Inhibition of LDHA to induce eEF2 release enhances thrombocytopoiesis.

Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet-specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell-derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy.

A Homozygotic Mutation in KDSR may Cause Keratinization Disorders and Thrombocytopenia: A Case Report.

Pathogenic mutations in 3-keto-dihydrosphingosine reductase (KDSR) gene are associated with keratinization disorders and impaired platelet function. However, no case with both homozygotic mutation of KDSR and hepatic hemangioendothelioma has ever been reported due to its low prevalence. Here we report a seven months old Chinese boy with a homozygotic missense mutation in KDSR and both of his parents carry a same heterozygous mutation. He was born with thick plate-like scales overlying erythrodermic skin, but the skin symptoms were resolved spontaneously over the first month of his birth. He was also diagnosed with hepatic hemangioendothelioma at birth and accepted a resection surgery at 2 months old. At birth, his platelet count was severely low (10-20×109/L) with recurrent skin and gingival bleeding. Meanwhile, he suffered a mild normocytic, normochromic anemia with normal iron and hematinic levels. The anemia spontaneously recovered over the first 6 months, while the platelet count keeped at a low level (4-20×109/L). Treatment with corticosteroids, immunoglobulin or thrombopoietin was all suboptimal.

The role of AGK in thrombocytopoiesis and possible therapeutic strategies.

Abnormal megakaryocyte development and platelet production lead to thrombocytopenia or thrombocythemia and increase the risk of hemorrhage or thrombosis. Acylglycerol kinase (AGK) is a mitochondrial membrane kinase that catalyzes the formation of phosphatidic acid and lysophosphatidic acid. Mutation of AGK has been described as the major cause of Sengers syndrome, and the patients with Sengers syndrome have been reported to exhibit thrombocytopenia. In this study, we found that megakaryocyte/platelet-specific AGK-deficient mice developed thrombocytopenia and splenomegaly, mainly caused by inefficient bone marrow thrombocytopoiesis and excessive extramedullary hematopoiesis, but not by apoptosis of circulating platelets. It has been reported that the G126E mutation arrests the kinase activity of AGK. The AGK G126E mutation did not affect peripheral platelet counts or megakaryocyte differentiation, suggesting that the involvement of AGK in megakaryocyte development and platelet biogenesis was not dependent on its kinase activity. The Mpl/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (Stat3) pathway is the major signaling pathway regulating megakaryocyte development. Our study confirmed that AGK can bind to JAK2 in megakaryocytes/platelets. More interestingly, we found that the JAK2 V617F mutation dramatically enhanced the binding of AGK to JAK2 and greatly facilitated JAK2/Stat3 signaling in megakaryocytes/platelets in response to thrombopoietin. We also found that the JAK2 JAK homology 2 domain peptide YGVCF617CGDENI enhanced the binding of AGK to JAK2 and that cell-permeable peptides containing YGVCF617CGDENI sequences accelerated proplatelet formation. Therefore, our study reveals critical roles of AGK in megakaryocyte differentiation and platelet biogenesis and suggests that targeting the interaction between AGK and JAK2 may be a novel strategy for the treatment of thrombocytopenia or thrombocythemia.

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets.

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

Role of Protein Kinase C-delta in regulating platelet activation and platelet-leukocyte interaction during sepsis.

