Thrombopoietin Levels Research Articles

The Potential Role of Thrombopoietin and Interleukin-6 in the Thrombocytosis Effect of Carica papaya Leaves

Dengue fever is endemic in tropical urban developing regions worldwide. Thrombocytopenia is an important clinical feature which may result in bleeding. However, there is no specific treatment for Dengue-induced thrombocytopenia. Carica papaya leaves (CPL) is a popular remedy in South East Asia to treat Dengue-induced thrombocytopenia. Development of CPL into pharmaceutical therapeutic agents is not forthcoming due to lack of rigorous scientific evidence and unknown mechanism of action. This study investigated the role of thrombopoietin (TPO) and interleukin (IL-6) in the thrombocytosis effect of CPL in vivo. These experiments were conducted using busulfan-induced thrombocytopenic rats. Treatment of aqueous and methanol extracts of CPL at 600mg/kg were administered orally for 7 consecutive days and serum platelet count was determined intermittently until day 15. At the end of experiments, serum Thrombopoietin (TPO) and IL-6 levels were determined by ELISA. Both aqueous and methanol extracts of CPL significantly increased platelet count compared to the control groups (x2 (2) = 25.373, P = 0.00). Investigations into the mechanism of thrombocytosis showed that TPO and IL-6 levels were increased compared to controls but was not statistically significant (H (3) = 5.339 P = 0.149) (H (3) = 4.412 P = 0.220) respectively. This study is the first to document the thrombocytosis effect of both aqueous and methanol CPL extracts in a rodent model system. Our findings showed that aqueous extract of CPL demonstrated an increase of TPO and IL-6 levels. We suggested that the possible mechanism could be linked with the upregulation of major thrombopoietic cytokines such as TPO and IL-6.

Abstract 15499: The Increase of Proplatelets and Tpo in Circulation May be a New Pathological Mechanism of Thrombosis

Introduction: Platelets are produced from megakaryocytes, which mainly regulated by thrombopoietin (TPO) and 5-HT. We hypothesized that the secretion of inflammatory cytokines increased in patients with thrombosis under stress or inflammation, leading to increased TPO levels, which in turn increased production capacity of platelet, and increased the number of platelet intermediates proplatelets into the circulation. Therefore, the increase in the number of proplatelets in the circulation, and the increase in plasma TPO levels may be a new pathological mechanism of thrombosis. Methods: TPO was detected by ELISA. Proplatelets from patients were stained with CD41-FITC and further confirmed by a fluorescence microscope and flow cytometry. The in vitro proplatelets were tested by CD41-FITC staining under a fluorescence microscope and flow cytometry detection. Results: Our clinical data demonstrated that patients' plasma TPO levels were significantly higher with acute inflammation state compared with healthy subjects (181.1 ± 35.38 vs 96.1 ± 9.7 pg/ml, p<0.001, n=65). The plasma TPO levels of cerebral infarction patients (186.2±12.5 pg/ml, n=20) were significantly higher than that of the control group (122.3±10.2 pg/ml, n=20). The number of platelets in patients with cerebral infarction (222.11±8.55 х10 9 /L, n=20) was slightly higher than that in the control group (206.55±8.83 х10 9 /L, n=20). More large platelets or proplatelets were found in the circulating blood of patients with cerebral infarction, which was significantly different from the control group (n=8, p<0.05). In vitro study confirmed that in 200 megakaryocytes (MKs), more proplatelets were produced in the TPO group (100 ng/ml, 18±2.5%) and in the 5-HT group (100 nM, 15±3.2%), while the control group was only (7±3.2) % (n=5). The effects of TPO/ 5-HT were interfered with by its receptor blockers, confirming that their effects were mediated by its receptors. Both TPO and 5-HT affect the cytoskeleton by activating p-ERK1/2, reorganizing F-actin to generate proplatelets, and its role was blocked by PD98059. Conclusions: The studies showed that the increase of proplatelets in circulation and TPO levels in plasma may be a new pathological mechanism of cerebral infarction or thrombosis.

