Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia.

Thrombocytopenia caused by long-term radiotherapy and chemotherapy exists in cancer treatment. Previous research demonstrates that 5-Hydroxtrayptamine (5-HT) and its receptors induce the formation of megakaryocytes (MKs) and platelets. However, the relationships between 5-HT1A receptor (5-HTR1A) and MKs is unclear so far. We screened and investigated the mechanism of vilazodone as a 5-HTR1A partial agonist in promoting MK differentiation and evaluated its therapeutic effect in thrombocytopenia. We employed a drug screening model based on machine learning (ML) to screen the megakaryocytopoiesis activity of Vilazodone (VLZ). The effects of VLZ on megakaryocytopoiesis were verified in HEL and Meg-01 cells. Tg (itga2b: eGFP) zebrafish was performed to analyze the alterations in thrombopoiesis. Moreover, we established a thrombocytopenia mice model to investigate how VLZ administration accelerates platelet recovery and function. We carried out network pharmacology, Western blot, and immunofluorescence to demonstrate the potential targets and pathway of VLZ. VLZ has been predicted to have a potential biological action. Meanwhile, VLZ administration promotes MK differentiation and thrombopoiesis in cells and zebrafish models. Progressive experiments showed that VLZ has a potential therapeutic effect on radiation-induced thrombocytopenia in vivo. The network pharmacology and associated mechanism study indicated that SRC and MAPK signaling are both involved in the processes of megakaryopoiesis facilitated by VLZ. Furthermore, the expression of 5-HTR1A during megakaryocyte differentiation is closely related to the activation of SRC and MAPK. Our findings demonstrated that the expression of 5-HTR1A on MK, VLZ could bind to the 5-HTR1A receptor and further regulate the SRC/MAPK signaling pathway to facilitate megakaryocyte differentiation and platelet production, which provides new insights into the alternative therapeutic options for thrombocytopenia.

Differentiation route determines the functional outputs of adult megakaryopoiesis.

Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.

A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility.

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

The bone marrow is the primary site of thrombopoiesis.

ABSTRACT: Megakaryocytes (MKs) generate thousands of platelets over their lifespan. The roles of platelets in infection and inflammation has guided an interest to the study of extramedullary thrombopoiesis and therefore MKs have been increasingly reported within the spleen and lung. However, the relative abundance of MKs in these organs compared to the bone marrow and the scale of their contribution to the platelet pool in a steady state remain controversial. We investigated the relative abundance of MKs in the adult murine bone marrow, spleen, and lung using whole-mount light-sheet and quantitative histological imaging, flow cytometry, intravital imaging, and an assessment of single-cell RNA sequencing (scRNA-seq) repositories. Flow cytometry revealed significantly higher numbers of hematopoietic stem and progenitor cells and MKs in the murine bone marrow than in spleens or perfused lungs. Two-photon intravital and light-sheet microscopy, as well as quantitative histological imaging, confirmed these findings. Moreover, ex vivo cultured MKs from the bone marrow subjected to static or microfluidic platelet production assays had a higher capacity for proplatelet formation than MKs from other organs. Analysis of previously published murine and human scRNA-seq data sets revealed that only a marginal fraction of MK-like cells can be found within the lung and most likely only marginally contribute to platelet production in the steady state.

Discovery of seven hox genes in zebrafish thrombopoiesis.

Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b, hoxb2a, hoxc5a, hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b, hoxb2a, hoxc5a, and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a, and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b, as a negative regulator for thrombocyte development.

The role of PKC in X-ray-induced megakaryocyte apoptosis and thrombocytopenia.

Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.

Wnt/ß-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System.

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/ß-catenin for MK and RBC differentiation, we activated ß-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, ß-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.

Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells.

The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.

Megakaryocyte- and erythroblast-specific cell-free DNA patterns in plasma and platelets reflect thrombopoiesis and erythropoiesis levels.

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.

Humanized mouse models for inherited thrombocytopenia studies.

Inherited thrombocytopenia (IT) is a group of hereditary disorders characterized by a reduced platelet count as the main clinical manifestation, and often with abnormal platelet function, which can subsequently lead to impaired hemostasis. In the past decades, humanized mouse models (HMMs), that are mice engrafted with human cells or genes, have been widely used in different research areas including immunology, oncology, and virology. With advances of the development of immunodeficient mice, the engraftment, and reconstitution of functional human platelets in HMM permit studies of occurrence and development of platelet disorders including IT and treatment strategies. This article mainly reviews the development of humanized mice models, the construction methods, research status, and problems of using humanized mice for the in vivo study of human thrombopoiesis.

Humanized mouse models (HMMs) refer to immunodeficient mice that have been used for the investigation of human hematopoiesis and immunity for years. With engrafted human hematopoietic stem cells (HSCs), the differentiation process of HSCs and re-construction of platelets can be monitored in the mice. Until now, several strains of HMMs have been used in the studies of inherited thrombocytopenia (IT), a genetic disorder associated with low platelet count in the blood. In this study, we reviewed the development of these HMMs in IT studies, compared the different sources of HSCs transplanted into HMMs and summarize the strategies of HSC transplantation in HMMs. The Kit^−/− immunodeficient mice showed effectively long-term and stable implantation of human HSC without irradiation and higher implantation levels, and they also support multilinear differentiation of human HSC, such as platelets and red blood cells. The source and count of HSCs and the transplantation strategy may also impact the result. This study provides a basis information for HMMs used in IT and will help other investigators in this field choosing the right research plan.

