Platelet Formation Research Articles

Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity.

Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbβ3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.

AUTOMATIC DETECTION OF ALZHEIMER'S DISEASE BASED ON ARTIFICIAL INTELLIGENCE

Alzheimer's disease is a neurodegenerative disease that progressively destroys neurons through the formation of platelets that prevent communication between neurons. The study carried out in this project aims to find a precise and relevant diagnostic solution based on artificial intelligence and which helps in the early detection of Alzheimer's disease in order to stop its progression. The study went through a process of processing MRI images followed by training of three deep learning algorithms (VGG-19, Xception and DenseNet121) and finally by a step of testing and predicting the results. The results of the accuracy metric obtained for the three algorithms were respectively 98%, 95%, 91%.

The relationship between structural evolution and high energy ball milling duration in tin reinforced Mg alloys

In this study, 9 wt% Sn reinforced Mg alloys are produced by high energy ball milling and conventional sintering method. Effect of ball milling time (30 min, 2 h, 4 h, 8 h, and 12 h) on particle and grain morphology is detected by SEM examination. Phase evolution and solid solution mechanism is monitored by XRD analysis. Sintering process is carried out at 400 °C with 2 h sintering duration under Ar atmosphere. Platelet structure in powder morphology is observed up to 4 h ball milling, which is linked with the texture formation on < 002 > direction in hexagonal structure of Mg. This platelet formation is broken into smaller and monodispersed particle morphology with further milling process due to excessive plastic deformation which eventually provide homogenous dispersion of reinforcement element. Furthermore, ball milling process after 4 h triggers solid solution along with formation of intermetallic phase (Mg2Sn) according to crystallographic approach. The highest relative density values is measured in 8 h milling duration. Besides, the highest hardness value is achieved along with proper distribution of reinforcement element in 8 h condition. The hardness values is increased almost 85% comparing with pure ball milled Mg and 74% increment comparing with as cast Mg. The highest elastic modulus values is measured at 8 h milling condition by ultrasound method and this value is found in reasonable range. By introducing 12 h ball milling process, density, hardness and elastic modulus properties of 9 wt% Sn-Mg alloy is diminished because excessive amount of intermetallic phase formation on grain and grain boundaries. Therefore, 8 h high energy ball milling process is chosen the most suitable condition for this materials system to obtain proper powder packing and sinterability along with superior physical and mechanical properties.

Improving pharmacological activities of thrombopoietin mimetic peptide by genetic fusion to albumin-binding domain.

Thrombopoietin mimetic peptide (TMP), an analog of natural thrombopoietin, can be used to treat primary immune thrombocytopenia. However, the short half-life of TMP limits its application in clinics. The present study aimed to improve the stability and biological activity of TMP in vivo via genetic fusion to the albumin-binding protein domain (ABD). TMP dimer was genetically fused to the N-terminal or C-terminal of ABD, denoted as TMP-TMP-ABD and ABD-TMP-TMP. A Trx-tag was used to improve the fusion proteins' expression levels effectively. ABD-fusion TMP proteins were produced in Escherichia coli and purified by Ni2+-NTA and SP ion exchange column. Albumin binding studies in vitro showed that the fusion proteins could effectively bind to serum albumin to extend their half-lives. The fusion proteins effectively induced platelet proliferation in healthy mice, and the platelet count was increased by more than 2.3-fold compared with the control group. The increased platelet count induced by the fusion proteins lasted 12days compared with the control group. The increasing trend was maintained for 6days before a decline occurred after the last injection in the fusion-protein-treated mice group. ABD can effectively improve the stability and pharmacological activity of TMP by binding to serum albumin, and the ABD-fusion TMP protein can promote platelet formation in vivo.

Dietary Arachidonate Enhances Tissue Arachidonate Levels and Eicosanoid Production in Syrian Hamsters

