ABSTRACT:Chronic kidney disease (CKD) is associated with an increased risk of thrombotic events, yet the underlying mechanisms driving platelet dysfunction remain incompletely understood. Historically, platelet abnormalities in CKD have been attributed to circulating uremic toxins; however, the contribution of the bone marrow microenvironment and megakaryocyte biology has not been fully explored. In this study, we used a murine model of CKD induced by aristolochic acid to investigate the impact of CKD on megakaryopoiesis and platelet production. CKD mice developed hallmark features of kidney dysfunction, including anemia, proteinuria, hypertension, and elevated creatinine. Histological analysis revealed a hypocellularity and decreased megakaryocyte density, whereas early hematopoietic progenitors were preserved. CKD-derived megakaryocytes displayed a distinct proteomic signature enriched for metabolic stress and hemostatic pathways. Functionally, bone marrow supernatant from CKD mice enhanced proplatelet formation in vitro, consistent with elevated platelet counts observed in vivo. Notably, CKD platelets exhibited a hyperreactive phenotype, characterized by increased integrin αIIbβ3 activation, degranulation, and enhanced aggregation in response to agonists. Together, these findings support a dual mechanism of platelet dysfunction in CKD: intrinsic reprogramming of megakaryocytes within the diseased bone marrow microenvironment and extrinsic priming by prothrombotic bone marrow factors. This study provides new insight into the hematopoietic origins of platelet abnormalities in CKD and underscores the importance of targeting bone marrow pathology to mitigate cardiovascular risk in this population.

Neutrophils are phenotypically heterogenous cells that mediate host defense and tissue homeostasis. Here, we identify emperipolesis - the evolutionarily conserved process by which neutrophils pass through megakaryocytes - as a phenotypically transformative route of egress from bone marrow. By intravital microscopy and 3-D histology, we show that the rapid form emperipolesis is markedly enhanced under inflammatory conditions. Neutrophils exit from megakaryocytes directly to the blood, acquiring exosomes enriched in proteins related to metabolism, migration, and immune function. This transfer induces a distinct neutrophil phenotype characterized by enhanced glycolysis, oxidative phosphorylation, cytokine release, and longevity. Correspondingly, emperipolesis-educated neutrophils display accelerated migration in vitro and in vivo. Disrupting emperipolesis does not alter circulating neutrophil abundance but impairs neutrophil infiltration into inflamed tissues, including Pseudomonas aeruginosa-infected lung. These findings establish emperipolesis as a mechanism by which megakaryocytes amplify neutrophil-mediated immunity.

AIMS:Hypoxia is associated with thrombosis, yet its effects on platelet function remain controversial due to variable experimental conditions. In this study, we systematically examined the effects of graded normobaric hypoxia on platelet function and arterial thrombosis in mice. METHODS AND RESULTS:Exposure to 8% O2 for 6 days increased red blood cell counts, haemoglobin, and haematocrit, recapitulating human hypoxic adaptation. Platelets from hypoxic mice show reduced activation and aggregation in response to multiple agonists, including thrombin and collagen-related peptide, with defects accentuated by more severe (8% vs. 12% O2) or prolonged (6-day vs. 4-day) hypoxia and most pronounced after glycoprotein VI (GPVI) stimulation. Similar platelet dysfunction occurs in humans after 4-day hypoxia (∼12% O2) followed by 4-day normoxia. Quantitative mass spectrometry identifies down-regulation of lysozyme, Hsp90 signalling molecules, and GPVI. Pathway and systems-level network analyses reveal significant down-regulation of Hsp90-involved protein folding and GPVI-mediated signalling pathways. Deletion of platelet lysozyme or Hsp90β, however, minimally affects platelet function, indicating regulation by multiple signalling pathways under hypoxia. We confirmed that GPVI levels are significantly reduced in platelets, while ADAM10 levels remain unchanged, suggesting hypoxia-induced GPVI down-regulation. Hypoxic blood forms smaller thrombi on collagen under arterial shear. Paradoxically, hypoxic mice exhibit enhanced arterial thrombosis and shortened tail bleeding times. Biochemical and mass spectrometry analyses of mouse plasma demonstrate elevated circulating von Willebrand factor under hypoxia. CONCLUSION:These results uncover a paradox under hypoxia: despite GPVI down-regulation and impaired agonist-induced platelet activation, elevated plasma von Willebrand factor drives enhanced arterial thrombosis. This multilayered regulation provides mechanistic insight into hypoxia-associated thrombotic risk and may inform therapeutic strategies.

