Megakaryocytic Activity Research Articles

The LAT Rheostat as a Regulator of Megakaryocyte Activation.

Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132-the binding site for PLC-γ2-is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known. To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences. iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced. These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.

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

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

Interleukins in Platelet Biology: Unraveling the Complex Regulatory Network.

Interleukins, a diverse family of cytokines produced by various cells, play crucial roles in immune responses, immunoregulation, and a wide range of physiological and pathological processes. In the context of megakaryopoiesis, thrombopoiesis, and platelet function, interleukins have emerged as key regulators, exerting significant influence on the development, maturation, and activity of megakaryocytes (MKs) and platelets. While the therapeutic potential of interleukins in platelet-related diseases has been recognized for decades, their clinical application has been hindered by limitations in basic research and challenges in drug development. Recent advancements in understanding the molecular mechanisms of interleukins and their interactions with MKs and platelets, coupled with breakthroughs in cytokine engineering, have revitalized the field of interleukin-based therapeutics. These breakthroughs have paved the way for the development of more effective and specific interleukin-based therapies for the treatment of platelet disorders. This review provides a comprehensive overview of the effects of interleukins on megakaryopoiesis, thrombopoiesis, and platelet function. It highlights the potential clinical applications of interleukins in regulating megakaryopoiesis and platelet function and discusses the latest bioengineering technologies that could improve the pharmacokinetic properties of interleukins. By synthesizing the current knowledge in this field, this review aims to provide valuable insights for future research into the clinical application of interleukins in platelet-related diseases.

Thioredoxin-Interacting Protein (TXNIP) Binding to GATA1 May Affect GATA1 Transcriptional Activity in Megakaryocytes

GATA1 is a crucial transcription factor for both megakaryopoiesis and erythropoiesis. In our previous work, we established a murine megakaryocyte-erythroid progenitor (MEP) cell line, named G1ME2 cells, which can be maintained with doxycycline (dox)-induced anti- Gata1 shRNA. These cells can differentiate into erythroid cells or megakaryocytes upon removal of dox; however, their megakaryocyte differentiation potential showed alterations after prolonged cell culture (90-100 days). Notably, CD41 expression in G1ME2 cells declined during long-term culture, prompting us to investigate the disparities between CD41+ and CD41- G1ME2 cells using total RNA sequencing. Interestingly, the western blotting analysis revealed a significant enrichment of thioredoxin-interacting protein (TXNIP) in the late G1ME2 cells compared to the early cells. TXNIP is known for its role in regulating cellular metabolism, and recently, we observed age-associated thrombocytopenia in TXNIP knockout mice, suggesting its involvement in hematopoiesis. Additionally, previous studies have identified its role in erythropoiesis through diverse mechanisms. In our current study, we made a novel discovery that TXNIP directly binds to GATA1 during megakaryopoiesis. Furthermore, deletion of GATA1 C-terminal enhanced binding affinity, implying that this interaction might occur when GATA1 is not localized to DNA. Consistently, TXNIP exhibited the ability to suppress GATA1 promoter activity, leading to changes in GATA1 localization in the presence or absence of TXNIP. In conclusion, our findings demonstrate the involvement of TXNIP in megakaryopoiesis, partially through the regulation of GATA1 transcriptional activity.

MAPK-interacting kinase 1 regulates platelet production, activation, and thrombosis

AbstractThe MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbβ3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.

Microrna-15a-5p Regulates Multiple Components of GPVI-Mediated Platelet and Megakaryocyte Activation Pathway

