Proplatelet Formation Research Articles

Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease

AbstractMegakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9–related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/−GFP+/−, NMIIAD1424N+/−, and NMIIAE1841K+/−) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.

Phospho‐inositide‐dependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets

BackgroundRegulated protein synthesis is essential for megakaryocyte (MK) and platelet functions, including platelet production and activation. PDK1 (phosphoinositide‐dependent kinase 1) regulates platelet functional responses and has been associated with circulating platelet counts. Whether PDK1 also directly regulates protein synthetic responses in MKs and platelets, and platelet production by MKs, remains unknown. ObjectiveTo determine if PDK1 regulates protein synthesis in MKs and platelets. MethodsPharmacologic PDK1 inhibitors (BX‐795) and mice where PDK1 was selectively ablated in MKs and platelets (PDK1−/−) were used. PDK1 signaling in MKs and platelets (human and murine) were assessed by immunoblots. Activation‐dependent translation initiation and protein synthesis in MKs and platelets was assessed by probing for dissociation of eIF4E from 4EBP1, and using m7‐GTP pulldowns and S35 methionine incorporation assays. Proplatelet formation by MKs, synthesis of Bcl‐3 and MARCKs protein, and clot retraction were employed for functional assays. ResultsInhibiting or ablating PDK1 in MKs and platelets abolished the phosphorylation of 4EBP1 and eIF4E by preventing activation of the PI3K and MAPK pathways. Inhibiting PDK1 also prevented dissociation of eIF4E from 4EBP1, decreased binding of eIF4E to m7GTP (required for translation initiation), and significantly reduced de novo protein synthesis. Inhibiting PDK1 reduced proplatelet formation by human MKs and blocked MARCKs protein synthesis. In both human and murine platelets, PDK1 controlled Bcl‐3 synthesis. Inhibition of PDK1 led to complete failure of clot retraction in vitro. ConclusionsPDK1 is a previously unidentified translational regulator in MKs and platelets, controlling protein synthetic responses, proplatelet formation, and clot retraction.

Melatonin enhances thrombopoiesis through ERK1/2 and Akt activation orchestrated by dual adaptor for phosphotyrosine and 3-phosphoinositides.

Melatonin (MT), endogenously secreted by the pineal gland, is closely related to multiple biological processes; however, its effect on thrombopoiesis is still not well illustrated. Here, we demonstrate that MT administration can elevate peripheral platelet levels. Analysis of different stages in thrombopoiesis reveals that MT has the capacity to promote the expansion of CD34+ and CD41+ cells, and accelerate proplatelet formation (PPF) and platelet production. Furthermore, in vivo experiments show that MT has a potential therapeutic effect on radiation-induced thrombocytopenia. The underlying mechanism suggests that both extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling are involved in the processes of thrombopoiesis facilitated by MT. Interestingly, in addition to the direct regulation of Akt signaling by its upstream phosphoinositide 3-kinase (PI3K), ERK1/2 signaling is also regulated by PI3K via its effector, dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), in megakaryocytes after MT treatment. Moreover, the expression level of DAPP1 during megakaryocyte differentiation is closely related to the activation of ERK1/2 and Akt at different stages of thrombopoiesis. In conclusion, our data suggest that MT treatment can promote thrombopoiesis, which is modulated by the DAPP1-orchestrated activation of ERK1/2 and Akt signaling.

SUN-038 HOMOCYSTEINE EXACERBATES IRI-INDUCED ACUTE KIDNEY INJURY VIA PROMOTING MEGAKARYOCYTE MATURATION AND PROPLATELET FORMATION

