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.

Contribution of human organic anion transporter 3-mediated transport of a major linezolid metabolite, PNU-142586, in linezolid-induced thrombocytopenia.

Thrombocytopenia, a common adverse effect of linezolid, often occurs in patients lacking typical risk factors. In this study, we investigated the key risk factors for linezolid-induced thrombocytopenia using two real-world clinical databases and explored its underlying mechanism through in vitro and in vivo experiments. In a retrospective analysis of 150 linezolid-treated patients, multivariate analysis identified coadministration of lansoprazole, a proton pump inhibitor, as a significant independent risk factor for thrombocytopenia (odds ratio: 2.33, p = 0.034). Additionally, analysis of the Food and Drug Administration Adverse Event Reporting System database revealed a reporting odds ratio of thrombocytopenia for lansoprazole of 1.64 (95% CI: 1.25-2.16). In vitro studies showed that the uptake of PNU-142586, a major linezolid metabolite, was significantly higher in human organic anion transporter 3-expressing HEK293 (HEK-hOAT3) cells compared to HEK-pBK cells. The apparent IC50 value of lansoprazole against hOAT3-mediated transport of PNU-142586 was 0.59 ± 0.38 µM. In a pharmacokinetic study using rats, coadministration of linezolid with lansoprazole intravenously resulted in approximately a 1.7-fold increase in the area under the plasma concentration-time curve of PNU-142586, but not linezolid and PNU-142300. Moreover, PNU-142586, but not linezolid, exhibited concentration-dependent cytotoxicity in a human megakaryocytic cell line. These findings suggest that linezolid-induced thrombocytopenia should be due to delayed elimination of PNU-142586. Furthermore, delayed elimination of PNU-142586 due to renal failure and hOAT3-mediated transport inhibition by lansoprazole should exacerbate linezolid-induced thrombocytopenia.

Notch1 regulates hepatic thrombopoietin production.

ABSTRACT: Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Inflammation modifies the platelet reactivity among thrombocytopenia patients undergoing percutaneous coronary intervention.

Percutaneous coronary intervention (PCI) patients combined with thrombocytopenia (TP) are usually considered to be at low ischemic risk, receiving less proper antiplatelet therapy. However, recent studies reported a paradoxical phenomenon that PCI patients with TP were prone to experience thrombotic events, while the mechanisms and future treatment remain unclear. We aim to investigate whether inflammation modifies platelet reactivity among these patients. Consecutive 10 724 patients undergoing PCI in Fuwai Hospital were enrolled throughout 2013. High-sensitivity C-reactive protein (hsCRP) ≥2 mg/L was considered inflammatory status. TP was defined as platelet count <150×109/L. High on-treatment platelet reactivity (HTPR) was defined as adenosine diphosphate-induced platelet maximum amplitude of thromboelastogram >47mm. Among 6617 patients finally included, 879 (13.3%) presented with TP. Multivariate logistic regression demonstrated that patients with TP were associated with a lower risk of HTPR (odds ratio [OR] 0.64, 95% confidence interval [CI] 0.53-0.76) than those without TP in the overall cohort. In further analysis, among hsCRP <2 mg/L group, patients with TP exhibited a decreased risk of HTPR (OR 0.53, 95% CI 0.41-0.68); however, in hsCRP ≥2mg/L group, TP patients had a similar risk of HTPR as those without TP (OR 0.83, 95% CI 0.63-1.08). Additionally, these results remain consistent across subgroups, including patients presenting with acute coronary syndrome and chronic coronary syndrome. Inflammation modified the platelet reactivity of PCI patients with TP, providing new insights into the mechanisms of the increased thrombotic risk. Future management for this special population should pay more attention to inflammation status and timely adjustment of antiplatelet therapy in TP patients with inflammation.

