Bone Marrow Megakaryocytes Research Articles

Eltrombopag for Treatment of Thrombocytopenia Following Hematopoietic Stem Cell Transplantation

Objective:This study aimed to evaluate the efficacy and safety of eltrombopag (ELT) in the treatment of thrombocytopenia following hematopoietic stem cell transplantation (HSCT).Materials and Methods:Forty-eight patients treated with ELT for thrombocytopenia after allogeneic or autologous transplantation at the Erciyes University Bone Marrow Transplantation Center between July 2017 and July 2021 were evaluated retrospectively.Results:Forty-eight HSCT recipients were included in this study. Thirty (62.5%) patients were evaluated as having experienced delayed platelet recovery (DPR) and 18 (37.5%) patients as having experienced secondary failure of platelet recovery (SFPR). The median platelet count before ELT treatment was 13x109/L (range: 3-20x109/L). Twenty-three patients responded to treatment and the cumulative incidence of successful platelet recovery was 48%. Patients with both DPR and SFPR responded, but patients with DPR had a higher response rate (50% vs. 44%). The median platelet count of the 23 responding patients was 12x109/L (5-19x109/L) before treatment and 68x109/L (52-266x109/L) after treatment (p<0.0001). While the number of bone marrow megakaryocytes before treatment was adequate in 22 (46%) cases, it was decreased in 26 (54%) cases. Patients with adequate bone marrow megakaryocytes had a better response rate than those without (77% vs. 23%, p<0.0001). The group with adequate megakaryocytes responded to treatment at a median of 33 days (range: 9-174 days). Patients with decreased megakaryocytes responded at a median of 55 days (30-164 days) (p=0.002). No drug-related side effects were observed in any patients.Conclusion:This real-life experience demonstrates that ELT is an effective and safe treatment option for thrombocytopenia after HSCT. The adequacy of bone marrow megakaryocytes before ELT treatment was an important factor affecting response to treatment.

Platelet and Megakaryocyte Roles in Innate and Adaptive Immunity.

Classically, platelets have been described as the cellular blood component that mediates hemostasis and thrombosis. This important platelet function has received significant research attention for >150 years. The immune cell functions of platelets are much less appreciated. Platelets interact with and activate cells of all branches of immunity in response to pathogen exposures and infection, as well as in response to sterile tissue injury. In this review, we focus on innate immune mechanisms of platelet activation, platelet interactions with innate immune cells, as well as the intersection of platelets and adaptive immunity. The immune potential of platelets is dependent in part on their megakaryocyte precursor providing them with the molecular composition to be first responders and immune sentinels in initiating and orchestrating coordinated pathogen immune responses. There is emerging evidence that extramedullary megakaryocytes may be immune differentiated compared with bone marrow megakaryocytes, but the physiological relevance of immunophenotypic differences are just beginning to be explored. These concepts are also discussed in this review. The immune functions of the megakaryocyte/platelet lineage have likely evolved to coordinate the need to repair a vascular breach with the simultaneous need to induce an immune response that may limit pathogen invasion once the blood is exposed to an external environment.

The Value of Combined Detection of Megakaryocyte and Platelet Parameters for the Diagnosis of Primary Immune Thrombocytopenia

Objective:To investigate the application value of bone marrow megakaryocyte count, the proportion of megakaryocytes at each stage, and the platelet parameter in the clinical diagnosis of primary immune thrombocytopenia (ITP). Methods: The megakaryocyte and platelet parameter level in 62 ITP and 40 control group patients were compared and analyzed. Linear correlation analysis, Pearson correlation analysis, and ROC curves were performed for the correlation between megakaryocytes and platelet parameters. Results: Compared to the control group, the total number of megakaryocytes, the promegakaryocytes the granular megakaryocytes (GMeg), and naked megakaryocytes (NMeg), MPV, and P-LCR% in the ITP group increased. All differences were statistically significant (P<0.05). While the proportion of platelet-producing megakaryocytes (PMeg), PLT, and PCT decreased in the ITP group. These differences were statistically significant (P < 0.05). PLT was strongly positively correlated with PCT (r = 0.921, p<0.01). PCT was weakly positively with MPV (r = 0.309, p<0.05). MPV was positively correlated with P-LCR (r = 0.856, p<0.01). PDW was weakly positively correlated with P-LCR (r = 0.296, p<0.05) and Meg (r = 0.301, p<0.05), and negatively correlated with PMeg (r = -0.336, p<0.05). ROC curve analysis showed that PLT, PCT MPV and P-LCR% gave a high sensitivity（100.0%，81.0%，74.6%，90.5%，respectively.) and specificity (100.0%, 92.5%, 80.0%, 77.5%, respectively.) in diagnosis of ITP. Conclusion: The combined analysis of bone marrow megakaryocyte count, the proportion of megakaryocyte classification at each stage, and platelet parameters have an important reference value for auxiliary diagnosis of ITP.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/μm), or high-LET 56Fe-ions (148 keV/μm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and 56Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, 56Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.