Platelets are crucial regulatory factors of acute kidney injury (AKI), as they are present in high numbers in the circulation and could release massive bioactive factors, which participate in intra-renal inflammation and haemostasis. After ischaemia-reperfusion injury (IRI), unbalanced macrocirculation and microcirculation lead to hypoxaemic injury and inflammatory response and haemostatic processes (platelet sequestration and thrombin). Homocysteine (Hcy), the important intermediate product of methionine cycle, is elevated as related genetic enzymatic deficiencies or nutritional defects which interfere metabolism of methionine. Moderate hyperhomocysteinemia (HHcy) is defined as total plasma Hcy in the range of 16-30uM, which is emerged as prothrombotic factor, influencing coagulative status and endothelial function and modulating platelet aggregation. We therefore aimed to evaluate the effects of hcy levels on platelet in IRI-induced AKI. Experiments were performed using 6 to 8 weeks old male C57BL/6 mice. To generate HHcy mice, mice were fed with high methionine diet containing 19.56g/kg methionine for 3 weeks. IRI induced AKI were performed by bilateral renal pedicles clamped for 30min in 37℃ with or without oral clopidogrel. After 48h, mice were euthanized and blood and kidney tissues were collected. For FeCl3 carotid artery injury, filter paper soaked in FeCl3 solution (3.5%) was placed on the right carotid artery for 90 seconds. Flow was recorded by Doppler Image scanner. As to deep venous thrombosis, the inferior vena cava was ligated for 48h and the length and weight of thrombus were recorded. A preparation of MKs was obtained from femora and tibiae BM of C57BL/6. Fetal liver-derived MKs was from the liver of mouse fetuses aged 13-15 days. The MKs was incubated with Hcy or not. In the present research, we found hyperhomocysteinemia mice developed more severe renal injury after IRI shown as more severe renal tubular damage and higher serum creatinine. Furthermore, the excess injury would be counteracted by oral clopidogrel compared with normal plasma Hcy level mice. Intriguingly, we observed that platelet counts rise by 30% in methionine-induced HHcy. The time required to form an occlusive thrombus in mice subjected to FeCl3 carotid artery injury was sharply shorter and the length and weight of deep venous thrombosis was significantly increased in HHcy model. We hypothesized that Hcy could regulate platelet production by affecting megakaryocytes. MKs cultured with Hcy or obtained from HHcy model increased proplatelet production and had higher ploidy. However, the amount of MKs and precursor cells were not change differently. In conclusion, the present study demonstrates that HHcy increases platelet counts by promoting megakaryocyte maturation and proplatelet formation. The results further suggest that mechanism of HHcy exacerbates IRI-induced acute Kidney Injury.

Genetic classification and confirmation of inherited platelet disorders: current status in Korea.

Inherited platelet disorders (IPDs), which manifest as primary hemostasis defects, often underlie abnormal bleeding and a family history of thrombocytopenia, bone marrow failure, hematologic malignancies, undefined mucocutaneous bleeding disorder, or congenital bony defects. Wide heterogeneity in IPD types with regard to the presence or absence of thrombocytopenia, platelet dysfunction, bone marrow failure, and dysmegakaryopoiesis is observed in patients. The individual processes involved in platelet production and hemostasis are genetically controlled; to date, mutations of more than 50 genes involved in various platelet biogenesis steps have been implicated in IPDs. Representative IPDs resulting from defects in specific pathways, such as thrombopoietin/MPL signaling; transcriptional regulation; granule formation, trafficking, and secretion; proplatelet formation; cytoskeleton regulation; and transmembrane glycoprotein signaling are reviewed, and the underlying gene mutations are discussed based on the National Center for Biotechnology Information database and Online Mendelian Inheritance in Man accession number. Further, the status and prevalence of genetically confirmed IPDs in Korea are explored based on searches of the PubMed and KoreaMed databases. IPDs are congenital bleeding disorders that can be dangerous due to unexpected bleeding and require genetic counseling for family members and descendants. Therefore, the pediatrician should be suspicious and aware of IPDs and perform the appropriate tests if the patient has unexpected bleeding. However, all IPDs are extremely rare; thus, the domestic incidences of IPDs are unclear and their diagnosis is difficult. Diagnostic confirmation or differential diagnoses of IPDs are challenging, time-consuming, and expensive, and patients are frequently misdiagnosed. Comprehensive molecular characterization and classification of these disorders should enable accurate and precise diagnosis and facilitate improved patient management.