Sepsis is characterized by an intense systemic inflammatory response activating a cascade of proinflammatory events resulting in leukocyte dysregulation and host tissue damage. The lung is particularly susceptible to systemic inflammation, leading to acute lung injury. Key to inflammation-induced lung damage is the excessive migration of neutrophils across the vascular endothelium. The mechanisms which regulate neutrophil activation and migration in sepsis are not well defined but there is growing evidence that platelets are actively involved and play a key role in microvascular permeability and neutrophil-mediated organ damage. We previously identified PKC-delta (PKCδ) as a critical regulator of the inflammatory response in sepsis and demonstrated PKCδ inhibition was lung protective. However, the role of PKCδ in sepsis-induced platelet activation and platelet-leukocyte interactions is not known. In this study, rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Following surgeries, a PKCδ inhibitor (200µg/kg) or vehicle (PBS) was administered intra-tracheally. At 24 hours post-surgeries, lung tissue, BAL fluid, and blood samples were collected. While sepsis caused thrombocytopenia, the remaining circulating platelets were activated as demonstrated by increased p-selectin expression, elevated plasma PF4, and enhanced platelet-leukocyte aggregate formation compared to Sham animals. Platelet activation was associated with increased platelet PKCδ activity. Inhibition of PKCδ attenuated sepsis-induced platelet activation, secretion and aggregate formation. Sepsis-induced thrombocytopenia was also significantly reduced and circulating platelet numbers were similar to sham animals. In the lung, sepsis induced significant influx of platelets and neutrophils and the development of lung injury. Administration of the PKCδ inhibitor decreased platelet and neutrophil influx, and was lung protective. Thus, PKCδ inhibition modulated platelet activity both locally and systemically, decreased neutrophil influx into the lung, and was lung protective. We demonstrate for the first time that PKCδ plays an important role in platelet activation and platelet-neutrophil interaction during sepsis.

PKC-epsilon deficiency alters progenitor cell populations in favor of megakaryopoiesis.

BACKGROUND: It has long been postulated that Protein Kinase C (PKC) is an important regulator of megakaryopoiesis. Recent contributions to the literature have outlined the functions of several individual PKC isoforms with regard to megakaryocyte differentiation and platelet production. However, the exact role of PKCε remains elusive. OBJECTIVE: To delineate the role of PKCε in megakaryopoiesis. APPROACH AND RESULTS: We used a PKCε knockout mouse model to examine the effect of PKCε deficiency on platelet mass, megakaryocyte mass, and bone marrow progenitor cell distribution. We also investigated platelet recovery in PKCε null mice and TPO-mediated signaling in PKCε null megakaryocytes. PKCε null mice have higher platelet counts due to increased platelet production compared to WT littermate controls (p<0.05, n = 8). Furthermore, PKCε null mice have more bone marrow megakaryocyte progenitor cells than WT littermate control mice. Additionally, thrombopoietin-mediated signaling is perturbed in PKCε null mice as Akt and ERK1/2 phosphorylation are enhanced in PKCε null megakaryocytes stimulated with thrombopoietin. Finally, in response to immune-induced thrombocytopenia, PKCε null mice recovered faster and had higher rebound thrombocytosis than WT littermate control mice. CONCLUSIONS: Enhanced platelet recovery could be due to an increase in megakaryocyte progenitor cells found in PKCε null mice as well as enhanced thrombopoietin-mediated signaling observed in PKCε deficient megakaryocytes. These data suggest that PKCε is a negative regulator of megakaryopoiesis.

Purinergic ecto-enzymes participate in the thromboregulation in acute in mice infection by Trypanosoma cruzi.

Coagulation disorders have been described in Chagas disease with thrombocytopenia as an important event. Several mechanisms may be related to this pathogenesis, such as enzymes of the purinergic system, purine, and receptors involved in the regulation and modulation of physiological events related to hemostasis. Therefore, the aim of this study was to evaluate the activities of E-NTPDase, E-5'nucleotidase, and ecto-adenosine deaminase (E-ADA) in platelets of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. Mice of infected group were intraperitoneally inoculated with 104 trypomastigotes of T. cruzi (strain Y). On day 12 post-infection (PI), blood samples were collected for quantitation and separation of platelets. A significant reduction in the number of platelets of infected mice (P < 0.05) was observed. The activities of E-NTPDase (ATP and ADP substrates), E-5'nucleotidase, and E-ADA in platelets increased significantly (P < 0.05) in mice infected by T. cruzi compared with uninfected animals. A negative correlation (P < 0.01)was observed between the number of platelets and ATP hydrolysis (r = -0.64), and ADP hydrolysis (r = -0.69) by E-NTPDase. Therefore, there is a response from the purinergic system activating ecto-enzymes in platelets of mice T. cruzi infected, as a compensatory effect of thrombocytopenia.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

OBJECTIVE: The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). APPROACH AND RESULTS: HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. CONCLUSIONS: Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Enhanced Megakaryopoiesis and Platelet Activity in Hypercholesterolemic, B6-Ldlr-/-, Cdkn2a-Deficient Mice.