ABSTRACT:Megakaryocytes (MKs) are large, hematopoietic cells with a polyploid, multilobulated nucleus. Although DNA replication in MKs (endomitosis) is well studied, limited investigations have examined the impact of DNA instability on megakaryopoiesis. Poly-adenosine diphosphate (ADP) ribose polymerase (PARP) inhibitors are chemotherapeutics that result in accumulation of DNA damage and are commonly associated with thrombocytopenia, presumably mediated through platelet progenitors, MKs. To explore PARP inhibitor-induced thrombocytopenia, we treated mice with the PARP inhibitor niraparib. Although high-dose niraparib treatment led to thrombocytopenia, consistent with clinical observations, lower-dose treatment led to a significant increase in bone marrow MKs, MK progenitors (MkPs), and circulating platelets. This increase was accompanied by elevated DNA damage in both MKs and MkPs, as measured by γH2AX accumulation and comet assays. Notably, platelets from niraparib-treated mice were functionally normal in their response to ADP, thrombin receptor activating peptide, and collagen. Treatment of mice with low-dose gamma irradiation similarly led to DNA damage in MKs and resulted in increased MK and platelet counts, suggesting that moderate DNA damage is a conserved mechanism that enhances megakaryopoiesis and platelet counts. These data reveal a previously unknown relationship between MKs and DNA damage and present a novel target for triggering enhanced platelet production in vivo.

Platelets are blood components not regularly analysed with proteomics due to the conventional wisdom that plasma for platelet research must be citrate-treated and freshly sampled to minimize artifactual changes. Information from platelets is complementary to plasma, specifically regarding immunophenotyping of patients with challenged immune systems due to infection or inflammation. We sought to develop a sample-sparing, high-throughput-compatible platelet proteomics workflow applicable to previously frozen, non-citrate platelet-rich plasma, in contrast to the field's standard, and test its efficacy by applying it to a COVID-19 cohort. We examined centrifugation of whole blood and platelet-rich plasma and volume requirements of plasma for platelet analysis. Platelet and platelet-poor plasma samples were analysed from a cohort of 79 patients, consisting of COVID-19 negative non-ICU and ICU controls and patients with COVID-19 over time. Conventional platelet count was successfully performed using flow cytometry on previously frozen plasma, showing minimal platelet aggregation and cell debris, demonstrating viability of previously frozen plasma for platelet proteomic analysis. Protein counts in platelets mostly mirrored trends in platelet count, except in severe COVID-19 patients within three days of admission to the ICU. Proteins dysregulated in this group compared to controls were enriched in terms related to platelet activation, phagosome, and efferocytosis. This agrees with prior reports using conventional platelet proteomics methods. Such similar findings suggest that the method developed here can utilize non-citrate, previously frozen plasma down to 0.5 μL per sample. This will make platelet proteomics studies on already collected, banked plasma samples more accessible and increase biomolecular information gained.

During inflammation, megakaryocytes (MKs) can expand from hematopoietic stem cells, yet the specific driving factors remain unclear. We investigated whether C-C Motif Chemokine Ligand 5 (CCL5) influences this process in murine and human iPSC-derived bone marrow organoid (BMO) models. Acute CCL5 administration to mice increased hematopoietic stem and progenitor cell (HSPC) populations and increased MKs, including the expansion of MHC II-expressing MKs, characteristic of the immune MK subpopulation. Mechanistically, MK progenitors upregulated CCR1 and CCR5, and MKs showed increased phosphorylation of JAK1, SRC, and STAT1-5. Phenotypically, transcriptomic profiling revealed enhanced expression of genes associated with the cell cycle, inflammatory pathways, and mitochondrial metabolism in MKs isolated from CCL5-treated mice, which was functionally supported by increased mitochondrial activity. Similarly, in human BMOs, CCL5 treatment increased MK numbers. Together, these findings implicate CCL5 as a direct mediator of acute megakaryopoiesis and immune MK expansion during inflammation.