Knock‐in mice bearing constitutively active αIIb(R990W) mutation develop macrothrombocytopenia with severe platelet dysfunction

BackgroundTo date, several mutations that induce constitutive activation of integrin αIIbβ3 have been identified in congenital macrothrombocytopenia. Of these, αIIb(R995W) is the most prevalent mutation observed in Japanese patients with αIIbβ3‐related congenital macrothrombocytopenia. Objective and MethodsThe present study aimed to explore the effects of constitutive activation of the αIIb(R995W) mutation on platelet production, morphology, and function. We generated αIIb(R990W) knock‐in (KI) mice corresponding to human αIIb(R995W). ResultsPlatelet counts of heterozygous (hetero) and homozygous (homo) KI mice were decreased by ~10% and ~25% relative to those of wild‐type (WT) mice, respectively, with increase in platelet size. Decrease in absolute reticulated platelet numbers in steady state, delayed recovery from thrombocytopenia induced by anti‐platelet antibody and impaired response to exogenous thrombopoietin administration suggested impaired platelet production in KI mice. WT and KI mice showed no significant differences in the number of megakaryocytes and ploidy of megakaryocytes, whereas proplatelet formation was significantly impaired in homo mice. We observed a slight but significant reduction in platelet lifespan in homo mice. The homo mice showed dramatic reduction in αIIbβ3 expression in platelets, which was accompanied by severe in vivo and in vitro platelet dysfunction. ConclusionThe αIIb(R990W) KI mice developed macrothrombocytopenia, which was primarily attributed to impaired proplatelet formation. In addition, homo KI mice showed marked downregulation in αIIbβ3 expression in platelets with severe impaired platelet function, similar to Glanzmann thrombasthenia.

The Pediatric Acute Leukemia Fusion Oncogene ETO2‐GLIS2 Increases Self‐Renewal and Alters Differentiation in a Human Induced Pluripotent Stem Cells‐Derived Model

Supplemental Digital Content is available in the text.

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia

AbstractTo identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.

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.

ATRA Could Correct the Defective S1P-Mediated Cytoskeletal Reorganization in Proplatelet Formation of ITP

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

The Mir-99b/Let-7e/Mir-125a cluster Is Associated with Platelet Count and Mean Platelet Volume and Regulates Megakaryocyte Proplatelet Formation

