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Ana C Glembotsky,1,2 Geraldine De Luca,1,2 Paula G Heller1,2 1Departamento Hematología Investigación, Instituto de Investigaciones Médicas “Dr. A. Lanari”, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina; 2Departamento Hematología Investigación, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Buenos Aires, ArgentinaCorrespondence: Paula G HellerDepartamento Hematología Investigación,Instituto de Investigaciones Médicas “Dr. A. Lanari”, Facultad de Medicina, Universidad de Buenos Aires, Combatientes de Malvinas 3150, Buenos Aires, 1427, ArgentinaTel +54 11 5287 3872Email paulaheller@hotmail.comAbstract: The gray platelet syndrome (GPS) is a rare platelet disorder, characterized by impaired alpha-granule biogenesis in megakaryocytes and platelets due to NBEAL2 mutations. Typical clinical features include macrothrombocytopenia, bleeding and elevated vitamin B12 levels, while bone marrow fibrosis and splenomegaly may develop during disease progression. Recently, the involvement of other blood lineages has been highlighted, revealing the role of NBEAL2 outside the megakaryocyte-platelet axis. Low leukocyte counts, decreased neutrophil granulation and impaired neutrophil extracellular trap formation represent prominent findings in GPS patients, reflecting deranged innate immunity and associated with an increased susceptibility to infection. In addition, low numbers and impaired degranulation of NK cells have been demonstrated in animal models. Autoimmune diseases involving different organs and a spectrum of autoantibodies are present in a substantial proportion of GPS patients, expanding the syndromic spectrum of this disorder and pointing to dysregulation of the adaptive immune response. Low-grade inflammation, as evidenced by elevation of liver-derived acute-phase reactants, is another previously unrecognized feature of GPS which may contribute to disease manifestations. This review will focus on the mechanisms underlying the pathogenesis of blood cell abnormalities in human GPS patients and NBEAL2-null animal models, providing insight into the effects of NBEAL2 in hemostasis, inflammation and immunity.Keywords: NBEAL2, gray platelet syndrome, α-granules, immune dysregulation, neutrophils

Correct interpretation of the pathogenicity of germline RUNX1 variants is essential for FPD/AML diagnosis, clinical management and leukaemia surveillance. We report two families with clear FPD/AML phenotypic features harbouring missense variants at RHD critical residue Gly168. Although classified as of unknown significance (VUS) by RUNX1-specific curation guidelines, these variants should rather be considered likely pathogenic, as supported by computational tools, structural modelling and dysregulated platelet expression of RUNX1-targets, adding Gly168 among amino acids currently recognised as mutational hotspots. Our data could help reduce the number of variants classified as VUS, providing evidence for updating RUNX1 guidelines, thus improving FPD/AML diagnosis., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players in MF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serum LDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11b and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1β and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1β and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo . In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 De Luca, Goette, Lev, Baroni Pietto, Marin Oyarzún, Castro Ríos, Moiraghi, Sackmann, Kamiya, Verri, Caula, Fernandez, Vicente, Pose Cabarcos, Caruso, Camacho, Larripa, Khoury, Marta, Glembotsky and Heller.)

Background: Multiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE., Materials and Methods: Thirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA., Results: In resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation., Conclusion: Our study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: In hemophilia and von Willebrand disease, the degree of alteration of laboratory assays correlates with bleeding manifestations. Few studies have assessed the predictive value for bleeding of laboratory assays in patients with inherited platelet function disorders (IPFDs)., Objectives: To assess whether there is an association between platelet function assay results and bleeding history, as evaluated by the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT)., Methods: Centers participating in the international ISTH-BAT validation study were asked to provide results of the diagnostic assays employed for the patients they enrolled, and the association with the individual patients' bleeding score (BS) was assessed., Results: Sixty-eight patients with 14 different IPFDs were included. Maximal amplitude of platelet aggregation was significantly lower in patients with a pathologic BS and correlated inversely with the BS, a finding largely driven by the subgroup of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency; after their exclusion, TRAP-induced aggregation remained significantly lower in patients with a pathologic BS. Bleeding time was significantly more prolonged in patients with a high BS than in those with a normal BS (27.1 ± 6.2 minutes vs 15.1 ± 10.6 minutes; P  < .01). Reduced α-granule content was significantly more common among patients with a pathologic BS than among those with a normal BS (80% vs 20%; P  < .05). Receiver operating characteristic curve analysis revealed a significant discriminative ability of all the aforementioned tests for pathologic BS ( P  < .001), also after exclusion of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency., Conclusion: This study shows that altered platelet laboratory assay results are associated with an abnormal ISTH-BAT BS in IPFD., (© 2023 The Author(s).)