While professional antigen-presenting cells drive adaptive immunity, atypical cell types can fulfill this role in the bone marrow. Megakaryocytes (MKs) are canonically recognized for platelet production, but recent studies indicate functional heterogeneity and immune potential. We found that ~20% of bone marrow MKs express Major Histocompatibility Complex (MHC) II and costimulatory receptors CD80, CD86, and CD40. These MKs process and present antigen to activate T cells in an MHC II-dependent manner. MK-T cell interactions induced TGF-β1 secretion and promoted induced regulatory T cell differentiation. Immunopeptidomics of MK MHC II receptor confirmed occupancy by exogenous peptides, demonstrating in vivo functionality. Using a model with MK-targeted deletion of MHC II (Pf4-MHC Δ/Δ mice), we observed altered TLR signaling, reduced bone marrow TGF-β1, and decreased numbers of hematopoietic stem cells. Together, these findings identify MHC II+ MKs as noncanonical antigen-presenting cells that modulate adaptive immunity and maintain the hematopoietic niche.

ABSTRACT:Megakaryocytes (MKs) produce platelets, and similar to other hematopoietic progenitors, they are involved in homeostatic aspects of their bone marrow niche. MKs release and endocytose various factors, such as platelet factor 4 (PF4)/CXCL4. Here, we show that the intra-α-granular proteoglycan, serglycin (SRGN), plays a key role in this process by retaining PF4, and perhaps other factors, during MK maturation. Immature, SRGN-/- MKs released ∼80% of their PF4, and conditioned media from these cells negatively affected wild-type MK differentiation in vitro. This was replicated in wild-type MKs by treatment with the polycation surfen, a known inhibitor of glycosaminoglycan (GAG)/protein interactions. In vivo, SRGN-/- mice had an interstitial accumulation of PF4, transforming growth factor β1, interleukin-1β, and tumor necrosis factor α in their bone marrow and increased numbers of immature MKs, consistent with their mild thrombocytopenia. SRGN-/- mice also had reduced numbers of hematopoietic stem cells and multipotent progenitors, reduced laminin, and increased collagen I deposition. These findings demonstrate that MKs depend on SRGN and its charged GAGs to balance the distribution of PF4 and perhaps other factors between their α-granules and their adjacent extracellular spaces. Disrupting this balance negatively affects MK development and bone marrow microenvironment homeostasis.

A common side effect of poly-ADP ribose polymerase (PARP) inhibitors is low platelet counts, or thrombocytopenia, presumably mediated through platelet progenitors, megakaryocytes (MKs). MKs are large, hematopoietic cells with a polyploid, multi-lobulated nucleus. While DNA replication in MKs (endomitosis) is well studied, limited investigations have examined the impact of DNA damage and repair inhibition on megakaryopoiesis. To explore PARP inhibitor-induced thrombocytopenia, we treated mice with PARP inhibitors (niraparib and olaparib), which are approved for the treatment of solid tumors. While high-dose niraparib treatment led to thrombocytopenia, consistent with clinical observations, treatment at a lower dosage led to a significant, >1.5-fold increase in both the number of bone marrow MKs and circulating platelets. This increase was accompanied by elevated DNA damage in both MKs and MK progenitors, as measured by both γH2AX accumulation and comet assays of MKs. Notably, platelets from niraparib-treated mice were functionally normal in their response to ADP, TRAP, and collagen. Gamma-irradiation treatment similarly increased MK and platelet counts in mice, suggesting that moderate DNA damage enhances megakaryopoiesis and increases platelet counts. These data reveal a previously unknown relationship between MKs and DNA damage and present a novel target for triggering enhanced platelet production in vivo.