The α2-adrenergic receptor pathway modulating depression influences the risk of arterial thrombosis associated with BDNFVal66Met polymorphism

Depression is associated with thrombotic risk and arterial events, its proper management is strongly recommended in coronary artery disease (CAD) patients. We have previously shown that the Brain-Derived Neurotrophic Factor (BDNF)Val66Met polymorphism, related to depression, is associated with arterial thrombosis in mice, and with an increased risk of acute myocardial infarction in humans. Herein, expanding the previous findings on BDNFVal66Met polymorphism, we show that desipramine, a norepinephrine reuptake-inhibitor, rescues behavioral impairments, reduces the arterial thrombosis risk, abolishes pathological coagulation and platelet hyper-reactivity, normalizes leukocyte, platelet, and bone marrow megakaryocyte number and restores physiological norepinephrine levels in homozygous knock-in BDNF Val66Met (BDNFMet/Met) mice. The in vitro data confirm the enhanced procoagulant activity and the alpha2A-adrenergic receptor (α2A-ADR) overexpression found in BDNFMet/Met mice and we provide evidence that, in presence of Met variant, norepinephrine is crucial to up-regulate procoagulant activity and to enhance platelet generation. The α2-ADR antagonist rauwolscine rescues the prothrombotic phenotype in BDNFMet/Met mice and reduces procoagulant activity and platelet generation in cells transfected with BDNFMet plasmid or exposed to pro-BDNFMet peptide. Finally, we show that homozygous BDNFMet/Met CAD patients have hyper-reactive platelets overexpressing abundant α2A-ADR. The great proplatelet release from their megakaryocytes well reflects their higher circulating platelet number compared to BDNFVal/Val patients. These data reveal an unprecedented described role of Met allele in the dysregulation of norepinephrine/α2A-ADR pathway that may explain the predisposition to arterial thrombosis. Overall, the development of α2A-ADR inhibitors might represent a pharmacological treatment for depression-associated thrombotic conditions in this specific subgroup of CAD patients.

Proanthocyanidin A1 promotes the production of platelets to ameliorate chemotherapy-induced thrombocytopenia through activating JAK2/STAT3 pathway

BackgroundChemotherapy-induced thrombocytopenia (CIT) is a severe adverse drug reaction, and the main reason for CIT is the destruction of megakaryocytes (MKs, precursor cells of platelet) in bone marrow by chemotherapy. Peanut skin, the seed coat of Arachis hypogaea L., is a traditional Chinese medicine commonly used to treat thrombocytopenia. However, its active compounds and the mechanisms remain unclear. PurposeThis study aims to clarify the active compounds of peanut skin to exhibit thrombogenic effects against CIT and their underlying mechanisms in vitro and in vivo. Study designThe bioassay-guided isolation based on the proliferation of MKs was used to explore the possible platelet-enhancing ingredients in peanut skin. HSCCC technique coupled with preparative HPLC was used to separate the active compounds. Dami cells and carboplatin-treated mice model were used to evaluate the thrombogenic effects of PS-1. Network pharmacology, molecular docking, dynamics simulation studies, kinase activity, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), isothermal dose-response fingerprint (ITDRFCETSA) and western blot analysis were performed to investigate the mechanisms of PS-1. ResultsProanthocyanidin A1 (PS-1) and its stereoisomers (PS-2–4) were demonstrated to promote the proliferation of MKs (Dami cells), especially PS-1 (EC50 = 8.58 μM). Further studies demonstrated that PS-1 could induce the differentiation of Dami cells in dose/time-dependent manner. Biological target analysis showed that PS-1 directly bound to JAK2 (KD = 2.06 μM) to exert potent activating effect (EC50 = 0.66 μM). Oral administration of PS-1 (25 or 50 mg/kg) significantly improved CIT, but this effect was confirmed to be inhibited by JAK2 inhibitor AG490, indicating that PS-1 exerted its efficacy through JAK2 in vivo. ConclusionProanthocyanins (PS-1–4) derived from peanut skin were first clarified as platelet-enhancing ingredients to improve CIT. The underlying mechanism of PS-1 was proved to promote the proliferation and differentiation of MKs via JAK2/STAT3 pathway both in vitro and in vivo.