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver ( JAK2 / CALR / MPL ) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1β and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 De Luca, Lev, Camacho, Goette, Sackmann, Castro Ríos, Moiraghi, Cortes Guerrieri, Bendek, Carricondo, Enrico, Vallejo, Varela, Khoury, Gutierrez, Larripa, Marta, Glembotsky and Heller.)

SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid ( Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.[Figure: see text].

Major surgery is associated with an increased risk of venous thromboembolism (VTE), thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of VTE in patients with inherited platelet disorders (IPD) undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in IPD patients undergoing surgery at VTE-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of IPD (VTE-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to VTE-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest VTE-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions use. Two thromboembolic events were registered, both occurring after high VTE-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that VTE incidence is low in patients with IPD undergoing surgery at VTE-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with IPD undergoing invasive procedures at VTE-risk and low-molecular-weight-heparin should be considered for major surgery., (Copyright© 2020 Ferrata Storti Foundation.)

Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK 2, CALR , and MPL , which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting., (Copyright © 2020 Marín Oyarzún, Glembotsky, Goette, Lev, De Luca, Baroni Pietto, Moiraghi, Castro Ríos, Vicente, Marta, Schattner and Heller.)

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 sh RUNX1 -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 (β)-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., (Copyright© 2019 Ferrata Storti Foundation.)

Mechanisms leading to low platelet count in immune thrombocytopenia (ITP) involves both decreased production and increased destruction of platelet. However, the contribution of these pathologic mechanisms to clinical outcome of individual patients is uncertain. Here we evaluated different pathogenic mechanisms including in vitro megakaryopoiesis, platelet/megakaryocyte (MK) desialylation and MK apoptosis, and compared these effects with thrombopoyesis and platelet apoptosis in the same cohort of ITP patients. Normal umbilical cord blood-CD34+ cells, mature MK derived cells or platelets were incubated with plasma from ITP patients. Despite inhibition of thrombopoiesis previously observed, megakaryopoiesis was normal or even increased. Plasma from ITP patients affected the sialylation pattern of control platelets and this effect occurred concomitantly with apoptosis in 35% ITP samples. However, none of these abnormalities were observed in control MKs incubated with ITP plasma. Addition of mononuclear cells as immune effectors did not lead to phosphatidylserine exposure in MK, ruling out an antibody-mediated cytotoxic effect. These results suggest that both desialylation and apoptosis may be relevant mechanisms leading to platelet destruction although, they do not interfere with MK function. Analysis of these thrombocytopenic factors in individual patients showed no specific distribution pattern. However, the presence of circulating antiplatelet autoantibodies was associated with higher incidence of abnormalities. In conclusion, the causes of thrombocytopenia are multifactorial and may occur together, providing a rational basis for the use of combination therapies targeting concomitant ITP mechanisms in patients with refractory disease.