Increased Circulating Proplatelets and Elevated Plasma TPO Levels: A Novel Pathological Mechanism of Cerebral Infarction

Background: Platelets are produced by megakaryocytes and are primarily regulated by thrombopoietin (TPO). In the conditions of inflammation or stress, increased 5-HT and other inflammatory cytokines may affect liver and bone marrow to increase TPO and platelet production (Yang et al., Stem Cells, 2007; 2014). Mature megakaryocytes are fragmented in pulmonary circulation to generate more platelet intermediate proplatelets, then entered the circulation. We hypothesized that in patients with cerebral infarction, the secretion of inflammatory cytokines increases under stress or inflammatory conditions, increasing circulating TPO levels, followed by an increase in platelet production, and an increase in the number of platelet intermediate proplatelets. At the same time, platelets also have varying degrees of activation. Therefore, the increase in the number of proplatelets in the circulation and the increase in TPO levels may be the new pathological mechanism of cerebral infarction. Methods: The plasma levels of TPO were detected by ELISA. The in vivo proplatelets from patients were stained with CD41-FITC, which was further confirmed under a fluorescence microscope and flow cytometry. The in vitro proplatelets were observed in a culture system by CD41-FITC staining under a fluorescence microscope and flow cytometry detection. Results: Our clinical data demonstrated that patients with acute inflammation states, plasma TPO levels were significantly higher compared with healthy subjects (181.1 ± 35.38 vs 96.1 ± 9.7 pg/ml, p<0.001, n=65), which may act as an acute response protein to protect the body. We also detected 20 patients with cerebral infarction and 20 normal controls and found that the plasma TPO levels of cerebral infarction patients (186.2±12.5 pg/ml) was significantly higher than that of the control group (122.3±10.2 pg/ml). The number of platelets in patients with cerebral infarction (222.11±8.55 ×109/L, n=20) was slightly higher than that in the control group (206.55±8.83 ×109/L, n=20). More importantly, we found more large platelets or proplatelets in the circulating blood of patients with cerebral infarction, which was significantly different from the control group (n=8). Furthermore, in vitro study confirmed that in 200 megakaryocytes (MKs), (18±2.5)% of MKs producing proplatelets in the TPO group (100 ng/ml), and in 5-HT group (100 nM), (15±3.2)% of MKs producing proplatelets, while the control group was only (7±3.2)% (n=5). At the same time, the effects of TPO and 5-HT were interfered by the corresponding receptor blockers, confirming that their effects were mediated by its receptors. The study further confirmed that both TPO and 5-HT affect the cytoskeleton by activating p-ERK1/2, reorganizing F-actin to generate proplatelets, and its role was blocked by PD98059. Conclusions: The study demonstrated that increased numbers of proplatelets in circulation and elevated plasma TPO levels may be novel pathological mechanisms of cerebral infarction Disclosures No relevant conflicts of interest to declare.

Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p<0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

Platelet-derived microparticles enhance megakaryocyte differentiation and platelet generation via miR-1915-3p

Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.

S2381 Evaluating Platelet Quantity in the Presence of Essential Thrombocythaemia With Cirrhosis

INTRODUCTION: Thrombocytopenia is normally a sequela of decompensated cirrhosis. Essential thrombocythaemia (ET), by contrast, is a myeloproliferative disorder involving an elevated platelet count that can manifest as thromboses of microvasculature. The rare concurrence of these two conditions is challenging for providers in that a normal platelet count may not be a proper indication of an adequate synthetic liver function, as a baseline value is unknown. CASE DESCRIPTION/METHODS: 66-year-old female with a history of ET and untreated chronic hepatitis B and C viral infections, who presented with intermittent left upper quadrant pain. On physical exam, the patient’s vital signs were within normal limits, and abdomen was non-tender on palpation. A complete metabolic panel was significant for elevated aminotransferases, with AST 78, ALT 78, and an alkaline phosphatase of 164. Additionally, a complete blood count was significant for thrombocytosis to 879 and hemoglobin of 10.6. Magnetic resonance imaging of the abdomen showed evidence of cirrhosis with sequelae of portal hypertension, including splenomegaly and upper abdominal varices, which were also seen on computed tomography of abdomen. She was diagnosed with new-onset cirrhosis secondary to chronic untreated viral hepatitis. Thrombopoietin (TPO) levels were not assessed as platelet count was markedly elevated. The patient was continued on her home hydroxyurea for continued management of ET. DISCUSSION: Platelet count is an important measure for patients with cirrhosis. Calculation like the AST to Platelet Ratio Index (APRI) score is used to predict disease severity. Assessing platelet production as an indicator of synthetic function in cirrhosis becomes difficult when treating patients with known ET. Prior cases have suggested that the thrombocytosis in ET may be attributed to an abnormality of the normal feedback inhibition responsible for platelet turnover. TPO levels are generally normal in ET and are notably reduced in decompensated cirrhosis. Providers, therefore, should consider direct measurement of TPO levels in patients with normal platelet counts who are diagnosed with ET and cirrhosis in order to assess whether synthetic liver function is impacted by liver disease. Furthermore, patients with cirrhosis have an increased risk of portal vein thrombosis (PVT); given that patients with both cirrhosis and ET may have an even higher risk of PVT, providers may consider prophylactically anticoagulating these patients to prevent such a complication.