BACKGROUND: Genome-wide association studies for coronary artery disease/myocardial infarction revealed a 58 kb risk locus on 9p21.3. Refined genetic analyses revealed unique haplotype blocks conferring susceptibility to atherosclerosis per se versus risk for acute complications in the presence of underlying coronary artery disease. The cell proliferation inhibitor locus, CDKN2A, maps just upstream of the myocardial infarction risk block, is at least partly regulated by the noncoding RNA, ANRIL, overlapping the risk block, and has been associated with platelet counts in humans. Thus, we tested the hypothesis that CDKN2A deficiency predisposes to increased platelet production, leading to increased platelet activation in the setting of hypercholesterolemia. METHODS AND RESULTS: Platelet production and activation were measured in B6-Ldlr(-/-)Cdkn2a(+/-) mice and a congenic strain carrying the region of homology with the human 9p21.3/CDKN2A locus. The strains exhibit decreased expression of CDKN2A (both p16(INK4a) and p19(ARF)) but not CDKN2B (p15(INK4b)). Compared with B6-Ldlr(-/-) controls, both Cdkn2a-deficient strains exhibited increased platelet counts and bone marrow megakaryopoiesis. The platelet overproduction phenotype was reversed by treatment with cyclin-dependent kinase 4/6 inhibitor, PD0332991/palbociclib, that mimics the endogenous effect of p16(INK4a). Western diet feeding resulted in increased platelet activation, increased thrombin/antithrombin complex, and decreased bleeding times in Cdkn2a-deficient mice compared with controls. CONCLUSIONS: Together, the data suggest that one or more Cdkn2a transcripts modulate platelet production and activity in the setting of hypercholesterolemia, amenable to pharmaceutical intervention. Enhanced platelet production and activation may predispose to arterial thrombosis, suggesting an explanation, at least in part, for the association of 9p21.3 and myocardial infarction.

G6PC3 Deficiency: Primary Immune Deficiency Beyond Just Neutropenia.

Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency was recently defined as a new severe congenital neutropenia subgroup remarkable with congenital heart defects, urogenital malformations, endocrine abnormalities, and prominent superficial veins. Here, we report 3 patients with G6PC3 deficiency presenting with recurrent diarrhea, failure to thrive, and sinopulmonary infections leading to bronchiectasis. In patient I and II, a combined immune deficiency was suspected due to early-onset disease with lymphopenia, neutropenia, and thrombocytopenia, along with variable reductions in lymphocyte subpopulations and favorable response to intravenous γ-globulin therapy. Apart from neutropenia, all 3 patients had intermittent thrombocytopenia, anemia, and lymphopenia. All patients had failure to thrive and some of the classic syndromic features of G6PC3 deficiency, including cardiac abnormalities and visibility of superficial veins in all, endocrinologic problems in PI and PIII, and urogenital abnormalities in PII. Our experience suggests that a diagnosis of congenital neutropenia due to G6PC3 may not be as straightforward in such patients with combined lymphopenia and thrombocytopenia. A high index of suspicion and the other syndromic features of G6PC3 were clues to diagnosis. Screening of all combined immune deficiencies with neutropenia may help to uncover the whole spectra of G6PC3 deficiency.

Protein kinase C δ deficiency enhances megakaryopoiesis and recovery from thrombocytopenia.

OBJECTIVE: We previously determined that protein kinase C δ (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown. APPROACH AND RESULTS: Using PKCδ(-/-) and wild-type littermate mice, we found that deficiency of PKCδ caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKCδ(-/-) mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKCδ deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKCδ(-/-) megakaryocytes compared with wild-type. Finally, PKCδ(-/-) mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. CONCLUSIONS: These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.

