committed to producing and proliferating platelets. In a cytoskeletal-driven process, mature MKs generate platelets by releasing thin cytoplasmic extensions, named proplatelets, into the sinusoids. Due to knowledge gaps in this process and mounting clinical demand for non-donor-based platelet sources, investigators are successfully developing artificial culture systems to recreate the environment of platelet biogenesis. Nevertheless, drawbacks in current methods entail elaborate procedures for stem cell enrichment, extensive growth periods, low MK yield, and poor proplatelet production. We propose a simple, robust method of primary MK culture that utilizes fetal livers from pregnant mice. Our technique reduces expansion time to 4 days, and generates ~15,000-20,000 MKs per liver. Approximately, 20-50% of these MKs produce structurally dense, high-quality proplatelets. In this review, we outline our method of MK culture and isolation.

PURPOSE OF REVIEW Platelets are small, anucleate cells that circulate within the blood and play essential roles in preserving vascular integrity. However, abnormalities in either platelet production or destruction can result in thrombocytopenia, clinically defined by a platelet count lower than 150 000/μL of whole blood. Thrombocytopenia is frequently associated with impaired hemostatic responses to vascular injury and can be life-threatening because of bleeding complications. Megakaryocytes are the precursor cells responsible for platelet production, a process commonly referred to as thrombopoiesis. This review specifically discusses how perturbation of molecular mechanisms governing megakaryocyte differentiation and development manifest in various forms of thrombocytopenia. RECENT FINDINGS This review highlights the identification of novel transcriptional regulators of megakaryocyte maturation and platelet production. We also provide an update into the essential role of cytoskeletal regulation in thrombopoiesis, and how both megakaryopoiesis and platelet production are altered by anticancer therapeutics. Lastly, we focus on recent investigative approaches to treat thrombocytopenia and discuss future prospects in the field of megakaryocyte research. SUMMARY In patients where thrombocytopenia is not due to heightened platelet destruction or clearance, defects in megakaryocyte development should be considered.

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

In this issue of Blood, Chiu et al report the presence of functional redundancy between the transcription factors lymphoblastic leukemia 1 protein (LYL1) and stem cell leukemia protein (SCL) in the process of megakaryocyte (MK) maturation and platelet production in mice.1 This discovery may provide an answer to the long-standing mystery of why mutations in SCL protein do not present with any phenotypic platelet disorder, whereas mutations in its binding partner proteins GATA1, FLI1, RUNX1, and GFI1B cause hereditary thrombocytopenia.

Actin reorganization regulates key processes in platelet activation. Here we examined the role of the Arp2/3 complex, an essential component in actin filament branching, in platelet function. The Arpc2 gene, encoding the p34 subunit of the Arp2/3 complex, was deleted in the megakaryocyte lineage ( Arpc2 fl/fl PF4-Cre ). Deletion of the Arp2/3 complex resulted in marked microthrombocytopenia in mice, caused by premature platelet release into the bone marrow compartment and impaired platelet survival in circulation. Arpc2 fl/fl PF4-Cre platelets exhibited alterations in their actin cytoskeleton and their peripheral microtubule coil. Thrombocytopenia was alleviated following clodronate liposome-induced macrophage depletion in Arpc2 fl/fl PF4-Cre mice. Arpc2 fl/fl PF4-Cre platelets failed to spread and showed a mild defect in integrin activation and aggregation; however, no significant differences in hemostasis or thrombosis were observed between Arpc2 fl/fl PF4-Cre and control mice. Thus, Arp2/3 is critical for platelet homeostasis but plays only a minor role for vascular hemostasis.