Abivertinib inhibits megakaryocyte differentiation and platelet biogenesis.

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton's tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34+ hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34+ HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (<100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa.Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy.Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane.The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%).The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts. DisclosuresPatel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.

The mTOR Pathway in Platelets Contributes to the Pathophysiology of Experimental Cerebral Malaria

Background: Cerebral malaria is a highly prevalent infectious disease in Sub-Saharan Africa caused by the Plasmodium parasite. The pathogenesis of cerebral malaria results from damaged vascular endothelium induced by parasite sequestration, inflammatory cytokine production and vascular leakage, which results in increased brain permeability and death. While maladaptive responses from immune cells are thought to contribute, growing evidence suggests a crucial role of platelets in malaria pathophysiology. The mammalian target of rapamycin (mTOR) pathway is critical in regulating outcomes in malaria. Previous studies have demonstrated an mTOR specific inhibitor, rapamycin, is protective in a mouse model of experimental cerebral malaria (ECM). However, if the mTOR pathway in platelets specifically contributes to the pathogenesis of malaria is unknown.Methods: Platelet-specific mTOR-deficient (mTOR plt-/-) mice and littermate controls were subjected to a well-established model of ECM, using Plasmodium berghei ANKA. In addition, platelets isolated from human malaria patients were examined for differential regulation of the mTOR pathway using RNA-seq.Results: Platelet RNA-seq and Ingenuity Pathway Analysis from patients infected with P. vivax demonstrated enrichment of mTOR-associated pathways in platelets, such as mTOR signaling and p70S6K signaling, indicating mTOR associated genes are upregulated in human platelets during malaria infection. In mice infected with P. berghei ANKA, the mTOR pathway was activated in bone marrow-megakaryocytes and platelets based on phosphorylation of mTOR and its downstream effector, 4E-BP1. As the mTOR pathway regulates protein translation in platelets, we examined de novo protein synthesis and observed increased protein translation in platelets isolated from mice infected with P. berghei ANKA compared to uninfected controls.To study the specific role of platelet mTOR during ECM pathogenesis, mTOR plt-/- mice and wild-type controls (mTOR plt+/+), were infected with P. berghei ANKA. Platelet deficient-mTOR mice had significantly (p=0.0336) prolonged survival compared to wild-type mice. Increased survival was independent of parasitemia, suggesting platelets did not alter parasite reproduction. While thrombocytopenia and anemia were similar in both genotypes, mTOR plt-/- mice had significantly reduced brain (p=0.0067) and lung (p<0.0001) vascular permeability during late-stage ECM. Interestingly, flow cytometric assessment of leukocyte recruitment to the brain demonstrated a 1.7-fold (p=0.0442) reduction in inflammatory monocytes in platelet-deficient mTOR mice. However, mTOR plt-/- mice had significantly (1.4-fold, p=0.007) more inflammatory monocytes in the blood. Interestingly, circulating platelet-monocytes aggregates were significantly less in mTOR plt-/- compared to mTOR plt+/+ (p=0.0433). Taken together, these results suggest that platelets assist in the recruitment of leukocytes to the brain vasculature during ECM, which is impaired when mTOR is ablated.Conclusions: Our data demonstrates that the mTOR pathway in platelets plays a significant role in malaria pathogenesis. Deletion of platelet mTOR reduces vascular permeability and prolongs survival during ECM. We hypothesize that altered platelet-inflammatory monocyte interactions drive this phenotype. DisclosuresRondina: Platelet Transcriptomics: Patents & Royalties; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding.

Ker-050, an Inhibitor of TGF- β Superfamily Signaling, Promoted Thrombopoiesis and Reversed Immune Thrombocytopenia in a Mouse Model of Disease