What is the context? Recent studies reported a paradoxical phenomenon that percutaneous coronary intervention (PCI) patients with thrombocytopenia (TP) were prone to experience thrombotic events. The potential mechanisms underlying the increased thrombotic risk and how to manage antiplatelet therapy in PCI patients with TP remain unclear.Growing attention has been paid to immunothrombosis. Inflammation is closely associated with high-on treatment platelet reactivity (HTPR) and thrombotic risk.HTPR is an independent risk factor of thrombosis and can provide information for guiding antiplatelet therapy.What is new? This prospective cohort study enrolled 10 724 patients undergoing PCI in Fuwai Hospital (National Center for Cardiovascular Diseases, Beijing, China), with HTPR risk being the study endpoint of interest.We first reported that inflammation significantly modified the platelet reactivity of PCI patients with TP.When hsCRP level <2 mg/L, PCI patients with TP had a decreased risk of HTPR. However, when hsCRP ≥2 mg/L, TP patients had similar HTPR risk as those without TP.HsCRP levels could modify the relationship between TP and HTPR risks both in patients with acute coronary syndrome and chronic coronary syndrome.What is the impact? These results provide insights into potential mechanisms of the increased thrombotic risk in PCI patients with TP. Specifically, inflammation might be involved in the thrombotic risk of PCI patients with TP by modifying the platelet reactivity.As for future management, personalized antiplatelet therapy should be administrated to TP patients with inflammation status.

Long non-coding RNA NORAD regulates megakaryocyte differentiation and proplatelet formation via the DUSP6/ERK signaling pathway.

Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia.

Alnustone promotes megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway.

OBJECTIVES: Thrombocytopenia is a disease in which the number of platelets in the peripheral blood decreases. It can be caused by multiple genetic factors, and numerous challenges are associated with its treatment. In this study, the effects of alnustone on megakaryocytes and platelets were investigated, with the aim of developing a new therapeutic approach for thrombocytopenia. METHODS: Random forest algorithm was used to establish a drug screening model, and alnustone was identified as a natural active compound that could promote megakaryocyte differentiation. The effect of alnustone on megakaryocyte activity was determined using cell counting kit-8. The effect of alnustone on megakaryocyte differentiation was determined using flow cytometry, Giemsa staining, and phalloidin staining. A mouse model of thrombocytopenia was established by exposing mice to X-rays at 4 Gy and was used to test the bioactivity of alnustone in vivo. The effect of alnustone on platelet production was determined using zebrafish. Network pharmacology was used to predict targets and signaling pathways. Western blotting and immunofluorescence staining determined the expression levels of proteins. RESULTS: Alnustone promoted the differentiation and maturation of megakaryocytes in vitro and restored platelet production in thrombocytopenic mice and zebrafish. Network pharmacology and western blotting showed that alnustone promoted the expression of interleukin-17A and enhanced its interaction with its receptor, and thereby regulated downstream MEK/ERK signaling and promoted megakaryocyte differentiation. CONCLUSIONS: Alnustone can promote megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway and thus provides a new therapeutic strategy for the treatment of thrombocytopenia.

Thrombopoietin, the Primary Regulator of Platelet Production: From Mythos to Logos, a Thirty-Year Journey.

Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.

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.

Platelet proteomic profiling in sitosterolemia suggests thrombocytopenia is driven by lipid disorder and not platelet aberrations.

ABSTRACT: Sitosterolemia is a rare autosomal recessive genetic disorder in which patients develop hypercholesterolemia and may exhibit abnormal hematologic and/or liver test results. In this disease, dysfunction of either ABCG5 or ABCG8 results in the intestinal hyperabsorption of all sterols, including cholesterol and, more specifically, plant sterols or xenosterols, as well as in the impaired ability to excrete xenosterols into the bile. It remains unknown how and why some patients develop hematologic abnormalities. Only a few unrelated patients with hematologic abnormalities at the time of diagnosis have been reported. Here, we report on 2 unrelated pedigrees who were believed to have chronic immune thrombocytopenia as their most prominent feature. Both consanguineous families showed recessive gene variants in ABCG5, which were associated with the disease by in silico protein structure analysis and clinical segregation. Hepatosplenomegaly was absent. Thrombopoietin levels and megakaryocyte numbers in the bone marrow were normal. Metabolic analysis confirmed the presence of strongly elevated plasma levels of xenosterols. Potential platelet proteomic aberrations were longitudinally assessed following dietary restrictions combined with administration of the sterol absorption inhibitor ezetimibe. No significant effects on platelet protein content before and after the onset of treatment were demonstrated. Although we cannot exclude that lipotoxicity has a direct and platelet-specific impact in patients with sitosterolemia, our data suggest that thrombocytopenia is neither caused by a lack of megakaryocytes nor driven by proteomic aberrations in the platelets themselves.

Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

The Y49H cytochrome c variant enhances megakaryocytic maturation of K-562 cells.

Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus.

High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury.

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Deficiency of thioredoxin-interacting protein results in age-related thrombocytopenia due to megakaryocyte oxidative stress.

BACKGROUND: Platelets are generated from megakaryocytes (MKs), mainly located in the bone marrow (BM). Megakaryopoiesis can be affected by genetic disorders, metabolic diseases, and aging. The molecular mechanisms underlying platelet count regulation have not been fully elucidated. OBJECTIVES: In the present study, we investigated the role of thioredoxin-interacting protein (TXNIP), a protein that regulates cellular metabolism in megakaryopoiesis, using a Txnip-/- mouse model. METHODS: Wild-type (WT) and Txnip-/- mice (2-27-month-old) were studied. BM-derived MKs were analyzed to investigate the role of TXNIP in megakaryopoiesis with age. The global transcriptome of BM-derived CD41+ megakaryocyte precursors (MkPs) of WT and Txnip-/- mice were compared. The CD34+ hematopoietic stem cells isolated from human cord blood were differentiated into MKs. RESULTS: Txnip-/- mice developed thrombocytopenia at 4 to 5 months that worsened with age. During ex vivo megakaryopoiesis, Txnip-/- MkPs remained small, with decreased levels of MK-specific markers. Critically, Txnip-/- MkPs exhibited reduced mitochondrial reactive oxygen species, which was related to AKT activity. Txnip-/- MkPs also showed elevated glycolysis alongside increased glucose uptake for ATP production. Total RNA sequencing revealed enrichment for oxidative stress- and apoptosis-related genes in differentially expressed genes between Txnip-/- and WT MkPs. The effects of TXNIP on MKs were recapitulated during the differentiation of human cord blood-derived CD34+ hematopoietic stem cells. CONCLUSION: We provide evidence that the megakaryopoiesis pathway becomes exhausted with age in Txnip-/- mice with a decrease in terminal, mature MKs that response to thrombocytopenic challenge. Overall, this study demonstrates the role of TXNIP in megakaryopoiesis, regulating mitochondrial metabolism.

Detection of circulating normal and tumor plasma cells in newly diagnosed patients of multiple myeloma and their associations with clinical and laboratory parameters.

INTRODUCTION: Circulating plasma cells (CPCs) are frequently noted in variable frequencies in the entire spectrum of plasma cells neoplasms. With advent of high sensitivity multi-parametric flow cytometry, it is not only possible to detect CPCs present in very low numbers, but also to categorise them into circulating tumor plasma cells (CTPCs) and circulating normal plasma cells (CNPCs), based on their marker-profile. This study used multi-colour flow cytometry to evaluate the load of both CTPCs & CNPCs at the time of diagnosis and at six months' time-point of therapy, and evaluated associations of both with clinical and laboratory parameters. METHODS: Twenty one newly diagnosed MM patients were enrolled. Six to nine millilitres of EDTA-anticoagulated peripheral blood sample was used for flow cytometry. A ten colour antibody panel was used for analysis of CPCs, which were categorised further into CTPCs and CNPCs. Approximately 4.8 million events were acquired for the analysis. The percentage &absolute numbers of CTPCs and CNPCs were noted and the proportion of CTPCs out of all CPCs (CTPCs + CNPCs) were also calculated for evaluating their statistical associations. RESULTS: All 21 patients of newly diagnosed MM showed presence of CPCs (CTPCs and/or CNPCs) at the time of diagnosis. The CTPCs were detected in 76 % of the study population. The median percentage and absolute counts of CTPCs were 0.52 % and 54.9 cells /µL, respectively. CNPCs were found in 95 % and the median percentage and absolute counts of CNPCs were 0.025 % and 2.66 cells/µL. After six months of therapy, CPCs (CTPCs and/or CNPCs) were found in all nine patients evaluated for this assay. CTPCs were found 33 %, with a median of 0.075 % and CNPCs were found in 89 % with a median of 0.01 %. Our study showed that the load of CTPCs was found to be higher in patients with presence of lytic bone lesions, plasmacytoma, presence of PCs on peripheral blood film by light microscopy, presence of Chr 1p32 deletion, expression of CD56 and CD81 on CTPCs, and in patients with absence of very good partial response (VGPR). Conversely, the load of CTPCs was significantly lower in patients with concomitant amyloidosis. Also, percentage of bone marrow plasma cells exhibited a significant positive correlation with the absolute count of CTPCs. We observed that the mean percentage of CNPCs was significantly higher in female patients. The load of CNPCs was lower in patients with thrombocytopenia and with hypoalbuminemia. CONCLUSION: Increased burden of CTPCs was associated with presence of lytic lesions, plasmacytomas, Chr 1p32 deletion, expression of CD56 and CD81 on tumor cells and with failure to achieve very good partial response. The CNPCs were lower in patients with thrombocytopenia and with hypoalbuminemia. To best ot our knowledge, this is the first study from India on the relevance of circulating tumor plasma cells and the first study in the world to analyse the associations of circulating normal plasma cells in newly diagnosed patients of multiple myeloma. The study also highlights the utility of multi-parametric flow cytometry in identification and enumeration of circulating plasma cells. MICRO ABSTRACT: Circulating plasma cells indicates poorer outcomes in patients of multiple myeloma. Twenty one newly diagnosed multiple myeloma patients were evaluated by flow cytometry to enumerate and characterise circulating tumor plasma cells (CTPCs) and circulating normal plasma cells (CNPCs). Higher load of CTPCs correlated with known poor prognostic markers and poor response to therapy.