Introduction: Platelet activation by collagen is a major effector ofnormal hemostasis. Mutations in GPVI, the major collagen signaling receptor, cause platelet-type bleeding. Two single nucleotide polymorphisms in GPVI have been identified as risk factors for myocardial infarction. Elevated levels of GPVI have been reported in patients with coronary syndromes. GPVI and its signaling pathway components for platelet activation have become potential antithrombotic targets. Reports have indicated microRNAs (miRs) regulate platelet activation, but none have considered the mechanisms involving miR-mRNA associations regulating the GPVI-mediated platelet activation pathway.Aims: To identify and characterize miRs regulating GPVI-mediated megakaryocyte (MK) integrin activation.Methods : Candidate miRs were identified via a platelet function association study. miR function was assessed by knockdown or overexpression studies in MKs derived from umbilical cord blood (CB) CD34+ stem cells. MK αIIbβ3 activation was measured by flow cytometric quantification of PAC-1 binding after collagen-related peptide (CRP) stimulation. miR and mRNA quantification were performed using quantitative polymerase chain reaction.Results: Our P latelet R NA A nd e X pression (PRAX1) study identified 20 miRs associated with CRP-induced platelet aggregation (p<0.008; n=154). To minimize false positives, we considered only miRs expressed in both platelets and cultured MKs, reducing the potential candidates to 10. We focused on miR-15a-5p because it is negatively correlated with platelet activation through CRP (p=0.0075) (Fig 1A) and because miR-15a-5p targets BCL2, which is known to enhance CRP-induced platelet activation. miR-15a-5p was not found to be associated with platelet aggregation in response to arachidonic acid, ADP, PAR1-AP or PAR4-AP, suggesting specificity of miR-15a-5p for GPVI-mediated integrin activation pathway. Knock down of miR-15a-5p using locked nucleic acid (LNA) in CBMKs caused a ~38% increase in CRP-induced integrin-activation (p=0.0035; n=7) compared to the scrambled (Scr) LNA control (Fig 1B), presumably by releasing miR-15a-5p repression of activation. Fig 1B shows lentiviral overexpression of miR-15a-5p decreased CRP-induced integrin-activation by ~33% (p=0.0154; n=4) as compared to empty vector (EV) control. To understand how miR-15a-5p regulates integrin activation, we bioinformatically identified putative mRNA targets FYN, SRGN, FCER1G, MYLK and PRKCQ, predicted to be regulated by miR-15a- 5p and known as signaling molecules in the GPVI activation pathway. Of note, GPVI was not predicted to be a target of miR-15a-5p and BCL2 was not included because it has been studied before. Importantly, these target genes are positively associated with CRP-induced platelet aggregation (all p<0.004; n=154). The transcript levels of FYN, FCER1G, MYLK and PRKCQ were derepressed upon miR-15a-5p knockdown (Fig 1C) as compared to Scr LNA (all p<0.05; n>4). In miR-15a-5p overexpressed CBMKs, transcript levels of FYN, FCER1G and PRKCQ were decreased compared to EV (Fig 1D) (all p<0.05; n=4). SRGN only showed trends upon miR-15a-5p manipulation. MYB and ACTIN were used as positive and negative controls, respectively. Initial studies of miR-15a-5p knockdown showed a trend for increased FYN, FCER1G and PRKCQ protein levels (Fig 1E; n=3). miR activity requires binding to Argonaute2 (Ago2), which catalyzes translational repression and mRNA degradation. We demonstrated that Ago2 immunoprecipitation enriches miR-15a-5p and its target FYN, SRGN, FCER1G, MYLK and PRKCQ mRNAs (Fig 1F). These data suggest miR-15a-5p and its targets are processed through the Ago complex and thereby, are potential miR-mRNA pairs together regulating GPVI-mediated integrin activation pathway.Conclusions:miR-15a-5p negatively regulates GPVI-mediated MK integrin activation. miR-15a-5p appears to act as a ‘master-miR’ regulating multiple components of GPVI-mediated platelet and MK activation. As miRs are increasingly becoming clinical therapeutic targets, miR-15a-5p overexpression shows potential for treating thrombosis, whereas its inhibition could be effective in bleeding disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Congenital Thrombocytopenia and Myelofibrosis Due to Germline Mutations in G6b-B— a Megakaryocyte-Specific Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Receptor

