FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

The Analysis of the Human Megakaryocyte and Platelet Coding Transcriptome in Healthy and Diseased Subjects.

Platelets are generated and released into the bloodstream from their precursor cells, megakaryocytes that reside in the bone marrow. Though platelets have no nucleus or DNA, they contain a full transcriptome that, during platelet formation, is transported from the megakaryocyte to the platelet. It has been described that transcripts in platelets can be translated into proteins that influence platelet response. The platelet transcriptome is highly dynamic and has been extensively studied using microarrays and, more recently, RNA sequencing (RNA-seq) in relation to diverse conditions (inflammation, obesity, cancer, pathogens and others). In this review, we focus on bulk and single-cell RNA-seq studies that have aimed to characterize the coding transcriptome of healthy megakaryocytes and platelets in humans. It has been noted that bulk RNA-seq has limitations when studying in vitro-generated megakaryocyte cultures that are highly heterogeneous, while single-cell RNA-seq has not yet been applied to platelets due to their very limited RNA content. Next, we illustrate how these methods can be applied in the field of inherited platelet disorders for gene discovery and for unraveling novel disease mechanisms using RNA from platelets and megakaryocytes and rare disease bioinformatics. Next, future perspectives are discussed on how this field of coding transcriptomics can be integrated with other next-generation technologies to decipher unexplained inherited platelet disorders in a multiomics approach.

Expanding the clinical and genetic spectrum of pathogenic variants in STIM1.

INTRODUCTION/AIMS: Stromal interaction molecule 1 (STIM1) is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1. METHODS: We performed a cross-sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene-targeted panel. RESULTS: We identified seven individuals (age range, 26-57 years) harboring variants in STIM1, including five novel changes: three located in the EF-hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store-operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients. DISCUSSION: Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1-related diseases.

Modeling genetic platelet disorders with human pluripotent stem cells: mega-progress but wanting more on our plate(let).

PURPOSE OF REVIEW: Megakaryocytes are rare hematopoietic cells that play an instrumental role in hemostasis, and other important biological processes such as immunity and wound healing. With the advent of cell reprogramming technologies and advances in differentiation protocols, it is now possible to obtain megakaryocytes from any pluripotent stem cell (PSC) via hematopoietic induction. Here, we review recent advances in PSC-derived megakaryocyte (iMK) technology, focusing on platform validation, disease modeling and current limitations. RECENT FINDINGS: A comprehensive study confirmed that iMK can recapitulate many transcriptional and functional aspects of megakaryocyte and platelet biology, including variables associated with complex genetic traits such as sex and race. These findings were corroborated by several pathological models in which iMKs revealed molecular mechanisms behind inherited platelet disorders and assessed the efficacy of novel pharmacological interventions. However, current differentiation protocols generate primarily embryonic iMK, limiting the clinical and translational potential of this system. SUMMARY: iMK are strong candidates to model pathologic mutations involved in platelet defects and develop innovative therapeutic strategies. Future efforts on generating definitive hematopoietic progenitors would improve current platelet generation protocols and expand our capacity to model neonatal and adult megakaryocyte disorders.

Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders.

Mutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene result in excessive extracellular Ca2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca2+ levels, promote major nuclear import of the Ca2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+ entry, highlighting that TAM and STRMK involve a common pathomechanism.

Regulation of Platelet Production and Life Span: Role of Bcl-xL and Potential Implications for Human Platelet Diseases.

Blood platelets have important roles in haemostasis, where they quickly stop bleeding in response to vascular damage. They have also recognised functions in thrombosis, immunity, antimicrobal defense, cancer growth and metastasis, tumour angiogenesis, lymphangiogenesis, inflammatory diseases, wound healing, liver regeneration and neurodegeneration. Their brief life span in circulation is strictly controlled by intrinsic apoptosis, where the prosurvival Bcl-2 family protein, Bcl-xL, has a major role. Blood platelets are produced by large polyploid precursor cells, megakaryocytes, residing mainly in the bone marrow. Together with Mcl-1, Bcl-xL regulates megakaryocyte survival. This review describes megakaryocyte maturation and survival, platelet production, platelet life span and diseases of abnormal platelet number with a focus on the role of Bcl-xL during these processes.

Recent advances in inherited platelet disorders.