KER-050 is a modified ActRIIA ligand trap that is designed to inhibit the activity of the TGF-β ligands, including activin A, activin B, GDF8 and GDF11, that act through the SMAD2/3 signaling cascade. Notably, in a Phase 1 clinical study we observed that, in addition to increases in red blood cells and hemoglobin, treatment with KER-050 elicited a robust and sustained increase in platelets (PLTs) in healthy volunteers. While ActRII ligand traps have been shown to increase erythropoiesis in preclinical and clinical studies, their role in thrombopoiesis has not yet been well-elucidated.A variety of conditions exist where hematopoiesis is impaired and cytopenia persists, including comorbidities associated with aging, diseases causing ineffective hematopoiesis such as myelodysplastic syndrome (MDS) and myelofibrosis (MF), and acute bleeding. Thrombocytopenia can arise from primary or secondary causes due to multiple etiologies and treatments are limited, aiming at treating the root cause of disease or replacing PLTs through transfusion. Therefore, there is an unmet medical need for more targeted treatments to correct thrombocytopenia. Here, in a series of preclinical studies, we investigated the mechanism of action of KER-050 on thrombopoiesis and evaluated its ability to accelerate recovery from acute platelet depletion.First, we examined how RKER-050 (a research form of KER-050) affected thrombopoiesis under homeostatic conditions. We observed that a single intraperitoneal dose of RKER-050 (10 mg/kg) to 11-week-old mice resulted in a 2-fold increase in PLTs compared to vehicle-treated mice 12 hours after treatment. The timing of the effect is suggestive of a direct effect of RKER-050 on terminal platelet maturation. Additionally, there was a 35% increase in the number of bone marrow (BM) megakaryocyte (Mk) progenitors (Lin -; sca1 -; cKit -; CD41 + cells), demonstrating that RKER-050 affected earlier stages of the PLT formation process. We also evaluated the effect of RKER-050 on the polyploidization of Mks, a hallmark of Mk differentiation. At 24 hours after treatment with RKER-050, there was an increase in BM CD41 + cells with ploidy greater than 16N compared to vehicle-treated mice, demonstrating that RKER-050 treatment resulted in a greater number of Mk that are potentially primed for platelet production. Taken together, these data are consistent with RKER-050 affecting multiple stages of thrombopoiesis in a preclinical model.We next tested whether RKER-050 affects PLTs in a mouse model of immune thrombocytopenia (IT) where antibodies directed against mouse GPIbα result in acutely reduced PLT numbers. In this model, mice receiving anti-GPIbα had a 25% reduction in PLT number at 4 days post-dose compared to IgG control-treated mice. At this point, the anti-GPIba cohort was divided into receiving either a single dose of vehicle or RKER-050. On day 7 following anti-GPIbα treatment, PLT counts in vehicle-treated mice were 62% lower compared to IgG-control-treated mice. In contrast, the GPIbα-mediated effect on PLT count was stabilized in the anti-GPIbα + RKER-050 group, which had 55% more platelets compared to the anti-GPIba + vehicle group. These data suggest that RKER-050 promoted thrombopoiesis in mice even under conditions when the system is acutely challenged and potentially could promote faster recovery from thrombocytopenia. Additionally, we observed a 25% increase in the number of CD41 + cells in the BM of the RKER-050-treated group compared to the vehicle-treated group at day 10 after PLT depletion, suggesting that under acute thrombocytopenia, RKER-050 treatment promoted differentiation of Mks as a mechanism of the accelerated recovery in platelet-depleted mice.In summary, our preclinical data demonstrate a potentially novel effect of RKER-050 on thrombopoiesis. RKER-050 treatment resulted in a rapid increase in PLTs, consistent with an effect on terminal maturation. Treatment also increased the number of Mk progenitors and increased the number of polyploid Mks, demonstrating an effect on early stages of thrombopoiesis. These findings support that RKER-050-targeted ligands regulate multiple stages of the thrombopoiesis pathway in mice. Additionally, our data demonstrate that KER-050 has the potential to accelerate the rate of PLT recovery due to acute depletion, and could represent a potential novel treatment option for thrombocytopenia in patients with MDS, MF and IT. DisclosuresFeigenson: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Nathan: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lamora: Keros Therapeutics: Current Employment. Fisher: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tseng: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Efficacy of Thrombopoietin Receptor Agonists in Evans Syndrome: An International Multicenter Experience