Unlike primary myelofibrosis (PMF) in adults, which is one of the three classical BCR-ABL-negative myeloproliferative neoplasms (MPN), myelofibrosis in children is rare, occurring most commonly in patients with acute leukemia, particularly acute megakaryoblastic leukemia. Congenital or inherited forms of myelofibrosis (cMF) are exceedingly rare, and may occur in the context of patients with inherited platelet disorders. Here we describe 3 families with inherited thrombocytopenia and cMF due to germline null mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM) receptor G6b-B.We studied 3 families with a total of 6 affected children, each of which presented with macrothrombocytopenia, mild anemia, mild leukocytosis and a distinctive pattern of bone marrow reticulin fibrosis centered around clusters of atypical megakaryocytes. The disease appeared to segregate as an autosomal recessive trait. All affected children had mild to moderate bleeding symptoms and required frequent platelet and occasional red cell transfusions. None showed evidence of extramedullary hematopoiesis, such as splenomegaly, and all were negative for MPN associated mutations in JAK2, MPL, and CALR.To identify the disease gene, we performed Affymetrix SNP 6.0 genotyping on peripheral blood DNA from three clinically affected children and 7 first and second-degree relatives from the index family, a complex consanguineous pedigree from Kuwait. Shared regions of homozygosity by descent were assessed using a custom designed platform; multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (MHC) on chromosome 6p (LOD=2.01), were identified as potential candidate genetic intervals. We focused on this region because affected individuals in the index family and one of the two other families, which were not known to be related, shared a common homozygous human leukocyte antigen (HLA) type and had congenital adrenal hyperplasia (CAH) due to the identical 21-hydroxylase (CYP21A2) mutation; the CYP21A2 and HLA loci are located at 6p21.33 and 6p21.32-6p22.1. Whole exome sequencing was performed on all three nuclear families and revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor G6b (MPIG6B, previously G6B-B, C6orf25), encoded within the candidate linkage region. We identified 5 individuals (4 affected, one previously unaffected) with a MPIG6B frameshift mutation (c.61_61+1dup; p.20fs) in two families from Kuwait. In addition we confirmed a second MPIG6B frameshift mutation at c.147insT (p.49fs) in two individuals (one proband, one mildly affected sibling) in a family from Saudi Arabia. No other homozygous mutations that segregated with the phenotype in all three families were identified. MPIG6B is a member of the immunoglobulin superfamily and is located in the MHC class III region on chromosome 6. To validate MPIG6B as a potential disease-causing gene, we evaluated G6b-B expression in bone marrow (BM) biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody specific to G6b-B. G6b-B was strongly and selectively expressed in megakaryocytes of control BM samples, but completely absent in megakaryocytes of the clinically affected individuals.Interestingly, a murine knockout that lacks G6b-B has a strikingly similar phenotype to these patients, the animals having macrothrombocytopenia, myelofibrosis and aberrant platelet production and function. Thus, in addition to uncovering the molecular basis of this rare disease, the clinical phenotype highlights the potential importance of MPIG6B and aberrant megakaryocytic activation in mediating other forms of BM fibrosis. DisclosuresNo relevant conflicts of interest to declare.

Thromboembolic involvement and its possible pathogenesis in COVID-19 mortality: lesson from post-mortem reports.

The emergence of Coronavirus Disease 19 (COVID-19) as a pandemic has claimed hundreds of thousands of lives worldwide since its initial breakout. With increasing reports from clinical observations and autopsy findings, it became clear that the disease causes acute respiratory distress syndrome (ARDS), as well as a broad spectrum of systemic and multiorgan pathologies, including angiopathy, endothelialitis, and thrombosis. Coagulopathy is associated with the activity of megakaryocytes, which play crucial roles in modulating the platelet homeostasis. Only a few autopsy reports include findings on thrombosis formation and the presence of megakaryocytes. Here we review and summarize the possible involvement and the pathophysiology of the thromboembolic events in COVID-19 patients based on post-mortem reports. We reviewed post-mortem reports from March 2020 to September 2020. Eleven autopsy reports that demonstrated thromboembolic involvement findings, either macroscopically or microscopically, were included in this review. All studies reported similar pulmonary gross findings. Not all studies described thrombi formation and megakaryocyte findings. Pulmonary embolism, coagulopathy, severe endothelial injury, and widespread thrombosis are frequent in COVID-19 patients, following many patients with high-level D-Dimer, increased fibrinogen, abnormal prothrombic coagulation, and thrombocytopenia. Reports showed that thrombus was also found in the lower extremities' deep veins and the prostatic venous plexus. In conclusion, a complex interaction of SARS-CoV-2 virus invasion with platelets, leukocytes, endothelial cells, inflammation, immune response, and the possible involvement of megakaryocytes may increase the cumulative risk of thrombosis by a yet unclear cellular and humoral interaction.