Eicosanoids are oxidative derivatives of arachidonic acid. When produced in excess many of them are proinflammatory agents. This study investigates whether dietary arachidonic acid enhances arachidonic acid phospholipid content of various tissues and whether this enrichment increases eicosanoid production. Male Syrian hamsters were divided into four groups and fed diets supplemented with ethyl esters of oleic acid, linoleic acid, arachidonic acid or eicosapentaenoic acid. Differences in the composition of the phospholipid fatty acids were monitored in liver, lung, heart, spleen, kidney, testes, macrophages and platelets. In all tissues analyzed, the phospholipid content of arachidonic acid was significantly higher in the arachidonic dietary group compared with all other dietary groups (average > 50% higher). In contrast, increasing dietary linoleic acid by 50% had little effect on altering tissue arachidonic acid levels. Following in vitro stimulation, macrophages and platelets from animals maintained on arachidonic acid produced, in general, the highest levels of eicosanoids compared with cells from animals fed the other diets. Significant differences were observed in prostaglandin E2 (macrophages) and thromboxane B2 (platelets) formation when compared with the oleic acid and eicosapentaenoic acid dietary groups. The data demonstrate that including low to moderate levels of arachidonic acid in the diet increases macrophage and platelet arachidonic acid levels and may augment eicosanoid production.

Real-time intelligent classification of COVID-19 and thrombosis via massive image-based analysis of platelet aggregates.

Microvascular thrombosis is a typical symptom of COVID-19 and shows similarities to thrombosis. Using a microfluidic imaging flow cytometer, we measured the blood of 181 COVID-19 samples and 101 non-COVID-19 thrombosis samples, resulting in a total of 6.3 million bright-field images. We trained a convolutional neural network to distinguish single platelets, platelet aggregates, and white blood cells and performed classical image analysis for each subpopulation individually. Based on derived single-cell features for each population, we trained machine learning models for classification between COVID-19 and non-COVID-19 thrombosis, resulting in a patient testing accuracy of 75%. This result indicates that platelet formation differs between COVID-19 and non-COVID-19 thrombosis. All analysis steps were optimized for efficiency and implemented in an easy-to-use plugin for the image viewer napari, allowing the entire analysis to be performed within seconds on mid-range computers, which could be used for real-time diagnosis.

Advanced materials engineering in historical gypsum plaster formulations

We show how historical gypsum plaster preparation methods affect the microstructure and the wettability properties of the final stucco materials. We reproduced a traditional Persian recipe (Gach-e Koshteh, ~14thcentury AD), which involves a continuous mechanical treatment during plaster hydration. These samples were compared with a laboratory-replicated historical recipe from Renaissance Italy (Gesso Sottile, ~15thcentury AD) and contemporary low-strength plaster. The Koshteh recipe induces the formation of gypsum platelets, which exhibit preferential orientation in the plaster bulk. In contrast, the Italian and low-strength plasters comprise a typical needle-like morphology of gypsum crystals. The platelets in Koshteh expose the more hydrophilic {010} face of gypsum in a much more pronounced manner than needles. Consequently, the Iranian plaster displays enhanced wettability, enabling its direct use for water-based decoration purposes, or as a fine finishing thin layer, without the need of mixing it with a binder material. Contrary, in Sottile, gypsum crystals are left to equilibrate in large excess of water, which promotes the growth of long needles at the expense of smaller crystals. Typically, such needles are several times longer than those found in a control regular plaster. For this crystal habit, the total surface of hydrophilic faces is minimized. Consequently, such plaster layers tend to repel water, which can then be used, e.g., as a substrate for oil-based panel paintings. These findings highlight the development of advanced functional materials, by tuning their microtexture, already during the premodern era.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Laboratory Markers of Platelet Production and Turnover

Platelets are formed from bone marrow megakaryocytes, circulate in blood for 7-10 days, and then are destroyed in the spleen and/or liver. Platelet production depends on the megakaryocyte population state in the bone marrow: number and size of the cells. The platelet turnover, i.e., the number of platelets passing through the bloodstream in a certain time, is determined by both the rate of their production and the rate of their destruction. The review considers laboratory markers, which are used to assess platelet production and turnover in the patients with hematologic and cardiovascular pathologies. These markers include some characteristics of platelets themselves: (i) content of reticulated ("young") forms in the blood detected by their staining with RNA dyes; (ii) indicators of the platelet size determined in hematology analyzers (mean volume, percentage of large forms) and in flow cytometers (light scattering level). Alterations of platelet production and turnover lead to the changes in blood plasma concentrations of such molecules as thrombopoietin (TPO, main mediator of megakaryocyte maturation and platelet formation in the bone marrow) and glycocalicin (soluble fragment of the membrane glycoprotein Ib detached from the surface of platelets during their destruction). Specific changes in the markers of platelet production and turnover have been observed in: (i) hypoproductive thrombocytopenias caused by suppression of megakaryocytes in the bone marrow; (ii) immune thrombocytopenias caused by accelerated clearance of the autoantibody-sensitized platelets; and (iii) thrombocytosis (both primary and reactive). The paper presents the data indicating that in patients with cardiovascular diseases an increased platelet turnover and changes in the corresponding markers (platelet size indexes and content of reticulated forms) are associated with the decreased efficacy of antiplatelet drugs and increased risk of thrombotic events, myocardial infarction, and unstable angina (acute coronary syndrome).

