What It Takes To Be a Platelet: Evolving Concepts in Platelet Production.

Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.

GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Cell cycle-dependent centrosome clustering precedes proplatelet formation.

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

Research progress of megakaryocytes and platelets in lung injury.

The lung is an important site of extramedullary platelet formation, and megakaryocytes in the lung participate in immune responses in addition to platelet production. In acute lung injury and chronic lung injury, megakaryocytes and platelets play a promoting or protective role through different mechanisms. The authors reviewed the role of megakaryocytes and platelets in common clinical lung injuries with different course of disease and different pathogenic factors in order to provide new thinking for the diagnosis and treatment of lung injuries.

What is the context?Platelets are specialized non-nucleated blood cells produced by cytoplasmic lysis of megakaryocytes.HSCs differentiate into granular mature megakaryocytes and produce platelets.Lung is a reservoir of megakaryocytes and a site where platelets are produced in addition to bone marrow and spleen.Lung injury can be divided into acute lung injury and chronic lung injury, and characterized by different pathogenesis.Platelets and megakaryocytes are involved in hemostasis and regulation of the body 's inflammatory response.The disease state of the lung affects the functions of megakaryocytes and platelets.The role of megakaryocytes and platelets in acute and chronic lung injury is poorly studied.What is new?Platelets in the lung are derived not only from the spleen and bone marrow, but also from megakaryocytes in the pulmonary circulation. In this study, we demonstrated that pulmonary megakaryocytes not only produce platelets to play a hemostatic role in lung injury, but also participate in inflammation and immune response with platelets to promote the process of lung injury or play a protective role.Therefore, it was suggested in our analysis that targeting lung megakaryocytes and platelets is currently a new direction for the treatment of a variety of lung injuries.What is the impact?This review intends to explain the relationship between megakaryocytes, platelets and many types of lung injury from the mechanism of platelet production in the lung, and make a prospect in the new progress in the diagnosis and treatment of lung injury.

Dysmorphic megakaryocytes in TAFRO syndrome: A case series from a single institute.

TAFRO syndrome is a rare systemic inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. The diagnosis of TAFRO syndrome can be challenging; however, prompt diagnosis is vital because TAFRO syndrome is a progressive and life-threatening disease. We have showcased five patients with TAFRO syndrome who had similar bone marrow (BM) findings that could be considered the findings that characterize TAFRO syndrome. All patients were treated with corticosteroids and tocilizumab; three of the five patients (60 %) responded positively to the treatment. The unique BM findings observed in this study were megakaryocytes with distinct multinuclei and three-dimensional and indistinct bizarre nuclei ("dysmorphic megakaryocyte"), similar to the megakaryocyte morphology observed in myeloproliferative neoplasms (MPNs). Notably, dysmorphic megakaryocytes were observed in all five cases, whereas only two of the five patients tested positive for reticulin myelofibrosis, and three of the five patients had megakaryocytic hyperplasia, which are considered typical findings of TAFRO syndrome. Thus, the BM findings of dysmorphic megakaryocytes could help in the correct and immediate diagnosis of TAFRO syndrome.

G2 arrest primes hematopoietic stem cells for megakaryopoiesis.

In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool.

A circle of life: platelet and megakaryocyte cytoskeleton dynamics in health and disease.

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation.

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

BACKGROUND: Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. METHODS: A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. RESULTS: Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. CONCLUSIONS: Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

SDF-1 promotes metastasis of NSCLC by enhancing chemoattraction of megakaryocytes through the PI3K/Akt signaling pathway.

Lung cancer (LC) is the leading cause of cancer-associated deaths worldwide, among which non-small-cell lung cancer (NSCLC) accounts for 80%. Stromal cell-derived factor-1 (SDF-1) inhibition results in a significant depletion of NSCLC metastasis. Additionally, SDF-1 is the only natural chemokine known to bind and activate the receptor CXCR4. Thus, we attempted to clarify the molecular mechanism of SDF-1 underlying NSCLC progression. Transwell migration, adhesion, and G-LISA assays were used to assess megakaryocytic chemotaxis in vitro and in vivo in terms of megakaryocytic migration, adherence, and RhoA activation, respectively. Western blotting was used to assess PI3K/Akt-associated protein abundances in MEG-01 cells and primary megakaryocytes under the indicated treatment. A hematology analyzer and flow cytometry were used to assess platelet counts in peripheral blood and newly formed platelet counts in Lewis LC mice under different treatments. Immunochemistry and flow cytometry were used to measure CD41+ megakaryocyte numbers in Lewis LC mouse tissue under different treatments. ELISA was used to measure serum TPO levels, and H&E staining was used to detect NSCLC metastasis.SDF-1 receptor knockdown suppressed megakaryocytic chemotaxis in Lewis LC mice. SDF-1 receptor inhibition suppressed megakaryocytic chemotaxis via the PI3K/Akt pathway. SDF-1 receptor knockdown suppressed CD41+ megakaryocyte numbers in vivo through PI3K/Akt signaling. SDF-1 receptor inhibition suppressed CD41+ megakaryocytes to hinder NSCLC metastasis. SDF-1 facilitates NSCLC metastasis by enhancing the chemoattraction of megakaryocytes via the PI3K/Akt signaling pathway, which may provide a potential new direction for seeking therapeutic plans for NSCLC.

Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Packaging of supplemented urokinase into alpha granules of in vitro-grown megakaryocytes for targeted nascent clot lysis.

ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

Acidification of α-granules in megakaryocytes by vacuolar-type adenosine triphosphatase is essential for organelle biogenesis.

BACKGROUND: Platelets coordinate blood coagulation at sites of vascular injury and play fundamental roles in a wide variety of (patho)physiological processes. Key to many platelet functions is the transport and secretion of proteins packaged within α-granules, organelles produced by platelet precursor megakaryocytes. Prominent among α-granule cargo are fibrinogen endocytosed from plasma and endogenously synthesized von Willebrand factor. These and other proteins are known to require acidic pH for stable packaging. Luminal acidity has been confirmed for mature α-granules isolated from platelets, but direct measurement of megakaryocyte granule acidity has not been reported. OBJECTIVES: To determine the luminal pH of α-granules and their precursors in megakaryocytes and assess the requirement of vacuolar-type adenosine triphosphatase (V-ATPase) activity to establish and maintain the luminal acidity and integrity of these organelles. METHODS: Cresyl violet staining was used to detect acidic granules in megakaryocytes. Endocytosis of fibrinogen tagged with the pH-sensitive fluorescent dye fluorescein isothiocyanate was used to load a subset of these organelles. Ratiometric fluorescence analysis was used to determine their luminal pH. RESULTS: We show that most of the acidic granules detected in megakaryocytes appear to be α-granules/precursors, for which we established a median luminal pH of 5.2 (IQR, 5.0-5.5). Inhibition of megakaryocyte V-ATPase activity led to enlargement of cargo-containing compartments detected by fluorescence microscopy and electron microscopy. CONCLUSION: These observations reveal that V-ATPase activity is required to establish and maintain a luminal acidic pH in megakaryocyte α-granules/precursors, confirming its importance for stable packaging of cargo proteins such as von Willebrand factor.

Decreasing circ_0014614 promotes the differentiation of bone marrow flineage cells into megakaryocytes in essential thrombocythemia via activiation of miR-138-5p/caspase3 axis.

BACKGROUND: Circular RNAs (circRNA) are pivotal in hematological diseases. Previous study showed that circ_0014614 (circDAP3) was significantly underexpressed in bone marrow-derived exosomes from essential thrombocythemia (ET) patients, affecting the differentiation of bone marrow lineage cells into megakaryocytes. METHODS: Fluorescence in situ hybridization (FISH) was used to display circ_0014614's primary cytoplasmic location in K562 cells. Cytoscape software was used to predict the circRNA-miRNA-mRNA networks, and their expression at the cellular level was detected by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR was utilized to detect the expression levels of circ_0014614，miR-138-5p and caspase3 mRNA. Western blot was used to determine the protein levels of GATA-1, RUNX-1, NF-E2, CD41 and caspase3. The proliferation of K562 cells was assessed using the Cell Counting Kit-8 (CCK-8) Assay. Furthermore, the interplay between miR-138-5p and circ_0014614 or caspase3 was elucidated through a Dual-luciferase reporter assay. RESULTS: FISH assay indicated circ_0014614's primary cytoplasmic location in K562 cells. In ET bone marrow and K562 cells, circ_0014614 and caspase3 were down-regulated, whereas miR-138-5p saw a significant surge. Overexpressing circ_0014614 curtailed K562 cells' proliferation and differentiation. Further, circ_0014614 targeted miR-138-5p, with heightened miR-138-5p levels counteracting circ_0014614's inhibition. MiR-138-5p further targeted caspase3, and caspase3 silencing neutralized suppressed miR-138-5p's effects on K562 cell differentiation. CONCLUSION: Circ_0014614 was down-regulated in ET bone marrow and bone marrow lineage cells, and upregulating circ_0014614 can inhibit bone marrow lineage cells' proliferation and differentiation into megakaryocytes. Mechanistically, circ_0014614 functioned as ceRNA via sponging miR-138-5p and alleviated the inhibitory effect of miR-138-5p on its target caspase3, which potentially deters tumor activity in ET.