CLINICAL RELEVANCE OF EXTENDED PLATELET INDICES IN THE DIAGNOSIS OF IMMUNE THROMBOCYTOPENIA.

SUMMARYImmune thrombocytopenia (ITP) is an autoimmune disorder. Besides platelet count, immature platelet fraction (IPF) can be used as a tool to predict megakaryocytic activity in ITP patients. The aim of the study was to evaluate the utility of extended platelet indices in ITP diagnosis and their association with disease persistence and severity. This case-control study (1:1), conducted from January 2015 to December 2017, included 111 ITP patients and 111 healthy controls. ITP patients were grouped as newly diagnosed ITP, persistent ITP, chronic ITP, and refractory ITP patients. Peripheral blood was collected and complete blood profile parameters were recorded using Sysmex XN 1000. Significant (p≤0.05) difference between the groups of ITP patients and healthy control subjects was determined by Fisher exact test, while Pearson correlation was used to evaluate platelet count correlation with IPF using SPSS ver. 23. Low hemoglobin and platelet counts with high total leukocyte count and IPF were detected in ITP patients as compared to healthy subjects (p≤0.001). Among all groups of ITP patients, very low platelet count (6.9±6.02.x109/L) with highest mean IPF (27.1±19.2%) was observed in newly diagnosed ITP group. Other platelet parameters including mean platelet volume (MPV), plateletcrit, platelet large cell ratio (P-LCR) and platelet distribution width values were also altered in patient groups. Pearson correlation revealed negative relationship between platelet count and IPF in all patient groups. With the advent of new, sophisticated hematologic analyzers, the IPF and other platelet parameters provide simple, reliable and easier tools for predicting platelet disorders such as ITP, and to some extent the disease severity. Besides IPF, the MPV and P-LCR seemed to predict disease severity, treatment responsiveness, and duration of the disease to some extent.

SARS-CoV-2 vaccination induced cerebral venous sinus thrombosis: Do megakaryocytes, platelets and lipid mediators make up the orchestra?

The COVID-19 vaccines comprised of adenoviral vectors encoding the Spike (S) glycoprotein of SARS-CoV-2 are highly effective but associated with rare thrombotic complications. The adenovirus vector infects epithelial cells expressing the coxsackievirus and adenovirus receptor (CAR). The S glycoprotein expressed locally stimulates neutralizing antibody and cellular immune responses. These vaccines have been associated with thromboembolic events including cerebral venous sinus thrombosis (CVST). S glycoprotein stimulates the expression of cyclooxygenase-2 (COX-2) and leads to massive generation of thromboxane A2 in COVID-19. Megakaryocytes express CAR and we postulate that S glycoprotein stimulated generation of thromboxane A2 leads to megakaryocyte activation, biogenesis of activated platelets and thereby increased thrombogenicity. Cerebral vein sinuses express podoplanin, a natural ligand for CLEC2 receptors on platelets. Platelets traversing through the cerebral vein sinuses could be further activated by thromboxane A2-dependent podoplanin-CLEC2 signaling, leading to CVST. A prothrombotic hormonal milieu, and increased generation of thromboxane A2 and platelet activation in healthy females compared to males is consistent with increased risk for CVST observed in women. We propose that antiplatelet agents targeting thromboxane A2 receptor signaling such as low-dose aspirin merit consideration for chemoprophylaxis when administering the adenovirus based COVID-19 vaccines to young adults at risk of thrombosis provided there are no contraindications.