Platelet count (PC) and mean platelet volume (MPV) are critical measurements when considering the role of platelets in hemostasis and thrombosis, abnormalities in PC and MPV have been shown to be predictors of ischemic cardiovascular disease. Ischemic vascular disease has a known genetic component, and we and others have shown high heritability for both PC and MPV. Many inherited, and most immune, thrombocytopenias show an inverse relationship between PC and MPV, and such a relationship has also been reported in healthy subjects. Although genome-wide association studies have identified numerous single nucleotide variants in protein-coding genes associated with PC and MPV (Geiger Nat Genet 2011), non-coding genes have had rather little consideration. The goal of our studies was to identify microRNAs (miRs) regulating PC and MPV and to begin to understand the molecular mechanisms of this inverse platelet number-volume relationship. Initially, we queried our Platelet RNA and Expression-1 data set and observed that PC and MPV are inversely correlated in these 154 healthy subjects (r=-0.45, p=3.98E-09). We next determined which of the 734 platelet-expressed miRs were significantly (p<0.05) correlated with both PC and MPV. Of these miRs, only miR-125a-5p, miR-99b-5p, let-7e-5p and miR-150-5p had an inverse relationship with PC and MPV. The Table shows the correlations between these miRs and complete blood count data. Subsequent studies focused on miR-125a-5p, miR-99b-5p, let-7e-5p because they (1) were associated with only platelet parameters and not WBC or Hb, (2) were more highly expressed than miR-150-5p and (3) showed the same directionality (e.g., positive r value for PC). We were also intrigued by the fact that these 3 miRs exist as a genomic cluster within 727 bp on chr19q13.41. The expression of each of the 3 miRs in this cluster was strongly correlated with each other in platelets (r=0.93, p=1.02E-68 for miR-125a and let-7e; r=0.91, p=8.53E-61 for miR-99b and let-7e; r=0.89, p=1.19E-52 for miR-125a and miR-99b), consistent with a single, polycistronic transcript containing all genes. To begin to address the potential functionality of this miR cluster, we overexpressed and knocked down their expression in megakaryocytes (MKs), since MK proplatelet formation (PPF) is a necessary step in the generation of peripheral blood platelets. We utilized human umbilical cord blood derived CD34-positive hematopoietic stem and progenitor cells differentiated along the MK lineage with thrombopoietin. Quantification of miR levels with qPCR demonstrated all 3 miRs in the cluster progressively increased from culture day 6 to day 9 to day 13 (the time during which MKs mature in culture). Compared to a scrambled control, when miR activity was knocked down with locked nucleic acids (LNAs), we observed a 61% reduction in PPF for miR-125a-5p (p<0.05), a 54% reduction for let-7e-5p (p<0.05) and a ~28% reduction for miR-99b-5p (p=0.33). PPF was quantified blinded as to treatment group. When all 3 miRs in the cluster were inhibited simultaneously by LNAs, we observed a 74% reduction in PPF (p<0.05) (Figure 1). LNA inhibition of two miRs not associated with PC or MPV did not alter MK PFF. To date, we have only tested the effect of overexpression of one miR, but observed that lentiviral overexpression of miR-125a-5p induced an increase in MK PPF (p<0.05). In summary, we have identified a genomic cluster of miRs (miR-99b/let-7e/miR-125a) that regulates PPF in cultured human MKs and whose expression is positively correlated with human PC and negatively correlated with MPV. We speculate that platelet mass homeostasis, which is determined by PC and MPV, may be controlled in part by this novel genomic locus. Disclosures No relevant conflicts of interest to declare.

KIFC1 Mediates Centrosome Clustering Prior to Proplatelet Formation from Megakaryocytes

Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

Strategies for Analysis of Novel Molecular Variants in the RUNX1 Gene As a Cause of Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML)

Introduction: Runx 1 is a key transcription factor (TF) in hematopoiesis. Germline mutations in RUNX1 (≈40 described) are associated with FPD/AML, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction and a high risk (40% before 35 yr) of developing acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Overestimating the pathogenicity of new molecular variants detected by high throughput sequencing (HTS) in the diagnosis of inherited thrombocytopenias (IT), even affecting well established candidate genes, could be more negative for the management and quality of life of patients than lacking such diagnosis (Lentaigne, Blood 2016). This is especially relevant for variants in TF such as Runx1, due to the risk of developing myeloid malignancies. Objective: To evaluate the pathogenicity of new variants in RUNX1 identified in patients enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with Congenital Platelet Disorders" (TPC-GT-PH-SETH). Methods: Three unrelated women aged 27 (P1), 46 (P2) and 50 (P3) years, were included in TPC-GT-PH-SETH because of their history of mild thrombocytopenia (≈105pl/uL) with normal volume (MPV) and moderate bleeding. A daughter and a brother of P2 died after developing MDS and LAM, respectively, and a sister of P3 had breast cancer. Only P1 and P2 had previously showed moderate functional platelet abnormalities (reduced aggregation, P-selectin secretion and number of δ-granules). By HTS (Bastida, Haematologica 2018) we identified new heterozygous variants in RUNX1: P1, de novo c.802C<T [p.Gln.268*]; P2, c.586A>G [p.Thr196Ala], segregated with thrombocytopenia and neoplasia in the family; P3, c.476A>G; [p.Asn159Ser], no segregation with thrombocytopenia, with 4 of 6 carriers in the family having normal platelet numbers. For this study, we obtained blood from patients and three controls, and isolated ultrapure platelets by filtration+immunoselection. Platelet RNA was analyzed using Clariom-D Array (≈540 000 transcripts) (Caparrós-Pérez, PLoS One 2017). In P1 we isolated CD34+ cells from blood, differentiated them in vitro to megakaryocytes (Mks), and evaluated the formation of proplatelets. Results: Analysis of transcriptomes showed that when considering the 13 genes well recognized as targets of RUNX1 (including MYL9, MYH9, ALOX12), the percentage of those with altered expression in patients, vs. controls, was 69.2% and 61.5% in P1 and P2, respectively, and only 7.7% in P3. Previously, the transcriptome of a single patient with RUNX1 variant (83aa deletion) (Sun, JTH 2007) has reported the infraexpression of 100 genes. Our cases showed low expression of 74.7%, 67.7% and 7.1% (P1, P2 and P3, respectively) of those genes. Principal component analysis of the array intensity signals, grouped together controls and the P3 case, while the P1 and P2 cases were clustered together. Additionally, in vitro culture of CD34-Mks from patient P1 showed reduced pro-platelet formation. Conclusion: Platelet phenotyping, family segregation studies, platelet transcriptome and in vitro pro-platelet formation, support the pathogenicity of Runx 1 variants p.Gln268* and p.Thr196Ala, but not of p.Asn159Ser. Platelet transcriptome analysis is a useful tool in the pathogenic characterization of new molecular variants affecting TF genes in patients with IT, such as RUNX1 and can help to identify new target genes of these TF. Disclosures No relevant conflicts of interest to declare.