ABSTRACT:Platelets modulate vascular microenvironments via the release of cargo molecules. Granule secretion is modulated by proteins called soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Secretion is complex and regulated by several protein-protein interactions; however, not all are characterized in platelets. We have identified cysteine string protein-α (CSPα; also known as, DNAJC5 or CLN4) as required for platelet secretion. CSPα is the only member from the CSP family present in platelets and has been proposed as a chaperone for the SNAP-23/25 t (Qb,c) SNAREs. To address CSPα's role, we analyzed platelets from CSPα-/- mice. The loss of CSPα significantly affected dense- and α-granule release with minimal effects on lysosomal secretion. Consistent with the secretion defects, in vivo and ex vivo assays showed that loss of CSPα caused significant bleeding and attenuated thrombosis under flow. Interestingly, loss of CSPα caused a reduction in glycoprotein VI (GPVI) levels and reduced αIIbβ3 activation, especially in response to GPVI-specific agonists. Deletion of CSPα did not affect proteins in the platelet secretory machinery, for example, the SNAP-23/25 proteins. Subcellular fractionation studies showed that CSPα, which is reported to be acylated, was present on membranes but not in lipid rafts. Immunofluorescence studies showed CSPα colocalized with α and lysosomal granule markers. CSPα-/- mice had reduced red blood cell, leukocyte, and megakaryocyte numbers, suggesting effects on bone marrow progenitor cells. Simultaneously, we detected increased collagen I deposition, but no fibrosis in the marrow of CSPα-/- mice. These results identify CSPα as another element of the platelet secretory machinery that significantly contributes to thrombosis and hemostasis.

Background Mechanical circulatory support (MCS) is a mainstay of therapy for advanced and end-stage heart failure. Accompanied by systemic anticoagulation, contemporary MCS has become less thrombogenic, with bleeding complications emerging as a major cause of readmission and 1-year mortality. Shear-mediated platelet dysfunction and thrombocytopenia of undefined etiology are primary drivers of MCS-related bleeding. Recently, it has been demonstrated that deprivation of platelet surface glycosylation is associated with the decline of hemostatic function, microvesiculation, and premature apoptosis. We test the hypothesis that shear stress induces remodeling of platelet surface glycosylation via upregulation of glycosidase activity, thus facilitating platelet count decline and intense microvesiculation. Methods Human gel-filtered platelets were exposed to continuous shear stress in vitro. Platelets and platelet-derived microparticles (PDMPs) were quantified via flow cytometry using size standard fluorescent nanobeads. Platelet surface glycosylation and NEU1 expression were evaluated using lectin- or immune-staining and multicolor flow cytometry; lectin blotting was utilized to verify glycosylation of individual glycoproteins. Platelet neuraminidase, galactosidase, hexosaminidase, and mannosidase activities were quantified using 4-methylumbelliferone-based fluorogenic substrates. Results We demonstrate that shear stress promotes selective remodeling of platelet glycosylation via downregulation of 2,6-sialylation, terminal galactose, and mannose, while 2,3-sialylation remains largely unchanged. Shear-mediated deglycosylation is partially attenuated by neuraminidase inhibitors, strongly suggesting the involvement of platelet neuraminidase in observed phenomena. Shear stress increases platelet NEU1 surface expression and potentiates generation of numerous NEU1+ PDMPs. Platelets exhibit high basal hexosaminidase and mannosidase activities; basal activities of platelet neuraminidase and galactosidase are rather low and are significantly upregulated by shear stress. Shear stress of increased magnitude and duration promotes an incremental decline of platelet count and immense microvesiculation, both being further exacerbated by neuraminidase and partially attenuated by neuraminidase inhibition. Conclusion Our data indicate that shear stress accumulation, consistent with supraphysiologic conditions of device-supported circulation, promotes remodeling of platelet glycosylation via selective upregulation of platelet glycosidase activity. Shear-mediated platelet deglycosylation is associated with platelet count drop and increased microvesiculation, thus offering a direct link between deglycosylation and thrombocytopenia observed in device-supported patients. Based on our findings, we propose a panel of molecular markers to be used for reliable detection of shear-mediated platelet deglycosylation in MCS.