The SDF-1-CXCR4 axis plays an essential role in the regulation of platelet production, by directing megakaryocyte (MK) migration toward the vascular niche, thus allowing terminal maturation and proplatelet formation, and also regulates platelet function in an autocrine manner. Inherited thrombocytopenias (IT) comprise a spectrum of diverse clinical conditions caused by mutations in genes involved in platelet production and function. We assessed CXCR4 expression and SDF-1 levels in a panel of well-characterized forms of IT. Decreased surface CXCR4 levels were found in 8 of 27 (29.6%) IT patients by flow cytometry, including 4 of 6 patients with ANKRD26-RT, 3 of 3 patients with GPS and 1 of 6 patients with FPD/AML. Low CXCR4 levels were associated with impaired SDF-1-triggered platelet aggregation, indicating that this decrease is functionally relevant, whereas a normal platelet response was shown in patients harbouring preserved membrane CXCR4. Reduced CXCR4 was not due to decreased gene expression, as platelet RNA levels were normal or increased, suggesting a post-transcriptional defect. Increased ligand-induced receptor internalization was ruled out, as circulating SDF-1 levels were similar to controls. MK CXCR4 expression was normal, indicating that the defect in CXCR4 arises after the step of platelet biogenesis. In conclusion, the finding of defective CXCR4 expression specifically associated with certain IT disorders highlights the fact that abnormalities in several megakaryocytic regulators underlie IT pathogenesis and further reveal the heterogeneous nature of these conditions.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence., (Copyright© 2017 Ferrata Storti Foundation.)

The mechanisms underlying increased thrombotic risk in chronic myeloproliferative neoplasms (MPN) are incompletely understood. We assessed whether neutrophil extracellular traps (NETs), which promote thrombosis, contribute to the procoagulant state in essential thrombocythemia, polycythemia vera and myelofibrosis (MF) patients. Although MPN neutrophils showed increased basal reactive oxygen species (ROS), enhanced NETosis by unstimulated neutrophils was an infrequent finding, whereas PMA-triggered NETosis was impaired, particularly in MF, due to decreased PMA-triggered ROS production. Elevated circulating nucleosomes were a prominent finding and were higher in patients with advanced disease, which may have potential prognostic implication. Histone-MPO complexes, proposed as specific NET biomarker, were seldomly detected, suggesting NETs may not be the main source of nucleosomes in most patients, whereas their correlation with high LDH points to increased cell turn-over as a plausible origin. Lack of association of nucleosomes or NETs with thrombosis or activation markers does not support their use as predictors of thrombosis although prospective studies in a larger cohort may help define their potential contribution to MPN thrombosis. These results do not provide evidence for relevant in vivo NETosis in MPN patients under steady state conditions, although availability of standardized NET biomarkers may contribute to further research in this field.

Mechanisms leading to decreased platelet count in immune thrombocytopenia (ITP) are heterogeneous. This study describes increased platelet apoptosis involving loss of mitochondrial membrane potential (ΔΨm), caspase 3 activation (aCasp3) and phosphatidylserine (PS) externalization in a cohort of adult ITP patients. Apoptosis was not related to platelet activation, as PAC-1 binding, P-selectin exposure and GPIb-IX internalization were not increased. Besides, ITP platelets were more sensitive to apoptotic stimulus in terms of aCasp3. Incubation of normal platelets with ITP plasma induced loss of ΔΨm, while PS exposure and aCasp3 remained unaltered. The increase in PS exposure observed in ITP platelets could be reproduced in normal platelets incubated with ITP plasma by adding normal CD3+ lymphocytes to the system as effector cells. Addition of leupeptin -a cathepsin B inhibitor- to this system protected platelets from apoptosis. Increased PS exposure was also observed when normal platelets and CD3+ lymphocytes were incubated with purified IgG from ITP patients and was absent when ITP plasma was depleted of auto-antibodies, pointing to the latter as responsible for platelet damage. Apoptosis was present in platelets from all patients carrying anti-GPIIb-IIIa and anti-GPIb auto-antibodies but was absent in the patient with anti-GPIa-IIa auto-antibodies. Platelet damage inversely correlated with platelet count and decreased during treatment with a thrombopoietin receptor agonist. These results point to a key role for auto-antibodies in platelet apoptosis and suggest that antibody-dependent cell cytotoxicity is the mechanism underlying this phenomenon.

