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Abstract: Clinical features in patients with the B-cell lymphoma, Waldenström macroglobulinemia (WM), include cytopenias, immunoglobulin M (IgM)-mediated hyperviscosity, fatigue, bleeding, and bruising. Therapeutics such as Bruton's tyrosine kinase inhibitors (BTKis) exacerbate bleeding risk. Abnormal hemostasis arising from platelet dysfunction, altered coagulation or vascular impairment have not yet been investigated in patients with WM. Therefore, this study aimed to evaluate hemostatic dysfunction in samples from these patients. Whole blood (WB) samples were collected from 14 patients with WM not receiving therapy, 5 patients receiving BTKis and 15 healthy donors (HDs). Platelet receptor levels and reticulation were measured by flow cytometry, plasma thrombin generation with or without platelets by fluorescence resonance energy transfer assay, WB clotting potential by rotational thromboelastometry, and plasma soluble glycoprotein VI (sGPVI) and serum thrombopoietin (TPO) by enzyme-linked immunosorbent assay. Donor platelet spreading, aggregation, and ability to accelerate thrombin generation in the presence of WM-derived IgM were assessed. WM platelet receptor levels, responses to physiological agonists, and plasma sGPVI were within normal ranges. WM platelets had reduced reticulation (P = .0012) whereas serum TPO levels were increased (P = .0040). WM plasma displayed slower thrombin generation (P = .0080) and WM platelets contributed less to endogenous thrombin potential (ETP; P = .0312). HD plasma or platelets incubated with IgM (50-60 mg/mL) displayed reduced spreading (P = .0002), aggregation (P < .0001), and ETP (P = .0081). Thus, alterations to thrombin potential and WB coagulation were detected in WM samples. WM IgM significantly impaired hemostasis in vitro. Platelet and coagulation properties are disturbed in patients with well-managed WM., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects., Objective: This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1., Methods: We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells., Results: A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1 c740 - 1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation., Conclusions: We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN -/- ) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1 -/- cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival.

Platelet adhesion and activation is fundamental to the formation of a hemostatic response to limit loss of blood and instigate wound repair to seal a site of vascular injury. The process of platelet aggregate formation is supported by the coagulation system driving injury-proximal formation of thrombin, which converts fibrinogen to insoluble fibrin. This highly coordinated series of molecular and membranous events must be routinely achieved in flowing blood, at vascular fluid shear rates that place significant strain on molecular and cellular interactions. Platelets have long been recognized to be able to slow down and adhere to sites of vascular injury and then activate and recruit more platelets that forge and strengthen adhesive ties with the vascular wall under these conditions. It has been a major challenge for the Platelet Research Community to construct experimental conditions that allow precise definition of the molecular steps occurring under flow. This brief review will discuss work to date from our group, as well as others that has furthered our understanding of platelet function in flowing blood., Competing Interests: Disclosures None.

The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo., (© 2022 by The American Society of Hematology.)

Ionotropic glutamate receptors include α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), kainate receptors (KAR), and N -methyl-D-aspartate receptors (NMDAR). All function as cation channels; AMPAR and KAR are more permeable to sodium and NMDAR to calcium ions. Compared to the brain, receptor assemblies in platelets are unusual, suggesting distinctive functionalities.There is convincing evidence that AMPAR and KAR amplify platelet function and thrombus formation in vitro and in vivo . Transgenic mice lacking GluA1 and GluK2 (AMPAR and KAR subunits, respectively) have longer bleeding times and prolonged time to thrombosis in an arterial model. In humans, rs465566 KAR gene polymorphism associates with altered in vitro platelet responses suggesting enhanced aspirin effect. The NMDAR contribution to platelet function is less well defined. NMDA at low concentrations (≤10 μM) inhibits platelet aggregation and high concentrations (≥100 μM) have no effect. However, open NMDAR channel blockers interfere with platelet activation and aggregation induced by other agonists in vitro ; anti-GluN1 antibodies interfere with thrombus formation under high shear rates ex vivo ; and rats vaccinated with GluN1 develop iron deficiency anemia suggestive of mild chronic bleeding. In this review, we summarize data on glutamate receptors in platelets and propose a unifying model that reconciles some of the opposing effects observed.