Особенности оказания трансфузиологической поддержки пациентам с гипопродукционной тромбоцитопенией в условиях дневного стационара ГБУЗ НСО «Новосибирский клинический центр крови»

Introduction. Hypoproductive thrombocytopenias caused by insufficient platelet formation in the bone marrow are usually a manifestation of the underlying process characterized by inhibition of thrombocytopoiesis. They accompany aplastic anemia, acute leukemia, primary myelofibrosis, myelodysplastic syndrome, may be the result of tumor metastasis to the bone marrow, with the substitution of the megakaryocyte lineage, as well as a complication of radiation therapy and chemotherapy. Aim. To analyze the features of the provision of transfusion support to patients with hypoproductive thrombocytopenia due to oncological or hematological diseases in the day hospital at the Novosibirsk Clinical Blood Center (NCBC). Materials and methods. The study included 222 patients of the NCBC day hospital who underwent replacement therapy with platelet concentrate to correct thrombocytopenia and hemorrhagic syndrome for the period 2017–2022. Results. Correction of thrombocytopenia due to malignant neoplasms (solid tumors) was performed in 69 patients and due to hematological diseases – in 153 patients. The average number of doses of platelet concentrate was 3.7 ± 2.4 for patients with solid tumors; the clinical efficacy of replacement therapy was achieved in this group of patients in all cases. For patients with hematologic-related thrombocytopenia, the average number of doses of platelet concentrate was 3.2 ± 1.3; the clinical efficacy in this group of patients was achieved in 131 cases out of 153. Conclusion. Replacement therapy with blood components in transfusion-dependent and comorbid patients in a day hospital allows in most cases (85.6%) to carry out effective clinical correction of thrombocytopenia and achieve complete reverse of manifestations of hemorrhagic syndrome (100% of cases).

Histone deacetylation-regulated cell surface Siglec-7 expression promoted megakaryocytic maturation and enhanced platelet-like particle release

BackgroundFunctioning as important hematologic cells for hemostasis, wound healing and immune defense platelets are produced before being released into the blood by cytoplasmic fragmentation at the end of the megakaryocyte (MK) differentiation, during which the involvement of both apoptosis and autophagy has been reported. Inhibitory sialic acid–binding immunoglobulin-like lectin-7 gene (Siglec-7) can be expressed on platelets and induce apoptosis on activation for uncharacterized function. ObjectiveWe aimed to investigate the regulatory mechanism for Siglec-7 activation along MK differentiation and its physiologic role during the MK maturation and platelet formation. MethodsBy using 2 well-established MK differentiation models (HEL and K562) and human primary CD34+ cell, we examined the upregulations of transcript and protein levels of Siglec-7 during MK differentiation, and the effect of Siglec-7 surface presence on MK differentiation and platelet-like particles (PLPs) release. ResultsWe show that both transcripts and surface Siglec-7 were elevated during MK differentiation, and the histone deacetylase 1 (HDAC1) acted as a negative regulator for Siglec-7 activation. By increasing Siglec-7 surface expression, we found that increased presence of Siglec-7 not only enhanced MK maturation but also the release of PLPs by activating caspase 3-dependent signaling, as evidenced in the observation of more CD41, polyploidy, and platelet factor 4 transcript formations. ConclusionIn this study, we demonstrated that Siglec-7 activation was subjected to epigenetic regulation, and the resulting induced expression of surface Siglec-7 played an important regulatory role in promoting MK differentiation, maturation, and PLP formation.