PURPOSE OF REVIEW: The increasing use of high throughput sequencing and genomic analysis has facilitated the discovery of new causes of inherited platelet disorders. Studies of these disorders and their respective mouse models have been central to understanding their biology, and also in revealing new aspects of platelet function and production. This review covers recent contributions to the identification of genes, proteins and variants associated with inherited platelet defects, and highlights how these studies have provided insights into platelet development and function. RECENT FINDINGS: Novel genes recently implicated in human platelet dysfunction include the galactose metabolism enzyme UDP-galactose-4-epimerase in macrothrombocytopenia, and erythropoietin-producing hepatoma-amplified sequence receptor transmembrane tyrosine kinase EPHB2 in a severe bleeding disorder with deficiencies in platelet agonist response and granule secretion. Recent studies of disease-associated variants established or clarified roles in platelet function and/or production for the membrane receptor G6b-B, the FYN-binding protein FYB1/ADAP, the RAS guanyl-releasing protein RASGRP2/CalDAG-GEFI and the receptor-like protein tyrosine phosphatase PTPRJ/CD148. Studies of genes associated with platelet disorders advanced understanding of the cellular roles of neurobeachin-like 2, as well as several genes influenced by the transcription regulator RUNT-related transcription factor 1 (RUNX1), including NOTCH4. SUMMARY: The molecular bases of many hereditary platelet disorders have been elucidated by the application of recent advances in cell imaging and manipulation, genomics and protein function analysis. These techniques have also aided the detection of new disorders, and enabled studies of disease-associated genes and variants to enhance understanding of platelet development and function.

CRAC channels and disease - From human CRAC channelopathies and animal models to novel drugs.

Ca2+ release-activated Ca2+ (CRAC) channels are intimately linked with health and disease. The gene encoding the CRAC channel, ORAI1, was discovered in part by genetic analysis of patients with abolished CRAC channel function. And patients with autosomal recessive loss-of-function (LOF) mutations in ORAI1 and its activator stromal interaction molecule 1 (STIM1) that abolish CRAC channel function and store-operated Ca2+ entry (SOCE) define essential functions of CRAC channels in health and disease. Conversely, gain-of-function (GOF) mutations in ORAI1 and STIM1 are associated with tubular aggregate myopathy (TAM) and Stormorken syndrome due to constitutive CRAC channel activation. In addition, genetically engineered animal models of ORAI and STIM function have provided important insights into the physiological and pathophysiological roles of CRAC channels in cell types and organs beyond those affected in human patients. The picture emerging from this body of work shows CRAC channels as important regulators of cell function in many tissues, and as potential drug targets for the treatment of autoimmune and inflammatory disorders.

Platelets retain inducible alpha granule secretion by P-selectin expression but exhibit mechanical dysfunction during trauma-induced coagulopathy.

Essentials Platelets in trauma-induced coagulopathy (TIC) are impaired, but the mechanism is not known. We performed comprehensive longitudinal platelet function testing in trauma patient samples. Platelets in TIC are widely impaired early after injury, but platelet activatability is intact. This suggests a mechanism of transient platelet cytoskeletal/integrin dysfunction during TIC. SUMMARY: Background Trauma-induced coagulopathy (TIC) is a common and deadly bleeding disorder. Platelet dysfunction is present during TIC, but its mechanisms remain unclear. Platelets are currently thought to become "exhausted," a state in which they have released their granule contents and can no longer aggregate or contract. Methods This prospective observational cohort study tested the hypothesis that platelet exhaustion is present during TIC and characterized the early time course of platelet dysfunction. Blood was collected from 95 adult trauma patients at a Level I trauma center at time of Emergency Department arrival and several time points over 72 h. Platelet activation state and function were characterized using CD62P (P-selectin) and PAC-1 surface membrane staining, platelet function analyzer (PFA-100), aggregometry, viscoelastic platelet mapping, and, to test for exhaustion, their ability to express CD62P after ex vivo adenosine diphosphate (ADP) agonism. Platelet function was compared between patients with and without TIC, defined by prothrombin time ≥18 s. Results Platelets in TIC showed no initial increase in their level of surface activation markers or impairment of their capacity to express CD62P in response to ADP stimulation. However, TIC platelets were impaired in nearly all functional assays, spanning adhesion, aggregation, and contraction. These effects largely remained after controlling for platelet count and fibrinogen concentration and resolved after 8 h. Conclusion The TIC platelets exhibit early impairment of adhesion, aggregation, and contraction with retained alpha granule secretion ability, suggesting a specific mechanism of cytoskeletal or integrin dysfunction that is not a result of more general platelet exhaustion.