MiRNA 146b mediates the regulation of nucleolar size and activity in polyploid megakaryocytes.

Megakaryocytes (MKs) follow a unique cell cycle duplication process, called endomitosis, resulting in polyploidisation of cells. It is hypothesised that polyploidy, as well as an increment in cytoplasm volume, allow more efficient platelets generation from MKs. Although polyploidy leads to an increase in the DNA amount, which impacts gene expression, little is known about ribosomal biogenesis in these polylobulated polyploid cells. The nucleolus acts as a hub for ribosomal biogenesis, which in turn governs the protein synthesis rate of the cells. We therefore estimated the size and activity of the nucleolus in polyploid cells during megakaryopoiesis in vitro. Polyploid megakaryocytic cell lines and in vitro cultured MKs, which were obtained from human cord blood-derived CD 34+ cells, revealed that miRNA 146b regulated the activity of nucleolar and coiled-body phosphoprotein 1, which plays an integral role in determining nucleolar size and activity. Additionally, miRNA-146b was up-regulated during endomitosis and was found to promote megakaryopoiesis. We propose that miRNA 146b regulates not only nucleolar size and activity, but also megakaryopoiesis. This study highlights the importance of nucleolar activity and miRNA in the progression of megakaryopoiesis and thrombopoiesis.

Regulation of platelet numbers and sizes by signaling pathways

Either the glycoprotein (GP) Ib deficiency or hyper-function in humans can cause macrothrombocytopenia, the molecular mechanisms of which remain unclear. Herein, the investigations for disease pathogenesis were performed in the human induced pluripotent stem cell (hiPSC) model. The hiPSCs carrying a gain-of-function GP1BA p.M255V mutation which was described in platelet-type von Willebrand disease (PT-VWD) were generated using CRISPR/Cas9. The GP1BA-null hiPSCs were previously derived from a Bernard–Soulier syndrome (BSS) patient. After full megakaryocyte differentiation in culture, both hiPSC mutations showed large proplatelet tips under fluorescence microscopy and yielded fewer but larger platelets compared with those of wild-type cells. The Capillary Western analyses revealed the lower ERK1/2 activation and higher MLC2 (Myosin light chain 2) phosphorylation in megakaryocytes with mutated GPIb. Adding a mitogen-activated protein kinase (MAPK) pathway inhibitor to wild-type hiPSCs recapitulated the phenotypes of GPIb mutations and increased MLC2 phosphorylation. Notably, a ROCK inhibitor which could inhibit MLC2 phosphorylation rescued the macrothrombocytopenia phenotypes of both GPIb alterations and wild-type hiPSCs with a MAPK inhibitor. In conclusion, the genetically modified hiPSCs can be used to model disorders of proplatelet formation. Both loss- and gain-of-function GPIb reduced MAPK/ERK activation but enhanced ROCK/MLC2 phosphorylation resulting in dysregulated platelet generation.

Bone marrow megakaryocytic activation predicts fibrotic evolution of Philadelphia-negative myeloproliferative neoplasms.

Philadelphia-negative chronic myeloproliferative neoplasms (MPN) have been traditionally considered as indistinctly slowly progressing conditions; recent evidence proves that a subset of cases have a rapid evolution, so that MPN prognosis needs to be personalized. We identified a new morphological parameter, defined as megakaryocytic activation (M-ACT) based on the coexistence of megakaryocytic emperipolesis, megakaryocytes (MK) cluster formation and evidence of arrangement of collagen fibers around the perimeter of MK. We retrospectively analyzed the bone marrow biopsy of two MPN cohorts of patients with polycythemia (PV) (n=64) and non-PV patients (including essential thrombocythemia, and early/prefibrotic primary myelofibrosis [PMF]) (n=222). M-ACT showed a significant correlation with splenomegaly, white blood cell count, and lactate dehydrogenase serum levels in both groups, with JAK2 V617F allele burden in PV patients, and with CALR mutations, and platelet count in non-PV patients. Progression-free survival, defined as PV-to-secondary MF progression and non-PV-to-overt PMF, was worse in both PV and early/prefibrotic PMF patients with M-ACT in comparison to those without M-ACT (P<0.0001). Interestingly, M-ACT was not found in the subgroup of essential thrombocythemia patients. In conclusion, M-ACT can be helpful in the differential diagnosis of MPN and can represent a new morphologic parameter with a predictive value for progression of MPN.