Background: Anagrelide represents a treatment option for essential thrombocythemia, although its place in therapy remains controversial., Aim: To assess the impact of mutational status in response rates and development of adverse events during long-term use of anagrelide., Methods: We retrospectively evaluated 67 patients with essential thrombocythemia treated with anagrelide during 68 (4-176) months., Results: Mutational frequencies were 46.3%, 28.3%, and 1.5% for JAK2V617F, CALR and MPL mutations. Anagrelide yielded a high rate of hematologic responses, which were complete in 49.25% and partial in 46.25%, without differences among molecular subsets. The rate of thrombosis during treatment was one per 100 patient-years, without excess bleeding. Anemia was the major adverse event, 30.3% at 5-yr follow-up, being more frequent in CALR(+) (P < 0.05). Myelofibrotic transformation developed in 14.9% (12.9%, 21%, and 12.5% in JAK2V617F(+), CALR(+), and triple-negative patients, respectively, P = NS) and those treated >60 months were at higher risk, OR (95% CI) 9.32 (1.1-78.5), P < 0.01, indicating the need for bone marrow monitoring during prolonged treatment., Conclusion: Although CALR(+) patients were at higher risk of developing anemia, anagrelide proved effective among all molecular subsets, indicating that mutational status does not seem to represent a major determinant of choice of cytoreductive treatment among essential thrombocythemia therapies., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Objectives: Anagrelide represents a treatment option for essential thrombocythemia patients. It lowers platelet counts through inhibition of megakaryocyte maturation and polyploidization, although the basis for this effect remains unclear. Based on its rapid onset of action, we assessed whether, besides blocking megakaryopoiesis, anagrelide represses proplatelet formation (PPF) and aimed to clarify the underlying mechanisms., Methods and Results: Exposure of cord blood-derived megakaryocytes to anagrelide during late stages of culture led to a dose- and time-dependent inhibition of PPF and reduced proplatelet complexity, which were independent of the anagrelide-induced effect on megakaryocyte maturation. Whereas anagrelide was shown to phosphorylate cAMP-substrate VASP, two pharmacologic inhibitors of the cAMP pathway were completely unable to revert anagrelide-induced repression in megakaryopoiesis and PPF, suggesting these effects are unrelated to its ability to inhibit phosphodiesterase (PDE) 3. The reduction in thrombopoiesis was not the result of down-regulation of transcription factors which coordinate PPF, while the myosin pathway was identified as a candidate target, as anagrelide was shown to phosphorylate the myosin light chain and the PPF phenotype was partially rescued after inhibition of myosin activity with blebbistatin., Conclusions: The platelet-lowering effect of anagrelide results from impaired megakaryocyte maturation and reduced PPF, both of which are deregulated in essential thrombocythemia. These effects seem unrelated to PDE3 inhibition, which is responsible for anagrelide's cardiovascular side-effects and antiplatelet activity. Further work in this field may lead to the potential development of drugs to treat thrombocytosis in myeloproliferative disorders with an improved pharmacologic profile., (© 2015 International Society on Thrombosis and Haemostasis.)

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L., (Copyright© Ferrata Storti Foundation.)

The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood-derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose-dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma-induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet-adsorbed plasma induced its reversion, suggesting the involvement of auto-antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti-GPIIb-IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC-1 binding and β3 integrin phosphorylation while the presence of anti-glycoprotein Ia-IIa auto-antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP., (© 2014 John Wiley & Sons Ltd.)

Background: Familial platelet disorder with a predisposition to acute myelogenous leukemia (FPD/AML) is an inherited platelet disorder caused by a germline RUNX1 mutation and characterized by thrombocytopenia, a platelet function defect, and leukemia predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype. In addition, by using a candidate gene approach, we sought to identify potential RUNX1-regulated genes involved in these defects., Methods: Lumiaggregometry, α-granule and dense granule content and release, platelet ultrastructure, αIIb β3 integrin activation and outside-in signaling were assessed in members of one FPD/AML pedigree. Expression levels of candidate genes were measured and luciferase reporter assays and chromatin immunoprecipitation were performed to study NF-E2 regulation by RUNX1., Results: A severe decrease in platelet aggregation, defective αIIb β3 integrin activation and combined αδ storage pool deficiency were found. However, whereas the number of dense granules was markedly reduced, α-granule content was heterogeneous. A trend towards decreased platelet spreading was found, and β3 integrin phosphorylation was impaired, reflecting altered outside-in signaling. A decrease in the level of transcription factor p45 NF-E2 was shown in platelet RNA and lysates, and other deregulated genes included RAB27B and MYL9. RUNX1 was shown to bind to the NF-E2 promoter in primary megakaryocytes, and wild-type RUNX1, but not FPD/AML mutants, was able to activate NF-E2 expression., Conclusions: The FPD/AML platelet function defect represents a complex trait, and RUNX1 orchestrates platelet function by regulating diverse aspects of this process. This study highlights the RUNX1 target NF-E2 as part of the molecular network by which RUNX1 regulates platelet biogenesis and function., (© 2014 International Society on Thrombosis and Haemostasis.)