The circadian clock is a specialised cell signalling circuit present in almost all cells. It controls the timing of key cell activities such as proliferation and differentiation. In osteoarthritis, expression of two components of the circadian clock, BMAL1 and PER2 is altered in chondrocytes and this change has been causally linked with the increase in proliferation and altered chondrocyte differentiation in disease. IL-1β, an inflammatory cytokine abundant in OA joints, has previously been shown to induce changes in BMAL1 and PER2 expression in chondrocytes. The purpose of this study is to identify the mechanism involved. We found IL-1β treatment of primary human chondrocytes led to activation of NMDA receptors as evidenced by an increase in phosphorylation of GluN1 and an increase in intracellular calcium which was blocked by the NMDAR antagonist MK801. Levels of phosphorylated CREB were also elevated in IL-1β treated cells and this effect was blocked by co-treatment of cells with IL-1β and the NMDAR antagonist MK-801. Knockdown of CREB or inhibition of CREB activity prevented the IL-1β induced increase in PER2 expression in chondrocytes but had no effect on BMAL1. Phosphorylated p65 levels were elevated in IL-1β treated chondrocytes indicating increased NF-κB activation. Inhibition of NF-κB activity prevented the IL-1β induced reduction in BMAL1 expression and partially mitigated the IL-1β induced increase in PER2 expression in chondrocytes. These data indicate that the NMDAR/CREB and NF-κB signalling pathways regulate the core circadian clock components PER2 and BMAL1 in chondrocytes. Given that changes in expression of these clock components have been observed in a wide range of diseases, these findings may be broadly relevant for understanding the mechanism leading to circadian clock changes in pathology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are micro and nanoscale packages that circulate in all bodily fluids and play an important role in intercellular communication by shuttling biomolecules to nearby and distant cells. However, producing sufficient amounts of EVs for many types of in vitro studies using standard culture methods can be challenging, and despite the success of some bioreactors in increasing EV-production, it is still largely unknown how individual culture conditions can alter the production and content of EVs. In this study, we demonstrate a simple and inexpensive micropatterning technique that can be used to produce polystyrene microtracks over a 100 mm diameter growth surface area. We then demonstrate that these microtracks can play a significant role in increasing EV production using a triple-negative breast cancer cell line (MDA-MB-231) and that these changes in EV production correlate with increases in cellular aspect ratio, alignment of the cells' long axes to the microtracks, and single-cell migration rates. These findings have implications in both biomanufacturing of EVs and potentially in enhancing the biomimicry of EVs produced in vitro., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The circadian clock is a specialized cell signalling pathway present in all cells. Loss of clock function leads to tissue degeneration and premature ageing in animal models demonstrating the fundamental importance of clocks for cell, tissue and organism health. There is now considerable evidence that the chondrocyte circadian clock is altered in OA. The purpose of this review is to summarize current knowledge regarding the nature of the change in the chondrocyte clock in OA and the implications of this change for disease development. Expression of the core clock component, BMAL1, has consistently been shown to be lower in OA chondrocytes. This may contribute to changes in chondrocyte differentiation and extracellular matrix turnover in disease. Circadian clocks are highly responsive to environmental factors. Mechanical loading, diet, inflammation and oxidative insult can all influence clock function. These factors may contribute to causing the change in the chondrocyte clock in OA., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The N -methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca 2+ ). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca 2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca 2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca 2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca 2+ overload, K + loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed., (Copyright © 2020 Kalev-Zylinska, Hearn, Makhro and Bogdanova.)

The release of calcium ions (Ca 2+ ) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N -methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca 2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01- GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca 2+ levels were higher in Meg-01- GRIN1 -/- cells, but ER Ca 2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca 2+ . Microarray analysis revealed that Meg-01- GRIN1 -/- cells had deregulated expression of transcripts involved in Ca 2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca 2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca 2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