Numerical Modelling of Erythrocyte Sticking Mechanics

The mechanics of thrombus formation includes the interaction of platelets, fibrin, and erythrocytes. The interaction was analyzed as the erythrocyte approaches the activated platelet and fibrin thrombus formation. The discrete element method (DEM) was used for the numerical experiment. Details of numerical experiments are presented by analyzing the dynamics of an erythrocyte in the process of interaction; a history of force, velocity, and displacement is given. It is usually assumed that the objects modeled by the DEM can oscillate during the sticking process. Modeling only this requires specialized knowledge and long-term research. However, by taking into account the influence of the fluid and modeling a soft biological cell, a completely different behavior can be achieved using the DEM method. The results of the numerical experiment show the different behavior of the erythrocyte when it interacts with a certain surface. Without taking into account the influence of the fluid in the sticking process, oscillations of the erythrocyte are observed. Meanwhile, after evaluating the influence of the liquid on the sticking process, there are no oscillations and unloading processes, which are typical for ultrafine objects. It is hoped that this will contribute to the study of the complex process of thrombus formation.

Assessment of microvesicles from different cell origins in patients with psoriasis: evidence of thrombogenic, proinflammatory microenvironment in the absence of established cardiovascular disease.

Psoriasis is associated with increased cardiovascular risk. Endothelial, platelet, and erythrocyte microvesicles (MVs) are novel biomarkers of endothelial dysfunction and thromboinflammation. We explored whether MVs of different cell types are elevated in patients with psoriasis, and investigated potential associations with disease severity and macrovascular function. Endothelial, platelet and erythrocyte MVs were measured using a standardized flow cytometry protocol in psoriasis patients and controls free from established cardiovascular disease. Carotid intima-media thickness (IMT) and pulse wave velocity (PWV) were measured as markers of subclinical atherosclerosis and arterial stiffness. Psoriasis severity was assessed with PASI (Psoriasis Area Severity Index). Both platelet (p < 0.001) and erythrocyte MVs (p = 0.046), yet not endothelial MVs, were significantly increased in patients with psoriasis (n = 41) compared with controls (n = 41). Patients with higher PASI (≥10) presented significantly higher levels of ErMVs compared to those with lower PASI (<10) (p = 0.047). Carotid IMT and PWV were comparable between psoriasis patients and controls and did not significantly correlate with MVs. In the multivariate analysis, psoriasis was identified as an independent predictor of both platelet (p < 0.001) and erythrocyte MVs (p = 0.043), while hypertension was independently associated with endothelial MVs (p < 0.001). Increased formation of platelet and erythrocyte MVs may be evident in psoriasis patients and is indicative of prothrombotic, proinflammatory microenvironment, even in the absence of subclinical macrovascular dysfunction and before the clinical onset of overt cardiovascular complications. Potential mechanistic links and prognostic implications of increased MVs in psoriasis warrant further investigation.

Insights into Regulatory Factors in Megakaryocyte Development and Function: Basic Mechanisms and Potential Targets.

Platelets are small, anucleate cellular fragments, which are produced by megakaryocytes, and play a key role in hemostasis and thrombus formation. The differentiation of megakaryocytes from hematopoietic stem cells in bone marrow and the development of megakaryocytes into platelets is a complex process. Various regulatory factorsin megakaryopoiesis including cytokines, growth factors, transcription factors, and gene expression, are all involved in the process of thrombocytopoiesis and play distinct roles in different stages of megakaryocytes development. In this review, we summarize the current state of knowledge ofmultiple regulatory factors including the TPO/Mpl signaling pathway, transcription factors, RasGTPases family, estrogen, and microRNAs. Altogether, we aimed to discuss more molecular mechanisms of megakaryocytes differentiation and maturation, and possess a better understanding of platelet formation.

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis

AbstractGlycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature β1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and β1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

Low-Dose Rhein Sensitizes Platelet Production to Thrombopoietin Receptor Agonists (TPO-RA) in Immune Thrombocytopenia