Harnessing a Novel Dyrk1a-Ablim2-MKL1 Regulatory Module in Megakaryocyte Morphogenesis to Enable Scalable Platelet “Pharming”

Growing clinical demands for platelet transfusions combined with supply limitations have created shortages which are trending toward a global crisis. Major efforts have been taken to address key issues of platelet sources, storage, and utilization. Recent progress in ex vivo culture-based production of megakaryocytes (Mk) and platelets, "pharming," has highlighted the potential for novel, donor-independent sources amenable to antigenic editing and cryo-stockpiling. Such cultures can be easily initiated from umbilical cord blood (CB) progenitors, induced pluripotent stem cells (iPSC), or directly re-programmed somatic cells. The major roadblock associated with these Mk sources consists of their fetal ontogenic status, which is beneficial for expansion but severely limits platelet production. The ability to elicit in pre-expanded Mk an adult program of morphogenesis (polyploidization, enlargement, and proplatelet formation) would enable circumvention of this scalability barrier. A master regulator of adult Mk morphogenesis consists of the transcriptional coactivator MKL1 which undergoes nuclear translocation in response to RhoA-mediated actin polymerization, stimulated by thrombopoietin and environmental mechano-sensing. Nuclear MKL1 associates with the transcription factor SRF1 to upregulate cytoskeletal remodeling factors, including filamin A and Hic-5, that act as morphogenesis effectors. Our previous studies identified in infantile CB Mk a failure in MKL1 upregulation resulting from repression by the oncofetal RNA-binding factor IGF2BP3. Pharmacologic suppression of IGF2BP3 with BET inhibitors rescued MKL1 expression and improved platelet production but caused cycle arrest preventing polyploidization. As an alternative approach to abrogate the fetal blockade in Mk morphogenesis, we sought to promote MKL1 activity by targeting a kinase, Dyrk1a, which had been shown to restrain MKL1 from nuclear translocation. Treatment of infantile CB Mk with a variety of Dyrk1-selective inhibitors including harmine and EHT 1610 strongly enhanced polyploidization (p = 0.015 and 0.009 respectively), enlargement (p < 0.005) , and in vitro platelet release (2 fold each, p = 0.001 and 0.007 respectively), attaining levels seen with adult Mk. When xenotransplanted into NSG mice, harmine-treated CB Mk demonstrated enhanced capability for in vivo platelet release (about 5 fold, p = 0.016). CB stem cells expanded with the AHR antagonist SR1 and an iPSC-Mk cell line also responded to Dyrk1 inhibition with robustly increased morphogenesis. Several findings implicated MKL1 in this response: 1) induction of nuclear translocation by the inhibitors, 2) induction of target genes (filamin A and Hic-5) by the inhibitors, and 3) loss of response to inhibitors in Mkl1-ko murine progenitors. Supporting Dyrk1a as a relevant target, mice with Mk-specific loss of one Dyrk1a allele (Dyrk1aflox/wt;Pf4-Cre) displayed increases in platelet counts (p = 0.037) and marrow Mk polyploidization (p = 0.02). In addition, retroviral expression in human progenitors of a dominant negative Dyrk1a mutant K188R promoted Mk enlargement (p = 0.014). shRNA knockdowns could not be obtained due to toxicity of > ~60% loss of Dyrk1a. To determine mechanisms for Dyrk1a control of morphogenesis, we analyzed the actin cytoskeleton, a key regulator of MKL1. Dyrk1 inhibition in all types of Mk progenitors (adult, infantile, and iPSC) induced assembly of cortical filamentous actin (F-actin), as detected by Alexa594-phalloidin staining. Prior studies showed cytoskeletal binding by Dyrk1a and direct phosphorylation of F-actin regulators N-WASP and Ablim1. A survey of human marrow expression patterns for candidate Dyrk1a substrates (The Human Protein Atlas) identified Ablim2, as showing a Mk-specific, cortical staining pattern. Dyrk1 inhibition increased Ablim2 levels ~5-fold in CB Mk (p < 0.005), and immunofluorescence displayed a cortical distribution similar to F-actin. Lentiviral shRNA knockdown of Ablim2 abrogated all effects of Dyrk1 inhibition, blocking: F-actin formation, MKL1 nuclear translocation, activation of the MKL1 targets, and Mk morphogenesis. These findings thus delineate a novel Dyrk1a-Ablim2-MKL1 regulatory module in Mk morphogenesis that can be manipulated to address the problem of scaling ex vivo production and might also serve as a future in vivo therapeutic target for thrombocytopenia. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Analysis of a New Haematopoietic Stem/Progenitor Cells with Megakaryocyte Differentiation Potential