Immature cell fractions after cessation of chronic P2Y12-inhibition in patients with coronary artery diseases

Changes in circulating cell populations may promote ischemic events that occur soon after discontinuation of P2Y12-inhibition. The aim of the study was to track the course of thrombopoietic and erythropoietic cells in patients with coronary artery diseases (CAD) after planned and physician-driven cessation of chronic P2Y12-inhibition (clopidogrel 75 mg OD, or prasugrel 10 mg OD, or ticagrelor 90 mg BID). Cell fractions were determined in 62 patients at baseline (the last day of P2Y12-inhibitor intake), on day-10, day-30, and day-180 thereafter. Immature platelet fraction (IPF), immature reticulocyte fraction (IRF), reticulocyte hemoglobin content (Ret-Hb) and red blood cell count (RBC) significantly increased from baseline to day-180 (IPF: p = .003; IRF: p = .013; Ret-Hb: p < .001; RBC: p = .044). Platelet count, leucocyte count and immature granulocyte fraction did not change over time (p = .561, p = .869, and p = .161, respectively). Fibrinogen levels significantly declined over time (p = .011), thrombopoietin levels increased in a non-significant manner (p = .379). We did not observe any significant interaction with choice of P2Y12-inhibitor, therefore suggesting a drug class-effect. Our data shows, that discontinuation of dual antiplatelet therapy is associated with raised thrombopoietic and erythropoietic activity in the bone marrow, without significant upregulation of thrombopoietin. This provides further evidence for a direct stimulation of precursor cells by P2Y12-inhibitors.

Megakaryocytes promote osteoclastogenesis in aging.

Megakaryocytes (MKs) support bone formation by stimulating osteoblasts (OBs) and inhibiting osteoclasts (OCs). Aging results in higher bone resorption, leading to bone loss. Whereas previous studies showed the effects of aging on MK-mediated bone formation, the effects of aging on MK-mediated OC formation is poorly understood. Here we examined the effect of thrombopoietin (TPO) and MK-derived conditioned media (CM) from young (3-4 months) and aged (22-25 months) mice on OC precursors. Our findings showed that aging significantly increased OC formation in vitro. Moreover, the expression of the TPO receptor, Mpl, and circulating TPO levels were elevated in the bone marrow cavity. We previously showed that MKs from young mice secrete factors that inhibit OC differentiation. However, rather than inhibiting OC development, we found that MKs from aged mice promote OC formation. Interestingly, these age-related changes in MK functionality were only observed using female MKs, potentially implicating the sex steroid, estrogen, in signaling. Further, RANKL expression was highly elevated in aged MKs suggesting MK-derived RANKL signaling may promote osteoclastogenesis in aging. Taken together, these data suggest that modulation in TPO-Mpl expression in bone marrow and age-related changes in the MK secretome promote osteoclastogenesis to impact skeletal aging.

Association of Immature Platelet Fraction and Trombopoietin Levels Based on the Grades of Sepsis Severity