Introduction: Primary immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by isolated thrombocytopenia and risk of hemorrhage. Impaired megakaryocyte maturation and insufficient platelet formation, induced by platelet-specific autoantibodies or ITP plasma, play vital roles in its pathogenesis. Although thrombopoietin receptor agonists (TPO-RA) is recommended the first choice among second-line treatment of ITP, clinical data have indicated that nearly half of ITP patients failed or relapsed during eltrombopag maintenance, and increasing drug dose cannot reverse therapeutic response. Diacerein, hydrolyzed into its active metabolite rhein after oral administration, is a non-steroidal anti-inflammatory drug used in osteoarthritis. Our team first observed that 3 ITP patients with low platelet levels (30-50 ×109/L) during eltrombopag treatment, achieved significant platelet response (≥100×109/L) after taking diacerein concomitantly due to the complaint of arthralgia. These data suggested that diacerein may have sensitization effect on TPO-RA, and this novel combination is expected to boost thrombopoiesis in ITP. Thus, in vitro and in vivo experiments were performed to explore the role of TPO-RA plus rhein in megakaryocyte growth and platelet formation in ITP. Methods: Twenty-eight eltrombopag-inefficient or relapsed ITP patients and 15 healthy volunteers were included in this study. We sorted CD34+ cells from healthy umbilical cord blood mononuclear cells, then cultured them in SFEM medium containing thrombopoietin, interleukin-3, stem cell factor and 10% serum from either ITP patients or healthy donors. Different doses of eltrombopag combined with rhein (0-100 μM) were added on the fourth day, and cell suspension was collected on the 14th day. Flow cytometry, RNA sequencing, proteomic sequencing and cytokine assay were performed to evaluate the overlaying effect and underlying mechanisms of rhein on eltrombopag. In addition, active ITP murine models were established to evaluate the therapeutic effects of romiplostim plus rhein in vivo. Results: Compared with eltrombopag, eltrombopag combined with low-dose rhein significantly increased the proportion of CD41+, CD41+CD42+, CD41+CD61+ megakaryocytes in vitro. We further found that eltrombopag plus low-dose rhein significantly promoted polyploid maturation and apoptosis of CD41+ megakaryocytes, as well as platelet production in vitro (Fig 1). The ITP mice exhibited profound thrombocytopenia, and their platelet counts decreased significantly by the second week after irradiation. Our data showed that romiplostim plus low-dose rhein significantly alleviated thrombocytopenia than romiplostim monotherapy on day 21 and day 28 (Fig 2). Mice receiving different doses of rhein monotherapy were comparable in platelet counts with control group. Flow cytometry analysis suggested that the combination therapy significantly increased megakaryocyte percentage and polyploidization than romiplostim in ITP mice. Subsequently, RNA sequencing and proteomic analysis revealed that the combination of eltrombopag and low-dose rhein promoted megakaryocyte growth and platelet formation, decreased proinflammatory cytokines, and upregulated phosphorylated PI3K in vitro. Cytokine analysis indicated that eltrombopag plus low-dose rhein significantly increased supernatant level of TGF-β, and reduced IFN-α, IFN-γ, IL-12p70, IL-17A and IL-18 than eltrombopag. Furthermore, it was confirmed that the addition of rhein to TPO-RA significantly promoted megakaryocyte PI3K phosphorylation in vitro and in vivo. PI3K inhibition offset the enhanced effect of eltrombopag plus low-dose rhein over eltrombopag in megakaryocyte maturation and platelet formation. Conclusions: In sum, our data reveal a sensitizing role of rhein to TPO-RA in megakaryocyte polyploidization and platelet formation in ITP, and this novel combination therapy warrants further investigation as an optimized strategy for ITP patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Eltrombopag Plus Diacerein Versus Eltrombopag in Adult Primary Immune Thrombocytopenia: Interim Analysis of a Multicenter, Randomized, Controlled, Phase 2 Trial