Background: Evans syndrome (ES) is a rare condition, defined as the presence of two autoimmune cytopenias (AIC), more frequently autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP), and rarely autoimmune neutropenia (AIN). ES can be classified as primary or secondary to various conditions, including lymphoproliferative disease (LPD), primary immune deficiencies (PID) or other systemic autoimmune diseases (AID). ES onset may be acute and life-threatening, whilst its clinical course is usually chronic and marked by several relapses of AIHA, ITP or both. First line therapy is based on steroids +/- intravenous immunoglobulins (IVIG), followed by rituximab and splenectomy in refractory/relapsing cases. Concerning ITP, splenectomy is not always feasible, and rituximab is poorly effective on the long term. Thrombopoietin receptors agonists (TPO-RA), such as romiplostim (ROMI) and eltrombopag (EPAG), are commonly used in primary ITP with high efficacy; however, their use in ES has never been systematically studied.Aim: to evaluate the efficacy and safety of TPO-RA in a multicentric cohort of patients with ES.Methods: all ES patients treated with TPO-RA at 8 European hematologic hospitals (3 Italian, 2 Danish, 1 United Kingdom and 2 Spanish) were retrospectively evaluated. Baseline hematologic parameters, associated conditions, previous treatments and those administered concomitantly to TPO-RA were registered. The time from diagnosis to first TPO-RA was collected. Response rates were evaluated at 1, 3, 6, and 12 months, and classified as partial (PR) or complete (CR), for platelets >30x10^9/L or >100x10^9/L, respectively. Treatment emergent adverse events (TAEs) were registered and graded according to CTCAE.R esults: As shown in Figure 1, 22 ES patients have been evaluated, 9 of whom secondary (40%). Almost all patients had received steroids +/- IVIG, and the majority at least one further line. The median time from diagnosis to TPO-RA start was 25,74 months (1-1390). Response rates to the first TPO-RA (16 EPAG and 6 ROMI) were: 82% at month 1, 84% at month 3, 83% at month 6 and 93% at month12. Eight patients started TPO-RA within 1 year from ES diagnosis. These patients displayed significantly lower platelets (p=0.01) as compared to others, however response rates were comparable. Of note, 73% of patients required concurrent therapies, including steroids +/- IVIG (N=13), danazol (N=2), rituximab (N=3), and immunosuppressants (N=3). Moreover, 7 patients required the addition of a rescue therapy to control ITP (steroids +/- IVIG N=4, rituximab N=1, danazol N=1, daratumumab N=1, immunosuppressants N=2, parsaclisib N=1), particularly in secondary ES (63% vs 33%). The latters less frequently showed increased bone marrow megakaryocytes (67% vs 92%) but had higher dysplasia (50% of patients vs 33% in primary ES). Interestingly, 5 subjects switched to the alternative TPO-RA (3 ROMI to EPAG and 2 vice versa) 2 because of no response (NR), and 3 for relapses. Three subjects responded but required additional therapies, including splenectomy, steroids +/- IVIG, or platelet transfusions. Ten out of 22 patients developed at least one TEAE: G1 thrombocytosis (N=1), G2 bone marrow fibrosis (N=1), G3/4 thrombosis (3 venous and 2 arterial: 1 pulmonary embolism, 1 cerebral vein thrombosis CVT, and 1 splanchnic thrombosis, 1 atrial thrombus and 1 acute myocardial infarction in the same APS patient experiencing CVT). Thrombosis was associated with the presence of secondary ES (p=0.03). Five patients are still receiving TPO-RA, whilst the other stopped because of persistent CR (N=12), thrombosis (N=3), increase in bone marrow reticulin fibrosis (N=1), or death for infectious complication.Conclusions: TPO-RAs were effective in more than 80% of ES patients, even heavily pre-treated. However, TPO-RA use was complicated by a high occurrence of thrombotic events that may be also favored by the underlying conditions. Additionally, TPO-RA required a concomitant therapy in the majority of patients, suggesting that in ES autoimmune platelet destruction cannot be completely overcome by bone marrow stimulation. [Display omitted] DisclosuresPatriarca: Incyte: Honoraria; Argenix: Honoraria; Pfizer: Honoraria; Amgen: Honoraria; Novartis: Honoraria; Takeda: Honoraria. Glenthøj: Sanofi: Research Funding; Bristol Myers Squibb: Consultancy; Saniona: Research Funding; Agios: Consultancy; Novo Nordisk: Honoraria; Novartis: Consultancy; Alexion: Research Funding; Bluebird Bio: Consultancy. Lund Hansen: Alexion: Research Funding; Novartis: Research Funding. Frederiksen: Gilead: Research Funding; Alexion: Research Funding; Novartis: Research Funding; Janssen Pharmaceuticals: Research Funding; Abbvie: Research Funding. Gonzalez-Lopez: Sobi: Other: Advisory board honoraria; Amgen: Other: Advisory board and speakers honoraria, Research Funding; Novartis: Other: Advisoryboard and speakers honoraria, Research Funding; Grifols: Other: Advisory board honoraria. Barcellini: Novartis: Honoraria; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees. Fattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Apellis: Speakers Bureau; Annexon: Consultancy; Amgen: Honoraria, Speakers Bureau. OffLabel Disclosure:Thrombopoietin receptor agonists in patients suffered from Evans syndrome when ITP is refractory/relapsing to standard therapies