GPIbα-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis.

ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

Platelets mediate the clearance of senescent red blood cells by forming prophagocytic platelet-cell complexes.

ABSTRACT: In humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

Platelet lifespan and mechanisms for clearance.

PURPOSE OF REVIEW: Activated or aged platelets are removed from circulation under (patho)physiologic conditions, the exact mechanism of platelet clearance under such conditions remains unclear and are currently being investigated. This review focuses on recent findings and controversies regarding platelet clearance and the disruption of platelet life cycle. RECENT FINDINGS: The platelet life span is determined by glycosylation of platelet surface receptors with sialic acid. Recently, it was shown that platelet activation and granule release leads to desialylation of glycans and accelerated clearance of platelets under pathological conditions. This phenomenon was demonstrated to be a main reason for thrombocytopenia being a complication in several infections and immune disorders. SUMMARY: Although we have recently gained some insight into how aged platelets are cleared from circulation, we are still not seeing the full picture. Further investigations of the platelet clearance pathways under pathophysiologic conditions are needed as well as studies to unravel the connection between platelet clearance and platelet production.

Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.

BACKGROUND: Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients. METHODS: In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice. RESULTS: In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (P=0.00013, P<0.0001, and P<0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P<0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets. CONCLUSIONS: ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.

Age-Dependent Surface Receptor Expression Patterns in Immature Versus Mature Platelets in Mouse Models of Regenerative Thrombocytopenia.

Aging is a multifaceted process that unfolds at both the individual and cellular levels, resulting in changes in platelet count and platelet reactivity. These alterations are influenced by shifts in platelet production, as well as by various environmental factors that affect circulating platelets. Aging also triggers functional changes in platelets, including a reduction in RNA content and protein production capacity. Older individuals and RNA-rich immature platelets often exhibit hyperactivity, contributing significantly to pathologic conditions such as cardiovascular diseases, sepsis, and thrombosis. However, the impact of aging on surface receptor expression of circulating platelets, particularly whether these effects vary between immature and mature platelets, remains largely unexplored. Thus, we investigated the expression of certain surface and activation receptors on platelets from young and old mice as well as on immature and mature platelets from mouse models of regenerative thrombocytopenia by flow cytometry. Our findings indicate that aged mice show an upregulated expression of the platelet endothelial cell adhesion molecule-1 (CD31), tetraspanin-29 (CD9), and Toll-like receptor 2 (TLR2) compared to their younger counterparts. Interestingly, when comparing immature and mature platelets in both young and old mice, no differences were observed in mature platelets. However, immature platelets from young mice displayed higher surface expression compared to immature platelets from old mice. Additionally, in mouse models of regenerative thrombocytopenia, the majority of receptors were upregulated in immature platelets. These results suggest that distinct surface receptor expressions are increased on platelets from old mice and immature platelets, which may partially explain their heightened activity and contribute to an increased thrombotic risk.