Background: Primary immune thrombocytopenia (ITP) is an autoimmune bleeding disorder. Eltrombopag is a preferred second-line treatment of ITP, but many patients still have no response or relapse due to unknown reasons. Our preclinical data indicate that rhein, the active metabolite of diacerein, can enhance megakaryocyte sensitivity to thrombopoietin receptor agonists, and promote megakaryocyte polyploidization and platelet formation in ITP. Thus, a prospective randomized controlled trial was conducted to compare the efficacy and safety of eltrombopag plus diacerein versus eltrombopag alone in adult ITP. Here we report the interim analysis of this study. Methods: This multicenter, open-label, randomized, phase 2 trial screened eltrombopag-inefficient or relapsed ITP patients from five tertiary medical hospitals in China. Eligible participants were randomly assigned into the combination arm (eltrombopag orally at an initial dose of 75 mg daily for 14 days, plus diacerein orally at an initial dose of 50 mg bid for 14 days) or the monotherapy arm (eltrombopag orally at an initial dose of 75 mg daily for 14 days) by masked statisticians. To maintain participants’ platelet counts at a safe range, individualized dosages were allowed to be adjusted by physicians according to the protocol. The primary outcome was initial response at day 15. Complete response was defined as a platelet count at or above 100×109/L and an absence of bleeding. Partial response was defined as a platelet count at or above 30×109/L but less than 100×109/L and at least a doubling of the baseline platelet count and an absence of bleeding. No response was defined as a platelet count of less than 30×109 cells per L, or less than two-times increase from baseline platelet count, or bleeding. Key secondary enpoints included response at day 28, time to response (TTR), duration of response, bleeding scores, health-related quality of life assessment and adverse events (AEs). This trial was registered with ClinicalTrials.gov (NCT04917679). Results: From September 2020 to December 2021, 90 patients were screened for eligibility, of whom 24 were ineligible, 66 were randomly assigned to receive either eltrombopag plus diacerein (n=32) or eltrombopag alone (n=34). Four patients did not receive allocated intervention and they were excluded from the analysis. Baseline characteristics were balanced between the two arms. The total population (female accounted for 51.6%) had a mean age of 42.6 years and a mean platelet count of 8.6 ×109/L. At day 15, a significantly higher proportion of participants in the eltrombopag plus diacerein arm (13 [43.3%] of 30) than in the eltrombopag monotherapy arm (5 [15.6%] of 32) had an initial response (p=0.0247). At day 28, the response rate in the combination arm was higher than that in the eltrombopag arm even though it did not reach statistical significance (10 [33.3%] of 30 vs 4 [12.5%] of 32, p=0.0699). There was no significant difference in median TTR between the two arms (p>0.05). During the follow-up period, the duration of response was longer in the eltrombopag plus diacerein arm than in the eltrombopag arm by the Kaplan Meier analysis (Hazard ratio 0.57, 95% CI 0.34-0.96, p=0.0178). Eltrombopag plus diacerein exhibited lower bleeding scores and better health-related quality of life scores than eltrombopag. Incidence of AEs was similar between the two arms. Most of AEs were mild and resolved spontaneously after treatment was completed. The incidence of gastrointestinal reactions in the combination arm was slightly higher, but no significant difference was observed. There were no grade 4 or 5 AEs. No treatment-related deaths occurred. Conclusions: In conclusion, our findings suggest that the addition of diacerein as a sensitizer to eltrombopag has improved initial response than eltrombopag alone for eltrombopag-inefficient or relapsed ITP patients. This oral combination therapy warrants further exploration. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sitagliptin Alleviates Excessive Autophagy in Macrophages in Immune Thrombocytopenia Patients to Promote Megakaryopoiesis Via the AMPK/mTOR Pathway