Is platelet responsiveness to clopidogrel attenuated in overweight or obese patients and why? A reverse translational study in mice

Overweight or obese patients exhibit poorer platelet responses to clopidogrel. However, the mechanisms behind this phenotype remain to be elucidated. Here, we sought to discover whether and why obesity could affect the metabolic activation of and/or platelet response to clopidogrel in obese patients and high-fat diet-induced obese mice. A post hoc stratified analysis of an observational clinical study was performed to investigate changes in residual platelet reactivity with increasing body weight in patients taking clopidogrel. Furthermore, high-fat diet-induced obese mice were used to reveal alterations in systemic exposure of clopidogrel thiol active metabolite H4, ADP-induced platelet activation and aggregation, the expression of genes involved in the metabolic activation of clopidogrel, count of circulating reticulated and mature platelets, and proliferation profiles of megakaryocytes in bone marrow. The relevant genes and potential signalling pathways were predicted and enriched according to the GEO datasets available from obese patients. Obese patients exhibited significantly attenuated antiplatelet effects of clopidogrel. In diet-induced obese mice, systemic exposure of clopidogrel active metabolite H4 was reduced but that of its hydrolytic metabolite was increased due to down-regulation of certain P450s but up-regulation of carboxylesterase-1 in the liver. Moreover, enhanced proliferation of megakaryocytes and elevated platelet count also contributed. Obesity attenuated metabolic activation of clopidogrel and increased counts of circulating reticulated and mature platelets, leading to impaired platelet responsiveness to the drug in mice, suggesting that clopidogrel dosage may need to be adjusted adequately in overweight or obese patients.

Efficiency and safety of eltrombopag for multi-line failed Chinese patients with immune thrombocytopenia: cases with decreased megakaryocyte response well from single-center experience.

Immune thrombocytopenia (ITP) was defined using the International Consensus Guidelines as a platelet count <100×109/L in the absence of other causes or disorders that may be associated with thrombocytopenia. For patients without response to first-line treatment or refractory, TPO receptor agonist (RA) is an ideal choice. This study was to evaluate the efficiency and safety of eltrombopag for multi-line failed Chinese patients with immune thrombocytopenia (ITP) and analyze the possible factors that may contribute to the differences based on personal characteristics. Thirty-five multi-line failed ITP patients who received eltrombopag treatment were enrolled retrospectively at the First Affiliated Hospital of Zhejiang Chinese Medical University from January 2018 to August 2020. The general information, peripheral hemogram changes, count of bone marrow megakaryocyte (MK), peripheral T cell subsets were recorded, the response, and adverse effects, was evaluated. Results showed that the overall, complete, and partial response rates were 54.3% (n=19), 48.6% (n=17), and 5.7% (n=2) respectively to eltrombopag in our center. The overall response rate of patients with decreased MK was 70%, which was unexpectedly higher than that of the patient with increased or normal MK count (52.9% and 40%, respectively). For patients with poorer eltrombopag response group, more NK cells were found in peripheral blood, andthe patient with decreased MK have a higher level of T helper (Th) cells and regulatory T (Treg) cells. Nine eltrombopag-related adverse events were reported, and most commonly were upper respiratory tract infection (8.6%), elevated alanine transaminase (ALT, 5.7%), and venous thrombosis (5.7%). In conclusion, this study revealed that ITP patients with decreased megakaryocyte respond well to eltrombopag, and the abnormality of NK cells may play a role in patients with a poor eltrombopag response.

Quantitative LC\u2013MS/MS uncovers the regulatory role of autophagy in immune thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is an autoimmune haemorrhagic disease whose pathogenesis is associated with bone marrow megakaryocyte maturation disorder and destruction of the haematopoietic stem cell microenvironment.MethodsIn this study, we report the qualitative and quantitative profiles of the ITP proteome. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was conducted to elucidate the protein profiles of clinical bone marrow mononuclear cell (BMMC) samples from ITP patients and healthy donors (controls). Gene Ontology (GO) and Kyoto Encyclopaedia Genes and Genome (KEGG) pathway analyses were performed to annotate the differentially expressed proteins. A protein–protein interaction (PPI) network was constructed with the BLAST online database. Target proteins associated with autophagy were quantitatively identified by parallel reaction monitoring (PRM) analysis.ResultsOur approaches showed that the differentially expressed autophagy-related proteins, namely, HSPA8, PARK7, YWHAH, ITGB3 and CSF1R, were changed the most. The protein expression of CSF1R in ITP patients was higher than that in controls, while other autophagy-related proteins were expressed at lower levels in ITP patients than in controls.ConclusionBioinformatics analysis indicated that disruption of the autophagy pathway is a potential pathological mechanism of ITP. These results can provide a new direction for exploring the molecular mechanism of ITP.