Sepsis is one of the major causes of morbidity and mortality around the world. Organ dysfunction in sepsis can be identified using Sepsis-related Organ Failure Assessment. The dysfunction of the coagulation cascade, the number of pro-inflammatory cytokines, and endothelial dysfunction promote the consumptive thrombocytopenia in sepsis patients, which triggers a compensatory response in the body by increasing thrombopoietin release. As a result, it increases platelet production which can be assessed by immature platelet fraction (IPF). This study is a preliminary research to see the relationship between IPF values and serum thrombopoietin (TPO) levels in terms of the clinical severity of patients with sepsis based on SOFA scores. A cross-sectional design was performed in this study with 49 samples of patients with sepsis, collected during June-July 2019, at Dr. Wahidin Sudirohusodo Hospital in Makassar. The study was conducted in August 2019. IPF values were measured by the fluorescent flow cytometry method. Meanwhile, serum thrombopoietin levels were calculated by the enzyme-linked immunosorbent assay (ELISA) method using human Thrombopoietin ELISA (RayBioTech, Australia). The results showed that the IPF value increased along with the SOFA score, followed by a decrease in platelet (p = 0.014). TPO levels presented an upward along with SOFA scores with a decrease in platelet (p = 0.008). There was a significant correlation between IPF values and TPO levels (r = 0.606, p &lt;0.001) where the Immature Platelet Fraction score and thrombopoietin levels increased with SOFA scores. In addition, there was also a positive correlation between IPF values and TPO levels in sepsis patients.

Mechanisms of anti-GPIbα antibody–induced thrombocytopenia in mice

AbstractImmune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody–induced ITP.

Eltrombopag monotherapy can improve hematopoiesis in patients with low to intermediate risk-1 myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by low blood counts and a propensity to develop acute myeloid leukemia. The management of lowerrisk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag, a thrombopoietin-receptor agonist, can improve platelet counts in LR-MDS and trilineage hematopoiesis in aplastic anemia. We conducted a phase II dose modification study to investigate the safety and efficacy of eltrombopag in LR-MDS. The eltrombopag dose was escalated from 50 mg/day to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a paroxysmal nocturnal hemoglobinuria clone, marrow hypocellularity, thrombocytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with that found in previous eltrombopag studies in aplastic anemia; no patients discontinued the drug due to adverse events. Three patients developed reversible grade 3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued eltrombopag after achieving a robust response (median time 16 months); four of them reinitiated eltrombopag because of declining blood counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to develop acute myeloid leukemia on study. In conclusion, eltrombopag was well-tolerated and effective in restoring hematopoiesis in some patients with low or intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.

Thrombopoietin is associated with a prognosis of gastric adenocarcinoma.

OBJECTIVE Thrombopoietin (THPO) is well-known as a megakaryocyte growth and development factor (MGDF) involved in megakaryocyte proliferation and maturation. To explore the biological effects of THPO in gastric adenocarcinoma, we conducted this study. Methods: By accessing the TCGA database, the expression level of THPO was determined in tumor tissues. The association between THPO expression and clinical features, or prognostic significance was described by Cox regression analysis and Kaplan-Meier. The SiRNA method was used to decline the THPO expression; then cell viability, invasion, and migration were detected to verify the effects of the knockdown of THPO. qPCR and western blotting were implemented to examine the expression level of THPO. Results: The expression of THPO was increased in tumor tissue and cells, its high-regulation was associated with a poor prognosis in patients with gastric adenocarcinoma. Cell viability, invasion, and migration were suppressed in AGS with the down-regulation of THPO. Furthermore, on the basis of si-THPO transfection, E-cadherin was promoted while N-cadherin and Vimentin were attenuated. CONCLUSION Our results revealed that THPO may be a potent marker of gastric adenocarcinoma, providing a novel potential screening method for gastric adenocarcinoma.

Different roles of soluble CD40 ligand in central nervous system damage

ABSTRACT Backgroud and purpose: Soluble CD40 ligand (sCD40L) plays an important role in inflammation and autoimmune disorders. There is still a controversy regarding sCD40L in neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS). Herein the aims of this study were to evaluate the levels of sCD40L in patients with NMOSD, MS, and other noninflammatory neurological diseases; to investigate its potential relationship with laboratory parameters, glial fibrillary acidic protein (GFAP), thrombopoietin (TPO) and IL-6; and to address whether serum sCD40L levels in acute attacks of NMOSD patients were decreased after treatment with immunoglobulins, plasma exchange, or methylprednisolone.Materials and methods: We enrolled 13 patients with NMOSD, 9 patients with MS, and 9 patients with other noninflammatory neurological diseases. The levels of sCD40L, IL-6 were measured by cytokine multiplex assay. GFAP levels were measured by ELISA.Results: Both serum and cerebrospinal fluid (CSF) sCD40L levels were increased in NMOSD and MS. No differences were found in serum and CSF sCD40L levels between NMOSD and MS. The CSF sCD40L levels were positively correlated with the CSF cell counts in NMOSD, whereas serum sCD40L levels were positively correlated with the albumin index in MS. Furthermore, the levels of CSF sCD40L were positively correlated with CSF GFAP levels in NMOSD. Serum sCD40L levels were correlated with serum TPO levels in MS. No correlation was found between levels of sCD40L and IL-6 in NMOSD and MS. No statistically meaningful difference between NMOSD patients with or without immunotherapy. Conclusions: Our study suggests that sCD40L can contribute to the destruction of the blood-brain barrier in MS, whereas it may contribute to CNS inflammation in NMOSD. The serum sCD40L concentrations were not changed after treatment with immunoglobulins, plasma exchange, or methylprednisolone in acute attacks of NMOSD.