Synergistic roles of DYRK1A and GATA1 in trisomy 21 megakaryopoiesis.

Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild-type GATA1 (wtGATA1) or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and derepression of its downstream targets. Notably, DYRK1A loss had no effect in T21 iPSCs or megakaryocytes with wtGATA1. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in T21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.

Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding.

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.

Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis.

Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b, and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b, and mir-223 knockdowns. These results suggested mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa, and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. We also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.

Diversity of Megakaryocytes.

Megakaryocytes are commonly known as large, polyploid, bone marrow resident cells that contribute to hemostasis through the production of platelets. Soon after their discovery in the 19th century, megakaryocytes were described in tissue locations other than the bone marrow, specifically in the lungs and the blood circulation. However, the localization of megakaryocytes in the lungs and the contribution of lung megakaryocytes to the general platelet pool has only recently been appreciated. Moreover, the conception of megakaryocytes as uniform cells with the sole purpose of platelet production has been challenged. Here, we review the literature on megakaryocyte cell identity and location with a special focus on recent observations of megakaryocyte subpopulations identified by transcriptomic analyses.

Decreased cyclooxygenase-2 associated with impaired megakaryopoiesis and thrombopoiesis in primary immune thrombocytopenia.

BACKGROUND: Cyclooxygenase (COX)-2 is a rate-limiting enzyme in the biosynthesis of prostanoids, which is mostly inducible by inflammatory cytokines. The participation of COX-2 in the maturation of megakaryocytes has been reported but barely studied in primary immune thrombocytopenia (ITP). METHODS: The expressions of COX-2 and Caspase-1, Caspase-3 and Caspase-3 p17 subunit in platelets from ITP patients and healthy controls (HC), and the expressions of COX-2 and CD41 in bone marrow (BM) of ITP patients were measured and analyzed for correlations. The effects of COX-2 inhibitor on megakaryopoiesis and thrombopoiesis were assessed by in vitro culture of Meg01 cells and murine BM-derived megakaryocytes and in vivo experiments of passive ITP mice. RESULTS: The expression of COX-2 was decreased and Caspase-1 and Caspase-3 p17 were increased in platelets from ITP patients compared to HC. In platelets from ITP patients, the COX-2 expression was positively correlated with platelet count and negatively correlated to the expression of Caspase-1. In ITP patients BM, the expression of CD41 was positively correlated with the expression of COX-2. COX-2 inhibitor inhibited the count of megakaryocytes and impaired the maturation and platelet production in Meg01 cells and bone marrow-derived megakaryocytes. COX-2 inhibitor aggravated thrombocytopenia and damaged megakaryopoiesis in ITP murine model. CONCLUSION: COX-2 plays a vital role in the physiologic and pathologic conditions of ITP by intervening the survival of platelets and impairing the megakaryopoiesis and thrombopoiesis of megakaryocytes.

Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

Development of an efficient, ready to use, blood platelet-release device based on two new flow regime parameters: The periodic hydrodynamic loading and the shear stress accumulation.

In vitro production of blood platelets for transfusion purposes is gaining interest. While platelet production is now possible on a laboratory scale, the challenge is to move towards industrial production. Attaining this goal calls for the development of platelet release devices capable of producing large quantities of platelets. To this end, we have developed a continuous-flow platelet release device composed of five spherical chambers each containing two calibrated cones placed in a staggered configuration. Following perfusion of proplatelet-bearing cultured megakaryocytes, the device achieves a high yield of about 100 bona-fide platelets/megakaryocyte, at a flow rate of ∼80 mL/min. Performances and operating conditions comply with the requirements of large-scale platelet production. Moreover, this device enabled an in-depth analysis of the flow regimes through Computational Fluid Dynamics (CFD). This revealed two new universal parameters to be taken into account for an optimal platelet release: i.e. a periodic hydrodynamic load and a sufficient accumulation of shear stress. An efficient 16 Pa.s shear stress accumulation is obtained in our system at a flow rate of 80 mL/min.

The role of CREG1 in megakaryocyte maturation and thrombocytopoiesis.

Abnormal megakaryocyte maturation and platelet production lead to platelet-related diseases and impact the dynamic balance between hemostasis and bleeding. Cellular repressor of E1A-stimulated gene 1 (CREG1) is a glycoprotein that promotes tissue differentiation. However, its role in megakaryocytes remains unclear. In this study, we found that CREG1 protein is expressed in platelets and megakaryocytes and was decreased in the platelets of patients with thrombocytopenia. A cytosine arabinoside-induced thrombocytopenia mouse model was established, and the mRNA and protein expression levels of CREG1 were found to be reduced in megakaryocytes. We established megakaryocyte/platelet conditional knockout (Creg1pf4-cre) and transgenic mice (tg-Creg1). Compared to Creg1fl/fl mice, Creg1pf4-cre mice exhibited thrombocytopenia, which was mainly caused by inefficient bone marrow (BM) thrombocytopoiesis, but not by apoptosis of circulating platelets. Cultured Creg1pf4-cre-megakaryocytes exhibited impairment of the actin cytoskeleton, with less filamentous actin, significantly fewer proplatelets, and lower ploidy. CREG1 directly interacts with MEK1/2 and promotes MEK1/2 phosphorylation. Thus, our study uncovered the role of CREG1 in the regulation of megakaryocyte maturation and thrombopoiesis, and it provides a possible theoretical basis for the prevention and treatment of thrombocytopenia.