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype-phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C-terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD., (© 2013 WILEY PERIODICALS, INC.)

The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.

MYH9-related disease (MYH9-RD) is a rare autosomal dominant syndromic disorder caused by mutations in MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (myosin-9). MYH9-RD is characterized by congenital macrothrombocytopenia and typical inclusion bodies in neutrophils associated with a variable risk of developing sensorineural deafness, presenile cataract, and/or progressive nephropathy. The spectrum of mutations responsible for MYH9-RD is limited. We report five families, each with a novel MYH9 mutation. Two mutations, p.Val34Gly and p.Arg702Ser, affect the motor domain of myosin-9, whereas the other three, p.Met847&#95;Glu853dup, p.Lys1048&#95;Glu1054del, and p.Asp1447Tyr, hit the coiled-coil tail domain of the protein. The motor domain mutations were associated with more severe clinical phenotypes than those in the tail domain., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34(+) was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.

Background: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource-limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina., Methods: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes., Results: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each., Conclusions: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases., (© 2012 International Society on Thrombosis and Haemostasis.)

The aim of this study was to evaluate cell maturation and the platelet production capacity of the megakaryoblastic DAMI cell line, to characterize platelet-like particles produced and to investigate the mechanisms involved in their production. DAMI cell maturation was induced by phorbol myristate acetate (PMA) and thrombopoietin (TPO). Expression levels of GATA-1, Fli-1 and NF-E2 were evaluated using real-time PCR and western blot. Platelet-like particles were characterized by the presence of GPIb and GPIIb by flow cytometry, while the soluble fragment of GPIb, glycocalicin, was detected by enzyme immunoassay. Dense and alpha granules were evaluated by mepacrine staining and thrombospondin-1 detection, respectively, and by electron microscopy. Functional capacity of platelet-like particles was studied by measuring P-selectin membrane after thrombin stimulation by flow cytometry and actin polymerization using phalloidin-FITC by immunofluorescence. We found that stimulation of DAMI cells with high concentration of PMA and TPO induced the expression of transcription factors GATA-1 and Fli-1 followed by an increase in the isoform a of NF-E2. Mature DAMI cells give rise to extensions resembling proplatelets and later, produce platelet-like particles expressing GPIIb and GPIb on their surface and containing dense and alpha granules, which were confirmed by electron microscopy. Platelet functionality was demonstrated by the increase in P-selectin membrane expression after thrombin stimulation and by their ability to spread on fibrinogen matrices. DAMI cell line induced to differentiate into mature megakaryocytes is able to produce functional platelets providing a suitable model to study the mechanisms involved in platelet generation.