Downregulation of TREM-like transcript-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia.

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.

A dual mechanism promotes switching of the Stormorken STIM1 R304W mutant into the activated state.

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.

Defective acid hydrolase secretion in RUNX1 haplodeficiency: Evidence for a global platelet secretory defect.

BACKGROUND: RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), α-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles. METHODS AND RESULTS: We studied two related patients with a RUNX1 mutation, easy bruising, and mild thrombocytopenia. Platelet aggregation and 14 C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), α-granules (ß-thromboglobulin) and AH-containing vesicles (ß-glucuronidase, ß-hexosaminidase, α-mannosidase) were normal or minimally decreased. Dense granules secretion on stimulation of gel-filtered platelets with thrombin and divalent ionophore A23187 (4-12 µmol L-1 ) were diminished. ß-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14 C -arachidonic acid into phospholipids and subsequent arachidonic acid release on thrombin activation was normal. Platelet thromboxane A2 production in whole blood serum and on thrombin stimulation of PRP was normal, suggesting that the defective secretion was not due to impaired thromboxane production. CONCLUSIONS: These studies provide the first evidence in patients with a RUNX1 mutation for a defect in AH (lysosomal) secretion, and for a global defect in secretion involving all three types of platelet granules that is unrelated to a granule content deficiency. They highlight the pleiotropic effects and multiple platelet defects associated with RUNX1 mutations.

Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease.

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

ORAI1 Mutations with Distinct Channel Gating Defects in Tubular Aggregate Myopathy.

Calcium (Ca2+ ) is a physiological key factor, and the precise modulation of free cytosolic Ca2+ levels regulates multiple cellular functions. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ homeostasis, and is mediated by the concerted activity of the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Dominant gain-of-function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss-of-function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation-dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non-muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca2+ homeostasis and leading to muscle dysfunction.

Inherited platelet dysfunction and hematopoietic transcription factor mutations.

Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for the defects in platelet production, morphology, and function. The hematopoietic TFs implicated in the patients with impaired platelet function include Runt related TF 1 (RUNX1), Fli-1 proto-oncogene, ETS TF (FLI1), GATA-binding protein 1 (GATA1), and growth factor independent 1B transcriptional repressor (GFI1B). These TFs act in a combinatorial manner to bind sequence-specific DNA within a promoter region to regulate lineage-specific gene expression, either as activators or as repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology and platelet production and function, culminating in thrombocytopenia, platelet dysfunction, and associated clinical features. Mutations in TFs may occur more frequently in the patients with inherited platelet dysfunction than generally appreciated. This review focuses on the alterations in hematopoietic TFs in the pathobiology of inherited platelet dysfunction.

Transcription factor defects causing platelet disorders.

Recent years have seen increasing recognition of a subgroup of inherited platelet function disorders which are due to defects in transcription factors that are required to regulate megakaryopoiesis and platelet production. Thus, germline mutations in the genes encoding the haematopoietic transcription factors RUNX1, GATA-1, FLI1, GFI1b and ETV6 have been associated with both quantitative and qualitative platelet abnormalities, and variable bleeding symptoms in the affected patients. Some of the transcription factor defects are also associated with an increased predisposition to haematologic malignancies (RUNX1, ETV6), abnormal erythropoiesis (GATA-1, GFI1b, ETV6) and immune dysfunction (FLI1). The persistence of MYH10 expression in platelets is a surrogate marker for FLI1 and RUNX1 defects. Characterisation of the transcription factor defects that give rise to platelet function disorders, and of the genes that are differentially regulated as a result, are yielding insights into the roles of these genes in platelet formation and function.

Acquired platelet disorders.

In contrast to congenital platelet disorders, which are rare, acquired platelet dysfunctions are more common in clinical practice. Their main causes are medications and systemic/hematologic diseases. Typical clinical manifestations are mucosal bleeding, epistaxis, or superficial epidermal bleeding normally of modest entity. In most cases, the molecular mechanisms underlying impaired platelet function are not fully established, making it difficult to optimize patient care. We here provide a short overview of the various forms of acquired platelet disorders, with a particular focus on recent mechanistic studies on platelet dysfunction in von Willebrand disease.