Thrombocytopenia is a common clinical condition and requires more effective treatments. Our previous original work confirmed that 5-HT contributes to the expansion of cord blood stem/progenitor cells (CD34+/CD41+CD61+) and haematopoietic reconstitution in NOD/SCID mice, and through 5-HT2R and Erk1/2 pathway, 5-HT remodel F-actin organization, promoting megakaryocytes to pro-platelet formation (Stem Cells, 2007; 2014). It is suggested that there is a subtype of haematopoietic stem cells (CD34+5HT2R+) with the potential to differentiate into megakaryocytes (CD41+5HT2R+). We studied the specific functions of cells through subsequent experiments, including receptor expression and signaling pathways. Results showed that: (1) The presence of 5-HT2 receptors on CD34+ and CD41+ cells; (2) 5-HT activated the PI3K/AKT pathway and the Wnt/β-catenin pathway via 5-HT2R in CD34+ and CD41+ cells, with anti-apoptotic and pro-proliferative functions; (3) These CD34+5HT2R+ differentiated CD41+5HT2R+ cells, 5-HT acted on 5-HT2R and Erk1/2 pathways, and promoted the formation of pro-platelets by F-actin reorganization; (4) CD34+5HT2R+ haematopoietic stem cells implanted in NOD/SCID mice reconstituted blood function, especially on thrombopoiesis. This study provides a more scientific basis for haematopoiesis and drug development, especially for the treatment of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

ANKRD26 Coding Variants Presenting with Giant Platelets and a Predisposition to Myeloid Neoplasia

ANKRD26 Coding Variants Presenting with Giant Platelets and a Predisposition to Myeloid Neoplasia

Shared roles for Scl and Lyl1 in murine platelet production and function

AbstractThe stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

Impact of Itga2-Gp6-double collagen receptor deficient mice for bone marrow megakaryocytes and platelets.

The two main collagen receptors on platelets, GPVI and integrin α2β1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2β1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.

Study of Bernard-Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells.

Bernard-Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.