BACKGROUND:Platelets are essential for hemostasis and thrombosis and play vital roles during metastatic cancer progression and infection. Hallmarks of platelet function are activation, cytoskeletal rearrangements, and the degranulation of their cellular contents upon stimulation. While α-granules and dense granules are the most studied platelet secretory granules, the dense tubular system (DTS) also functions as a secretory system for vascular thiol isomerases. However, how DTS cargo is packaged and transported from megakaryocytes (MKs) to platelets is poorly understood. OBJECTIVES:To underpin the mechanisms responsible for DTS cargo transport and leverage those for therapeutic protein packaging into platelets. METHODS:A retroviral expression system combined with immunofluorescence confocal microscopy was employed to track protein DTS cargo protein disulfide isomerase fused to enhanced green fluorescent protein (eGFP-PDI) during platelet production. Murine bone marrow transplantation models were used to determine the release of therapeutic proteins from platelets. RESULTS:We demonstrated that the endoplasmic reticulum retrieval motif Lys-Asp-Glu-Leu (KDEL) located at the C-terminus of protein disulfide isomerase was essential for the regular transport of eGFP-PDI-containing granules. eGFP-PDIΔKDEL, in which the retrieval signal was deleted, was aberrantly packaged, and its expression was upregulated within clathrin-coated endosomes. Finally, we found that ectopic transgenic proteins, such as tissue factor pathway inhibitor and interleukin 2, can be packaged into MKs and proplatelets by adding a KDEL retrieval sequence. CONCLUSION:Our data corroborate the DTS as a noncanonical secretory system in platelets and demonstrate that in vitro-generated MKs and platelets may be used as a delivery system for transgenic proteins during cellular therapy.

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 lightsheet 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.

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.

Background: Despite the importance of platelets in thrombosis and hemostasis, the mechanism by which megakaryocytes (MKs) differentiate and initiate platelet release is incompletely understood. Although thrombopoietin (TPO) has been identified as the most important regulator of MK maturation, recent reports indicate that MK maturation and platelet biogenesis can occur independently of TPO. For instance, in inflammatory conditions, platelet counts can rapidly elevate within hours. We previous identified CCL5 [chemokine ligand 5 (CCL5, also known as RANTES)] as an additional regulator of MK differentiation and maturation during inflammation. In an in vitro model, we found that CCL5 enhanced proplatelet production in a CCR5-dependent manner. In addition, other work has shown that exposing isolated hematopoietic stem cells (HSCs) to CCL5 before transplantation can directly affect fate choices of them and their progenitor cells. Building upon these observations, we aimed to investigate whether CCL5 affects megakaryo- and thrombopoiesis using in vivo mouse models, and in a novel human bone marrow organoid. Results: To determine the effect of CCL5 under steady state, we administered acute CCL5 to mice and verified that cytokine levels were elevated in both plasma and bone marrow fluid 24 hours post administration. Quantification of MKs by flow cytometry revealed an increase in both MK progenitors [CD41+] and mature MKs [CD41+CD42d+] 24 hours after treatment with CCL5. Additionally, immunofluorescence imaging of femoral cryosections confirmed an increase in MK numbers, suggesting that acute exposure to CCL5 enhanced megakaryopoiesis. Expanding our analysis to HSPCs, we observed that CCL5 also induced the expansion of MK-biased CD41+ long-term (LT)-HSCs [Lin-cKithighSca-1+Flt3-CD48-CD150+CD41+] and MkPs [Lin-cKithighCD150+CD41+], 2.8- and 2.1-fold respectively, suggesting a potential non-canonical pathway triggered by CCL5. To elucidate the mechanism responsible for increased MK numbers, we aimed to determine the in vivo expression patterns of the CCL5 receptors CCR1, CCR3, and CCR5 with and without CCL5 treatment. While no differences were found in LT- and short-term (ST)-HSCs, CCR1 and CCR5 expression was increased in MkPs 24 hours after CCL5 administration, suggesting that CCL5 signaling through CCR1 and CCR5 represents a pathway by which CCL5 signals to drive megakaryopoiesis. To investigate the effect of CCL5 administration upon stress, i.e. during platelet recovery following anti-GPIba antibody-induced platelet depletion, mice were treated with either vehicle or CCL5 48 hours post platelet depletion. After 6 days, platelets counts were higher in CCL5-treated mice compared to the vehicle-treated group. Due to the faster recovery observed in CCL5-treated mice, we aimed to determine if the increase in platelet counts occurred via non-canonical pathways. Quantification of MKs by flow cytometry revealed an increase in MK-biased CD41+ LT-HSCs while we did not observe differences in multipotent progenitors (MPPs) compared to the vehicle-treated group, indicating that CCL5 facilitated the expansion of MKs in the bone marrow post depletion. To substantiate our findings in a human system, we utilized an iPCs-derived human bone marrow organoid, which recapitulates essential features of the adult human bone marrow and offers a platform to study cellular behavior within a bone marrow microenvironment. We treated bone marrow organoids with CCL5 for 24 hours and quantified populations including endothelial cells, HSPCs, erythroid cells and MKs using flow cytometry. Consistent with our observations in the murine model, the organoids exhibited 2-fold increase in MKs [CD41+]. Conclusions: Using murine and human models, we demonstrate a CCL5-induced expansion of MkPs, revealing a role for CCL5 in promoting megakaryopoiesis in the bone marrow. These data suggest that CCL5 may be one component of the inflammatory milieu that triggers elevated platelet counts in inflammatory thrombocytosis.