Megakaryopoiesis and Platelet Biology: Roles of Transcription Factors and Emerging Clinical Implications.

Platelets play a critical role in hemostasis and thrombus formation. Platelets are small, anucleate, and short-lived blood cells that are produced by the large, polyploid, and hematopoietic stem cell (HSC)-derived megakaryocytes in bone marrow. Approximately 3000 platelets are released from one megakaryocyte, and thus, it is important to understand the physiologically relevant mechanism of development of mature megakaryocytes. Many genes, including several key transcription factors, have been shown to be crucial for platelet biogenesis. Mutations in these genes can perturb megakaryopoiesis or thrombopoiesis, resulting in thrombocytopenia. Metabolic changes owing to inflammation, ageing, or diseases such as cancer, in which platelets play crucial roles in disease development, can also affect platelet biogenesis. In this review, I describe the characteristics of platelets and megakaryocytes in terms of their differentiation processes. The role of several critical transcription factors have been discussed to better understand the changes in platelet biogenesis that occur during disease or ageing.

CD226 Is Required to Maintain Megakaryocytes/Platelets Homeostasis in the Treatment of Knee Osteoarthritis With Platelet-Rich Plasma in Mice.

Platelet-rich plasma (PRP) is a platelet-based application used to treat osteoarthritis (OA) clinically. The co-stimulatory molecule CD226 is expressed in T cells, NK cells, and also platelets. However, exact effects of CD226 on platelets and whether its expression level influences PRP efficacy are largely unknown. Here, CD226fl/flPF4-Cre mice were obtained from mating CD226 fl/fl mice with PF4-Cre mice. Blood samples and washed platelets were collected from the mice eyeballs to undergo routine blood tests and transmission electron microscopy. Differentially expressed proteins were detected by iTRAQ-based proteomics analysis. Animal OA models were established through surgical destabilization of the medial meniscus (DMM) for C57BL/6 wildtype mice, followed by PRP injection to evaluate the effects of platelet CD226 on PRP efficacy. The results showed that deletion of platelet CD226 increased the number of megakaryocytes (MKs) in bone marrow (BM) but reduced MKs in spleen, combined with significantly decreased platelet amounts, α-granule secretion, and reduced immature platelets; indicating that absence of platelet CD226 may disrupt MK/platelet homeostasis and arrested platelet release from MKs. Sequencing analysis showed abnormal ribosomal functions and much downregulated proteins in the absence of platelet CD226. Autophagy-related proteins were also reduced in the CD226-absent MKs/platelets. Moreover, deletion of platelet CD226 diminished the protective effects of PRP on DMM-induced cartilage lesions in mice, and PDGF restored it. Therefore, deficiency of platelet CD226 inhibited platelet maturation, secretion, and normal ribosomal functions, which may lead to depressed PRP efficacy on OA, suggesting that CD226 is required to regulate platelet growth, functions, and its application.

MPN-343: The BET Inhibitor Pelabresib Decreases Inflammatory Cytokines, Improves Bone Marrow Fibrosis and Function, and Demonstrates Clinical Response Irrespective of Mutation Status in Myelofibrosis Patients in the Phase 2 MANIFEST Trial