Thrombosis in Patients with Immune Thrombocytopenia ,Review of Literature

Introduction ITP is characterized by low platelet count due to immune mediated destruction and bleeding tendency. However, during last few decades thromboembolic events have been reported in patients with ITP. This review is done to study the reported cases of thromboembolic phenomenon in patient with ITP in an attempt to assess the patient characteristics and to understand the underlying mechanism. Methods We searched google Scholar, PubMed about cases with ITP and thrombosis the summary is presented in the following table (Table 1). Results Around 30 reported cases of ITP with thrombotic events were identified and a total of 36 events were recognized in last 10 years. The ages ranged from 3 years to 81 years with a mean of 51 years. Most of the patients were young and middle aged (18-65 years of age), meanwhile around 9 patients were elderly (age &amp;gt; 65 years). Only 3 cases were observed in pediatric age. Almost equal incidence in both genders was recognized. Half of the patient had chronic ITP while in the rest it was diagnosed less than a year. 20 out of 36 (55.6%) events happened at platelet count less than 100*10^9. While 16 events were reported with platelet count higher than this or unknown. Majority of the patients (around 64%) developed arterial events while fewer developed venous thrombosis. For treatment, most of the patients (44%) were not receiving any particular treatment for ITP at the time of thrombotic event. While 6 events (17%) happened while being treated with IVIG and 10 events (28%') happened while on TPO-RA. Only 3 patients were treated with corticosteroids prior to the event. In patients treated with TPO-RAs arterial and venous events were almost similar (57% vs 43% respectively) while majority of the events happened at lower than normal platelet count (7/10 events). Almost half of the patients had one or more underlying risk factor predisposing to atherosclerosis and thrombosis. Most of the patients were treated appropriately for the events with either antiplatelet agents or anticoagulation while simultaneously treatment for ITP was given. Corticosteroids were most frequently used for ITP during the episode followed by IVIG (52% and 28% of total treated patients respectively). Only 1 patient was treated with TPO-RA after the event for low platelet counts while others received other treatments (Rituximab, Danazol and splenectomy). Discussion Thrombosis is a complex process involving arteries and veins. Accelerated atherosclerosis and plaque rupture is the underlying event for arterial thrombosis. While in venous thrombosis immobility and procoagulant states are the main factors. Immune thrombocytopenia is characterized by immune mediated destruction and impaired production of platelets predisposing to bleeding mostly. However, it is a unique pathological process that is linked to both bleeding and thrombosis. Multiple factors predispose patients to thrombosis in ITP. The patients with chronic and active disease are particularly at risk of paradoxical thrombosis due to accelerated atherosclerosis as in other autoimmune conditions, predisposing to arterial thrombotic events. Active disease is also characterized by increased turnover of platelets in bone marrow and higher levels of circulating platelets microparticles (PMPs) which promote thrombin formation and promote venous thrombosis. The patients treated with IVIG and TPO-RA are at higher risk as compared to other forms of treatment. IVIG is used in acute states as it prevents the destruction of platelets but simultaneously promotes thrombosis by increasing blood viscosity and thrombin production. TPO-RAs are agents which mimic the action of thrombopoietin on megakaryocytes promoting their growth and differentiation and increasing platelet production. Increasing platelet count above the normal target might contribute to thrombosis however megakaryocyte activation itself leads to increased risk of thrombosis, despite low platelet count. In patients with ITP and thrombotic events, judicious use of antiplatelet therapy and anticoagulation is indicated along with simultaneous therapy directed at improving platelet count. Conclusion Patient with active ITP are predisposed to thrombosis in addition to bleeding. A treating physician needs to be vigilant to diagnose early the events and then to institute proper use of antiplatelets and anticoagulation along with therapy directed at ITP. Figure Disclosures No relevant conflicts of interest to declare.

Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN

AbstractPrimary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic–stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis

AbstractDuring thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1−/−) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1−/− BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1−/− BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1−/− BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis.

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

S832 A PHASE 2A STUDY OF THE LSD1 INHIBITOR IMG‐7289 FOR THE TREATMENT OF MYELOFIBROSIS

Background:Myeloproliferative neoplasms share a common pathophysiology of constitutive activation of the JAK/STAT pathway. Ruxolitinib, the only approved agent for the treatment for myelofibrosis (MF), can reduce spleen volume and symptom scores, but is not curative; disease will progress underscoring the need for therapies with a mode of action distinct from JAK inhibition.IMG‐7289 is an inhibitor of a lysine‐specific demethylase, LSD1, an epigenetic regulator critical for self‐renewal of malignant myeloid cells and differentiation of myeloid progenitors. LSD1 bound to GFI1b licenses the maturation of progenitors to megakaryocytes and enables their normal function. In mouse models of MPN (Mpl W515L , JAK2 V617F ), LSD1 inhibition reduced elevated peripheral cell counts, spleen volumes, inflammatory cytokines, mutant allele frequencies, and marrow fibrosis (Jutzi et al. 2018) supporting clinical investigation.Aims:This multi‐center, open‐label Phase 1/2a study evaluated the safety, tolerability, steady‐state pharmacokinetics and pharmacodynamics of IMG‐7289 administered orally once‐daily in adult patients with high‐ or intermediate‐2 risk MF who were resistant to or intolerant of approved therapy.Methods:The primary objectives were safety, PK and spleen volume reduction. Exploratory endpoints included reductions in total symptoms scores (TSS) using the MPN‐SAF instrument and bone marrow (BM) fibrosis. Key inclusion criteria included platelet count ≥100K/μL and circulating blasts ≤10%. Patients were treated daily for 12 weeks followed by a washout period of up to 28 days. BM biopsies and imaging studies were conducted prior to treatment and after the 12 week dosing cycle. The MPN‐SAF was self‐administered weekly. Patients for whom clinical benefit was demonstrable could resume treatment for additional 12 week cycles. Dosing was tailored using thrombocytopenia as a biomarker of megakaryocyte activity. All patients were started at the presumed sub‐therapeutic dose of 0.25 mg/kg/d and up‐titrated weekly until the platelet count rested between 50 and 100K/μL.Results:This preliminary analysis includes an enrolled cohort of 16 patients. All but one patient had received one or more prior treatments including ruxolitinib. 44% had PMF, 38% PET‐MF, 18% PPV‐MF. The median patient age was 65 (48–89) with 63% males. 56% were classified as high risk, the remainder, intermediate risk‐2. All patients were up‐titrated from the starting dose; 75% were able to sustain a platelet count within the target zone with an average dose of 0.81 mg/kg. 88% (14/16) completed the 85 day study; eight remain on‐study. Two patients withdrew, one for fatigue (Day 33), one for accelerating disease (Day 39). In evaluable patients (N = 9), 66% demonstrated a reduction in spleen volume by imaging; 56% recorded a ≥50% reduction in TSS. Two had an improved BM fibrosis score. There were 239 AEs in 87% of the patients of which 15 were SAEs. Of the SAEs, only one, painful splenomegaly, was deemed related to IMG‐7289. There have been no safety signals, DLTs, or deaths. The median duration of treatment stands at 156 days (33–498) in this ongoing study.Summary/Conclusion:This is the first report of the clinical use of an LSD1 inhibitor for MPNs. IMG‐7289 was well‐tolerated in a heterogeneous population of patients with MF and limited therapeutic options. IMG‐7289 was effective in reducing spleen volumes and substantially improved symptom scores in a majority of patients. On the basis of these results, this study has expanded into a Phase 2b trial to include EU and UK sites now open for enrollment.image

CD226 is involved in megakaryocyte activation and early-stage differentiation

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N–4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA‐binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.