Objective: In a previous study, we found increased plasma soluble receptor for interleukin-6 (sIL-6R) levels in patients with essential thrombocythemia (ET) that could promote megakaryopoiesis through IL-6 binding and further interaction with the signal transducer gp130. Here we have searched for the cell source of sIL-6R within mononuclear cells in these patients and the underlying abnormalities involved in its overproduction., Materials and Methods: Thirty patients with the diagnosis of ET were studied. sIL-6R levels were measured by enzyme-linked immunosorbent assay technique in the supernatants of peripheral monocyte and lymphocyte cultures. Expression of membrane-anchored IL-6R was determined by flow cytometry. In order to study the mechanism of sIL-6R production, tumor necrosis factor-α protease inhibitor was added to specifically block IL-6R shedding. Gene expression of sIL-6R levels were evaluated by reverse transcription polymerase chain reaction., Results: Monocytes were the main source of sIL-6R. Besides, in ET patients, monocyte sIL-6R release was higher than that of controls (p = 0.0014). Lymphocytes enhanced monocyte sIL-6R production by cell-mediated contact in normal controls, but this cooperation could not be seen in patients. Membrane expression of IL-6R was increased after monocyte adhesion in ET. sIL-6R synthesis was upregulated in most patients, while messenger RNA was normal., Conclusions: Our results indicate that ET monocytes are responsible for sIL-6R overproduction within mononuclear cells through synthesis upregulation. In addition, the lack of cooperation of lymphocytes in monocyte sIL-6R production in ET could be due to a monocyte abnormality. The agonistic effect of sIL-6R on IL-6 action could contribute to the exacerbated megakaryocytic growth in ET., (Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO)., Methods: Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA., Results: MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was <50% in platelets from the ET patient, who also showed the mutation in granulocytes, monocytes and B cells. Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced., Conclusions: The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.

Objective: JAK2V617F mutation rate in granulocytes from essential thrombocythemia (ET) patients ranges from 12% to 57%. Our aim was to evaluate the frequency of this mutation in the megakaryocyte/platelet lineage, and to analyze its clinical associations in ET. In addition, we determined whether this mutation leads to constitutive phosphorylation of STAT5 in platelets., Materials and Methods: Consecutive patients with ET were included and clinical features were retrospectively reviewed. Mutation detection was performed by allele specific RT-PCR (AS-RT-PCR) and Restriction fragment length polymorphism (RFLP) analysis of platelet RNA. Constitutive phosphorylation of STAT5 in platelets was studied by Western blot., Results: Fifty patients were included, 24 (48%) were JAK2V617F-positive by both AS-RT-PCR and RFLP. Patients with the mutation were older, had significantly higher hemoglobin levels, and lower platelet counts. Besides, higher frequency of thrombotic events was found in JAK2V617F-positive patients younger than 60, 53% vs. 4%, P = 0.0008. In addition, constitutive STAT5 phosphorylation was not detected in platelets from 12 patients., Conclusions: The frequency of the JAK2V617F mutation in platelets was similar to that reported in granulocytes in the literature, suggesting this mutation does not occur as an isolated event in the megakaryocyte lineage. If confirmed in a larger study, the observed higher frequency of thrombosis in patients younger than 60 might be a useful predictive marker for thrombosis in this subset of patients. Even though this mutation has been predicted to constitutively activate the JAK2 kinase, spontaneous phosphorylation of STAT5 does not seem to be a frequent finding in platelets from ET patients.

Germ-line heterozygous mutations in the hematopoietic transcription factor AML1 (RUNX1) have been identified in patients with familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), which is characterized by thrombocytopenia, abnormal platelet function, and propensity to myeloid malignancies. We identified a novel mutation in the AML1 gene in an FPD/AML pedigree characterized by a single nucleotide deletion that generates a frameshift and premature chain termination (Pro218fs-Ter225). Both wild-type and mutant transcripts were expressed in affected individuals by allele-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Thrombopoietin (TPO) binds to the Mpl receptor and is the major regulator of megakaryopoiesis. To explore the mechanisms underlying thrombocytopenia, we studied the TPO/Mpl pathway in this newly identified pedigree. TPO levels were mildly to moderately elevated. On flow cytometry and immunoblotting, Mpl receptor expression was decreased and TPO-induced signaling was impaired. While no mutations were identified in the MPL gene by sequence analysis, low MPL mRNA levels were found, suggesting decreased gene expression. Of particular interest, several AML1-binding motifs are present in the MPL promoter, suggesting MPL is an AML1 target. In conclusion, we identified a C-terminal AML1 mutation that leads to a decrease in Mpl receptor expression, providing a potential explanation for thrombocytopenia in this FPD/AML pedigree.