Introduction: The pathogenesis of ITP is complex and mainly manifests as autoantibody-mediated platelet destruction and impaired platelet production. Autophagy is a highly conserved cellular process that eliminates damaged organelles and promotes the degradation of misfolded proteins. Appropriate autophagy exerts a protective effect, while excessive autophagy may lead to cell death and increased inflammation. The specific role of bone marrow macrophages (BM MΦ) in regulating megakaryopoiesis in ITP patients has not been elucidated. Sitagliptin, a DPP-4 inhibitor, was reported to play a key role in the production and engraftment of hematopoietic cells by modulating SDF-1α and several colony-stimulating factors, thus enhancing hematopoietic stem and hematopoietic progenitor cell recovery. However, whether sitagliptin has the potential to promote megakaryocyte maturation and platelet production in ITP is unclear. Therefore, this article explores the effect of macrophage autophagy on megakaryopoiesis in ITP patients and investigates its underlying role in the pathogenesis of ITP in order to explore whether sitagliptin could be a potential treatment for ITP. Methods: BM MΦ and CD34+ cells were isolated from ITP patients and healthy control subjects. Excessive autophagy in bone marrow macrophages from ITP patients was confirmed by Western blotting and transmission electron microscopy. PCR and Western blotting were used to assess the expression of A20 and autophagy-related AMPK/mTOR/ULK1 pathway components in ITP-MΦ. Vectors carrying A20 cDNA/TNFAIP3-specific shRNA were transduced into the BM MΦ from either healthy control subjects or ITP patients. The different BM MΦ populations were separately cocultured with CD34+ cells. We compared the ability of BM MΦ to support megakaryocytopoiesis by determining the relative percentages of MKs at different stages using flow cytometry as well as CFU counts of megakaryocytes and platelets in culture. The mechanism by which sitagliptin normalized the impaired function of ITP-MΦ was evaluated in vitro. A prospective single-arm clinical cohort study was designed to explore the efficacy and safety of sitagliptin in the treatment of corticosteroid-resistant or relapsed ITP. Results: The expression of LC3-II/LC3-I and Beclin1 increased in ITP-MΦ, and the expression of P62 decreased. The number of autophagic vesicles also increased, indicating enhanced autophagy in ITP-MΦ. Transcriptome sequencing and untargeted metabolomics revealed that macrophage autophagy-related pathways were involved in the pathogenesis of ITP. Further studies confirmed that ITP-MΦ regulated megakaryopoiesis, which was dependent on the A20-mediated autophagy-related AMPK/mTOR/ULK1 pathway. Inhibition of A20 expression with A20-shRNA inhibited the AMPK/mTOR/ULK1 pathway and alleviated excessive autophagy in ITP-MΦ, thus increasing the number of megakaryocytes, the proportion of hyperploid megakaryocytes and the number of platelets produced in the ITP-MΦ coculture system. Inhibition of autophagy with the autophagy inhibitor 3-MA reversed the A20 overexpression-induced excessive autophagy in MΦ and normalized the differentiation and maturation of megakaryocytes. Inhibiting the AMPK/mTOR/ULK1 signaling pathway with the AMPK inhibitor Compound C reversed the A20 overexpression-induced increase in the expression of AMPK/mTOR/ULK1 autophagy pathway components in MΦ and normalized megakaryopoiesis. Sitagliptin was found to inhibit the expression of A20 and AMPK/mTOR/ULK1 autophagy pathway components in ITP-MΦ in vitro. Sitagliptin improved the ability of ITP-MΦ to promote the differentiation and development of megakaryocytes and platelet production. Sitagliptin was administered to a total of 56 patients with corticosteroid-resistant or relapsed ITP. Complete response and overall response were observed in 11 (19.6%) and 21 patients (37.5%), respectively. Conclusions: A20 mediated the AMPK/mTOR/ULK1 signaling pathway to regulate autophagy in ITP-MΦ and affected megakaryopoiesis and platelet formation. Sitagliptin could restore the proper function of the A20-mediated AMPK/mTOR/ULK1 signaling pathway in ITP-MΦ, thus promoting megakaryopoiesis. Sitagliptin resulted in a sustained response in patients with corticosteroid-resistant/recurrent ITP and exhibited good safety. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SETD2 Regulates Nuclear Division to Achieve Polyploidization in Megakaryocyte

Megakaryocytes (MKs) undergo a fascinating endomitosis process to become naturally large multilobulated, polyploid nuclei cells that give rise to platelets. Two key steps involving in this process are the controlled failure in cytokinesis (cytoplasm division) and karyokinesis (nuclear division). The cytokinesis defect has been reported to be regulated by factors related to abnormal actomyosin ring and cleavage furrow formation such as RhoA, MYH9 and Rac1. In contrast, it remains largely unknown how nuclear division defect is regulated during MK endomitosis. Using a mouse model and an in vitro differentiation system, we found SETD2 expression markedly declined when MKs underwent polyploidization. Knockdown of SETD2 increased the high ploidy of primary bone marrow (BM) murine MKs, as well as MKs derived from cultured human cord-blood CD34+ cells and MEG-01 cells. Furthermore, the higher ploidy of MKs was accompanied by the increase of platelet formation both in the mouse model and the in vitro differentiation system. With forced overexpression of SETD2, the proportion of high ploidy MKs (≥8N) was remarkably decreased. These data suggest that SETD2, an α-tubulin methyltransferase, negatively regulates polyploidization during megakaryopoiesis. To investigate how SETD2 regulates endomitosis, we employed live-cell imaging to monitor the whole process of endomitosis at the single-cell level by observing the spindle geometry. We found SETD2 deficient MKs were capable of completing the endomitotic cycle, allowing cells to reach a higher ploidy in a significantly shorter time compared to control cells (2.9 hours vs. 4.9 hours). In comparison, overexpression of SETD2 significantly increased endomitosis cycle time compared to control conditions (5.2 hours vs. 4.3 hours). These results show that SETD2 deficiency accelerates the endomitotic cell cycle. Moreover, loss of SETD2 increased the percentage of cells within the presence of nucleoplasmic bridges (NPBs), a thin DNA link referred as a characteristic structure for karyokinesis defect. These results suggest that SETD2 may regulate endomitosis through facilitating the defect of karyokinesis. During karyokinesis, microtubules localized near spindle poles are need to be depolymerized as force-generating apparatus to separate nuclei. Thus, we measured the amount of polymerized and depolymerized microtubule forms in the presence and absence of SETD2. We found that loss of SETD2 decreased the proportion of the depolymerized form of tubulin in spindles, while SETD2 overexpression increased depolymerized microtubule forms. These data suggest that loss of SETD2 promotes the defect of karyokinesis by reducing depolymerized microtubule state. Mechanistically, as SETD2 could trimethylate α-tubulin at lysine 40 (α-tubK40me3), we found that the levels of α-TubK40me3 near spindle poles were inversely correlated with the degree of karyokinesis defect and MK polyploidization. Moreover, SETD2-deficiency resulted in an absence of α-TubK40me3 with an increase of the MK ploidy. And this ploidy increase was largely reversed by overexpressing a mutant tubulin protein that mimics the α-tubK40me3. Together, these data indicate that SETD2-mediated α-TubK40me3 regulates MK polyploidization by influencing microtubule dynamics. In summary, our study reveals that SETD2 is an important, novel regulator of MK endomitosis by regulating α-TubK40me3 to influence microtubule dynamics. These findings help fill current knowledge gaps in the mechanism of the karyokinesis defect during MK endomitosis. Targeting SETD2 may improve high ploidy MKs and platelet production that could promote the regeneration of platelets in vitro and help treat megakaryocytic dysplasia diseases with abnormal polyploidization in the future.