<h3>Context:</h3> Pelabresib (CPI-0610) is a potent, selective BET bromodomain inhibitor under investigation in the ongoing Phase 2 MANIFEST trial, given as monotherapy to ruxolitinib (rux)-intolerant/refractory myelofibrosis (MF) pts in arm 1, as an "add-on" to rux in MF pts who have suboptimal/lost response to rux in arm 2, and as combination therapy with rux in JAKi-naïve MF pts in arm 3. <h3>Objective:</h3> We assessed activity of pelabresib, including changes in inflammatory cytokines (Ck) in blood, improvement of bone marrow (BM) biology, including fibrosis (BMF), erythroid (Ery) progenitor and megakaryocyte (MK) numbers and clustering, and impact of mutational status on clinical response. <h3>Results:</h3> Rapid PD response was demonstrated by median blood <i>IL8</i> mRNA level reduced to 55% of baseline (BL) levels 4 hours post-1st dose of pelabresib in 102 pts, with similar reduction across arms. Elevated blood Ck levels were observed at BL across arms. Reduction of NF-κB and non-NF-κB regulated inflammatory Ck was observed on cycle 1 day 14 (C1D14) across arms and maintained through C9D1 (24 weeks). BMF grading was assessed by local pathologists for BL and post-treatment (most at 24 weeks) biopsies from 116 evaluable pts. Relative BMF improvement of ≥1 grade was observed in 33% (38/116) of all pts, with 21% (6/29) in arm 1, 41% (16/39) in arm 2, and 33% (16/48) in arm 3. BMF grade worsening was observed in only 6% (7/116) pts. Exploratory analyses of Ery and MK lineages by immunohistochemical staining for CD71 and CD61, respectively, on BM biopsy pairs (BL and week 24) revealed an increase in Ery progenitors in 59% (22/37) of pts. Tight clusters of MK, characteristic in MF BM, were observed at BL, and improvement in MK numbers and/or clustering was observed in 65% (24/37) pts. Mutation analysis revealed similar profiles at BL across arms. SVR35 at 24 weeks was similar, regardless of mutational status for each of the 14 genes analyzed. <h3>Conclusions:</h3> Our data demonstrate a robust PD effect of pelabresib in MF pts, indicate broad clinical responses irrespective of mutational status, and suggest the disease-modifying potential of pelabresib by improving BM fibrosis and function.

HDAC1 is required for GATA-1 transcription activity, global chromatin occupancy and hematopoiesis.

The activity of hematopoietic factor GATA-1 is modulated through p300/CBP-mediated acetylation and FOG-1 mediated indirect interaction with HDAC1/2 containing NuRD complex. Although GATA-1 acetylation is implicated in GATA-1 activation, the role of deacetylation is not studied. Here, we found that the FOG-1/NuRD does not deacetylate GATA-1. However, HDAC1/2 can directly bind and deacetylate GATA-1. Two arginine residues within the GATA-1 linker region mediates direct interaction with HDAC1. The arginine to alanine mutation (2RA) blocks GATA-1 deacetylation and fails to induce erythroid differentiation. Gene expression profiling and ChIP-seq analysis further demonstrate the importance of GATA-1 deacetylation for gene activation and chromatin recruitment. GATA-12RA knock-in (KI) mice suffer mild anemia and thrombocytopenia with accumulation of immature erythrocytes and megakaryocytes in bone marrow and spleen. Single cell RNA-seq analysis of Lin− cKit+ (LK) cells further reveal a profound change in cell subpopulations and signature gene expression patterns in HSC, myeloid progenitors, and erythroid/megakaryocyte clusters in KI mice. Thus, GATA-1 deacetylation and its interaction with HDAC1 modulates GATA-1 chromatin binding and transcriptional activity that control erythroid/megakaryocyte commitment and differentiation.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing. Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

Immature platelet levels correlate with disease activity and predict treatment response of thrombocytopenia in lupus patients.

The immature platelet fraction (IPF) represents recently produced platelets in bone marrow and this parameter is increased in patient with primary immune thrombocytopenia (ITP). We investigated the associations between IPF and absolute immature platelet count (AIPC), and clinical parameters in systemic lupus erythematosus (SLE), which has more complex pathological mechanisms than in primary ITP. Patients with SLE were retrospectively reviewed at the University of Tokyo Hospital from May, 2012 to January, 2021. The correlations between clinical parameters and the number of immature platelets were assessed with Spearman's rank correlation coefficients. A multiple logistic regression model was used to identify the independent clinical parameters for IPF and AIPC. The difference in the distribution of time for a complete response (CR) after prednisolone (PSL) administration was also evaluated by log-rank test. A total of 282 SLE patients were enrolled, and 12.41% of those patients showed thrombocytopenia. IPF correlated with clinical parameters such as platelet count (r = -0.58), AIPC (r = 0.64) and systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) (r = 0.24). SLEDAI-2K [odds ratio (OR) (per unit increase), 1.07; 95% CI, 1.013 - 1.13] and thrombocytopenia (OR, 32.23; 95% CI, 11.072 - 93.80) were independent clinical parameters to account for IPF increase. IPF correlated with the number of bone marrow megakaryocytes (n = 19, r = 0.57). Notably, the probability of CR in response to PSL in AIPC-high patients was higher than in AIPC-low patients (hazard ratio, 4.62; 95% CI, 1.07 - 20.02). IPF correlated with disease activity of SLE and represented platelet production in the bone marrow, whereas AIPC predicted a rapid response to steroids in thrombocytopenic patients with SLE.