Effect of ketoprofen on lactic dehydrogenase from human platelets.

BACKGROUND: In different clinical investigations of thrombocytopenia, ketoprofen was found to be the associated cause. Ketoprofen alone or in combination with other therapeutic regimens leads to a decrease in platelet count. Thrombocytopenia due to ketoprofen use can be a threatening condition to the patients who require uncompromised platelet function. OBJECTIVES: In order to establish a mechanism for thrombocytopenia associated with ketoprofen use, the enzyme inhibition effects of ketoprofen on lactic dehydrogenase (LDH) were investigated in this study. LDH is essentially involved in platelet energy production. MATERIAL AND METHODS: LDH isolated from human platelets was subjected to different concentrations of ketoprofen (250, 500, 750, 1000 and 1500 µg/mL) and pyruvate as a substrate (45, 60 and 90 µM/mL) to gain insight into the enzyme inhibition effects for forward reaction. Oxidation of nicotinamide adenine dinucleotide (NADH) was measured at 340 nm to evaluate enzyme activity. Enzyme inhibition kinetics were studied via Lineweaver Burk plot. RESULTS: Ketoprofen was found to be a competitive inhibitor of LDH in human platelets. 89% of enzyme activity was inhibited by a 1500 µg/mL concentration of the drug and the enzyme inhibition constant was 882 µg/mL. CONCLUSIONS: The possible main cause of thrombocytopenia due to ketoprofen use is LDH inhibition in platelets, which are essential for platelet energy metabolism. So patients who require uncompromised platelet function and are receiving ketoprofen in their prescription should be monitored for platelet count and blood clotting.

Three-year efficacy, safety, and survival findings from COMFORT-II, a phase 3 study comparing ruxolitinib with best available therapy for myelofibrosis.

Ruxolitinib is a potent Janus kinase (JAK)1/JAK2 inhibitor that has demonstrated rapid reductions in splenomegaly and marked improvement in disease-related symptoms and quality of life in patients with myelofibrosis (MF). The present analysis reports the 3-year follow-up (median, 151 weeks) of the efficacy and safety of Controlled Myelofibrosis Study With Oral Janus-associated Kinase (JAK) Inhibitor Treatment-II (the COMFORT-II Trial), comparing ruxolitinib with the best available therapy (BAT) in 219 patients with intermediate-2 and high-risk MF. In the ruxolitinib arm, with continued therapy, spleen volume reductions of ≥35% by magnetic resonance imaging (equivalent to approximately 50% reduction by palpation) were sustained for at least 144 weeks, with the probability of 50% (95% confidence interval [CI], 36-63) among patients achieving such degree of response. At the time of this analysis, 45% of the patients randomized to ruxolitinib remained on treatment. Ruxolitinib continues to be well tolerated. Anemia and thrombocytopenia were the main toxicities, but they were generally manageable, improved over time, and rarely led to treatment discontinuation (1% and 3.6% of patients, respectively). No single nonhematologic adverse event led to definitive ruxolitinib discontinuation in more than 1 patient. Additionally, patients randomized to ruxolitinib showed longer overall survival than those randomized to BAT (hazard ratio, 0.48; 95% CI, 0.28-0.85; log-rank test, P = .009).

Successful liver transplantation in a patient with splanchnic vein thrombosis and pulmonary embolism due to polycythemia vera with Jak2v617f mutation and heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatment resulting in a severe acquired thrombophilic condition with an associated mortality of about 10 %. We report the first case of successful urgent liver transplantation (LT) in a patient with end-stage liver disease due to a Budd-Chiari syndrome, portal vein thrombosis and pulmonary embolism due to acquired thrombophilia associated to polycythemia vera carrying JAK2V617F gene mutation and HIT in the acute phase. Lepirudin was used to provide anticoagulation in the LT perioperative period that was performed without haemorrhagic and thrombotic complications despite the donor received heparin during liver explantation.