Objectives Sepsis is a life-threatening condition implicating an inadequate activation of the immune system. Platelets act as modulators and contributors to immune processes. Indeed, altered platelet turnover, thrombotic events, and changes in thrombopoietin levels in systemic inflammation have been reported, but thrombopoietin-levels in sepsis and septic-shock have not yet been systematically evaluated. We therefore performed a meta-analysis of thrombopoietin (TPO)-levels in patients with sepsis.Methods Two independent reviewers screened records and full-text articles for inclusion. Scientific databases were searched for studies examining thrombopoietin levels in adult sepsis and septic-shock patients until August 1st 2022.Results Of 95 items screened, six studies met the inclusion criteria, including 598 subjects. Both sepsis and severe sepsis were associated with increased levels of thrombopoietin (sepsis vs. control: standardized mean difference 3.06, 95 % CI 1.35-4.77; Z=3.50, p=0.0005) (sepsis vs. severe sepsis: standardized mean difference -1.67, 95 % CI -2.46 to -0.88; Z=4.14, p<0.0001). TPO-levels did not show significant differences between severe sepsis and septic shock patients but differed between sepsis and inflammation-associated non-septic controls. Overall, high heterogeneity and low sample size could be noted.Conclusions Concluding, increased levels of thrombopoietin appear to be present both in sepsis and severe sepsis with high heterogeneity but thrombopoietin does not allow to differentiate between severe sepsis and septic-shock. TPO may potentially serve to differentiate sepsis from non-septic trauma and/or tissue damage related (systemic) inflammation. Usage of different assays and high heterogeneity demand standardization of methods and further large multicenter trials.

Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation.

Blood platelets are anucleate cells essential for normal blood hemostasis. To maintain a normal platelet count of 150 000 to 400 000 per μL of blood, 1011 platelets must be released each day from precursor cells called megakaryocytes. In this review, we aim to provide an overview of platelet production and evaluate the proposed mechanisms of platelet generation. We will discuss novel cytoskeletal mechanisms of platelet production, including microtubule and actin-based systems. We present new evidence that supports a cytoplasmic trigger for platelet production, discuss centrosome clustering as a new mechanism to trigger proplatelet production, and review new data supporting the bone marrow as the major location of platelet production.

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 G 1 of interphase. Forced cell cycle arrest in G 1 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.