The chondrocyte circadian clock is altered in osteoarthritis. This change is implicated in the disease-associated changes in chondrocyte phenotype and cartilage loss. Why the clock is changed is unknown. N-methyl-D-aspartate receptors (NMDAR) are critical for regulating the hypothalamic clock. Chondrocytes also express NMDAR and the type of NMDAR subunits expressed changes in osteoarthritis., Objective: To determine if NMDAR regulate the chondrocyte clock and phenotype., Design: Chondrocytes isolated from macroscopically-normal (MN) and osteoarthritic human cartilage were treated with NMDAR antagonists or transfected with GRIN2A or GRIN2B-targetting siRNA. H5 chondrocytes were transfected with GluN2B-expression plasmids. Clock genes and chondrocyte phenotypic markers were measured by RT-qPCR., Results: PER2 amplitude was higher and BMAL1 amplitude lower in osteoarthritic compared to MN chondrocytes. In osteoarthritic chondrocytes, NMDAR inhibition restored PER2 and BMAL1 expression to levels similar to MN chondrocytes, and resulted in reduced MMP13 and COL10A1. Paradoxically, NMDAR inhibition in MN chondrocytes resulted in increased PER2, decreased BMAL1 and increased MMP13 and COL10A1. Osteoarthritic, but not MN chondrocytes expressed GluN2B NMDAR subunits. GluN2B knockdown in osteoarthritic chondrocytes restored expression of circadian clock components and phenotypic markers to levels similar to MN chondrocytes. Ectopic expression of GluN2B resulted in reduced BMAL1, increased PER2 and altered SOX9, RUNX2 and MMP13 expression. Knockdown of PER2 mitigated the effects of GluN2B on SOX9 and MMP13., Conclusions: NMDAR regulate the chondrocyte clock and phenotype suggesting NMDAR may also regulate clocks in other peripheral tissues. GluN2B expression in osteoarthritis may contribute to pathology by altering the chondrocyte clock., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: N -methyl-d-aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro- and anti-differentiating effects have been shown in different contexts., Objectives: The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells., Methods: Meg-01, Set-2, and K-562 leukemic cell lines were differentiated using phorbol-12-myristate-13-acetate (PMA, 10 nmol L -1 ) or valproic acid (VPA, 500 μmol L -1 ). Normal megakaryocytes were grown from mouse marrow-derived hematopoietic progenitors (lineage-negative and CD41a-enriched) in the presence of thrombopoietin (30-40 nmol L -1 ). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK-801 (100 μmol L -1 ); their effects compared against appropriate controls., Results: The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK-801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg-01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves., Conclusions: NMDAR-mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

GluN1 is a mandatory component of N-methyl-D-aspartate receptors (NMDARs) best known for their roles in the brain, but with increasing evidence for relevance in peripheral tissues, including platelets. Certain anti-GluN1 antibodies reduce brain infarcts in rodent models of ischaemic stroke. There is also evidence that human anti-GluN1 autoantibodies reduce neuronal damage in stroke patients, but the underlying mechanism is unclear. This study investigated whether anti-GluN1-mediated neuroprotection involves inhibition of platelet function. Four commercial anti-GluN1 antibodies were screened for their abilities to inhibit human platelet aggregation. Haematological parameters were examined in rats vaccinated with GluN1. Platelet effects of a mouse monoclonal antibody targeting the glycine-binding region of GluN1 (GluN1-S2) were tested in assays of platelet activation, aggregation and thrombus formation. The epitope of anti-GluN1-S2 was mapped and the mechanism of antibody action modelled using crystal structures of GluN1. Our work found that rats vaccinated with GluN1 had a mildly prolonged bleeding time and carried antibodies targeting mostly GluN1-S2. The monoclonal anti-GluN1-S2 antibody (from BD Biosciences) inhibited activation and aggregation of human platelets in the presence of adrenaline, adenosine diphosphate, collagen, thrombin and a protease-activated receptor 1-activating peptide. When human blood was flowed over collagen-coated surfaces, anti-GluN1-S2 impaired thrombus growth and stability. The epitope of anti-GluN1-S2 was mapped to α-helix H located within the glycine-binding clamshell of GluN1, where the antibody binding was computationally predicted to impair opening of the NMDAR channel. Our results indicate that anti-GluN1-S2 inhibits function of human platelets, including dense granule release and thrombus growth. Findings add to the evidence that platelet NMDARs regulate thrombus formation and suggest a novel mechanism by which anti-GluN1 autoantibodies limit stroke-induced neuronal damage.

Chemotherapy with taxanes such as paclitaxel (PTX) is a key component of triple negative breast cancer (TNBC) treatment. PTX is used in combination with other drugs in both the adjuvant setting and in advanced breast cancer. Because a proportion of patients respond poorly to PTX or relapse after its use, a greater understanding of the mechanisms conferring resistance to PTX is required. One protein shown to be involved in drug resistance is Y-box binding protein 1 (YB-1). High levels of YB-1 have previously been associated with resistance to PTX in TNBCs. In this study, we aimed to determine mechanisms by which YB-1 confers PTX resistance. We generated isogenic TNBC cell lines that differed by YB-1 levels and treated these with PTX. Using microarray analysis, we identified EGR1 as a potential target of YB-1. We found that low EGR1 mRNA levels are associated with poor breast cancer patient prognosis, and that EGR1 and YBX1 mRNA expression was inversely correlated in a TNBC line and in a proportion of TNBC tumours. Reducing the levels of EGR1 caused TNBC cells to become more resistant to PTX. Given that PTX targets cycling cells, we propose a model whereby high YB-1 levels in some TNBC cells can lead to reduced levels of EGR1, which in turn promotes slow cell cycling and resistance to PTX. Therefore YB-1 and EGR1 levels are biologically linked and may provide a biomarker for TNBC response to PTX., (© 2016 UICC.)