Cyclic Thrombocytopenia: A Case Report

Introduction Cyclic thrombocytopenia (CTP) is a rare disorder characterized by episodes of oscillating platelet counts. It is usually associated with another primary hematologic disorder but there are idiopathic cases in the literature. Patients with CTP have similar clinical features and symptoms with idiopathic thrombocytopenic purpura (ITP), so they are often misdiagnosed as ITP and treated accordingly. In general, CTP does not respond to ITP therapies, so it should always be considered when treatments are not effective. It is more common in females. However, in our case report we present a 93-year-old male with CTP who was initially mistaken for ITP. Case description A 93-year-old male patient with a past medical history of hypertension was initially thought to have idiopathic thrombocytopenia (ITP) presenting with mucosal gum bleeding and a petechial rash. He was found to have isolated thrombocytopenia, and no evidence of other cytopenias. He did trials of multiple medications such as corticosteroids and intravenous immunoglobulin (IVIG) with no response. He was not a surgical candidate for splenectomy. He also deferred a bone marrow biopsy, though peripheral blood testing with flow cytometry showed a small monoclonal B cell population. Peripheral blood myeloid sequencing panel showing ASXL1 and EZH2. He was started on Eltrombopag, a thrombopoietin (TPO) agonist with significant lability in the platelet count, from 8,000 per microliter (mcL) to over 1,000,000 per mcL. Romiplostim, another TPO agonist also gave similar results. A diagnosis of cyclic thrombocytopenia was suspected. A Helicobacter pylori antibody was positive, and treated with antibiotics with no improvement in platelet count oscillations. Thyroid function was within normal limits. T-cell gene rearrangement was negative. Thrombopoetin (TPO) levels were checked and these were found to be in discordance with the platelet count, consistent with a CTP diagnosis. He was started on cyclosporine 150 milligrams twice daily with significant improvement in fluctuations of platelet counts. Discussion Cyclic hematopoietic disorders are rare and have regular predictable oscillations of one or more cellular components of the blood. Physiologically, there is a complex interplay between thrombopoietic stimulation and feedback inhibition in order to maintain the circulating platelets within the normal range. An error in any of these steps results in oscillation of the platelet counts (cyclic thrombocytopenia). Many mechanisms are behind the pathophysiology of this disease, such as: autoimmune destruction of platelets, hypoplasia of megakaryocytes, a defect in regulating platelet formation and an infectious or hormonal etiology. There are many reports linking T-lymphocytes proliferation and clonal TCR gene rearrangement to cyclic thrombocytopenia. We noticed in the literature that there can be variable responses to TPO agonists, but often can result in very high platelet counts. Due to its rarity, there are only case reports which show reports of remission after hormone therapy with danazol and immune suppressant therapies, such as cyclosporine. Further studies are needed to determine the mechanism responsible for cycling phenomena and optimal treatments for this disorder. In conclusion, any presumed ITP case that does not respond well to the standard therapy of ITP should be reevaluated for the possibility of cyclic thrombocytopenia.