\u03b24GALT1 controls \u03b21 integrin function to govern thrombopoiesis and hematopoietic stem cell homeostasis

Glycosylation is critical to megakaryocyte (MK) and thrombopoiesis in the context of gene mutations that affect sialylation and galactosylation. Here, we identify the conserved B4galt1 gene as a critical regulator of thrombopoiesis in MKs. β4GalT1 deficiency increases the number of fully differentiated MKs. However, the resulting lack of glycosylation enhances β1 integrin signaling leading to dysplastic MKs with severely impaired demarcation system formation and thrombopoiesis. Platelets lacking β4GalT1 adhere avidly to β1 integrin ligands laminin, fibronectin, and collagen, while other platelet functions are normal. Impaired thrombopoiesis leads to increased plasma thrombopoietin (TPO) levels and perturbed hematopoietic stem cells (HSCs). Remarkably, β1 integrin deletion, specifically in MKs, restores thrombopoiesis. TPO and CXCL12 regulate β4GalT1 in the MK lineage. Thus, our findings establish a non-redundant role for β4GalT1 in the regulation of β1 integrin function and signaling during thrombopoiesis. Defective thrombopoiesis and lack of β4GalT1 further affect HSC homeostasis.

Avatrombopag for the management of thrombocytopenia in patients with chronic liver disease.

Severe thrombocytopenia (platelet count < 50 x 109/l) is the most frequent hematological disorder in patients with chronic liver disease, affecting 64-84 % of individuals with cirrhosis. The pathophysiological mechanisms that underlie thrombocytopenia are complex, but the reduction of thrombopoietin levels is considered to play a key role. Until recently, there was a continuous debate about the optimal management of patients with chronic liver disease and whether low platelet counts require a scheduled invasive procedure. Transfusion of platelet concentrates is considered to be the standard treatment, but has major limitations such as its short lifespan, limited efficacy and relevant adverse effects. Avatrombopag is an oral thrombopoietin receptor agonist that induces megakaryocytic proliferation and differentiation and platelet production. It has been approved by the Spanish Agency of Medicines and Health Products for the treatment of severe thrombocytopenia in adult patients with chronic liver disease before scheduled procedures, to decrease the risk of bleeding. Randomized trials have demonstrated that this agent is effective in maintaining platelet counts above 50 × 109/l over a period of more than two weeks, with a similar safety profile to the placebo. In this article, we review avatrombopag in the treatment of thrombocytopenia in patients with chronic liver disease. Furthermore, the main differences of this intervention in comparison to the previous standard of care therapy are discussed.

Biomarkers of Response to Romiplostim in Patients with Lower-Risk Myelodysplastic Syndrome (MDS) and Thrombocytopenia - Results of the Europe Trial By the Emsco Network

Biomarkers of Response to Romiplostim in Patients with Lower-Risk Myelodysplastic Syndrome (MDS) and Thrombocytopenia - Results of the Europe Trial By the Emsco Network

Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

Rebound Thrombocytosis after Induction Chemotherapy Is a Strong Biomarker for Favorable Outcome in AML Patients

Background: Whereas the molecular events underlying acute myeloid leukemia (AML) are increasingly identified, dynamics of hematologic suppression and recovery following induction chemotherapy remain enigmatic. In addition, thrombocytopenia at diagnosis is related to deregulated cytokine expression in specific leukemia subtypes rather than on the degree of bone marrow infiltration per se. Similarly, recovery of platelets after the first induction cycle among individual patients can vary from incomplete to excess recovery even if morphologic remission is achieved. In particular, excess platelet rebound (EPR) with values exceeding 500 G/L can be observed in some patients. However, this clinical observation of EPR is poorly understood so far, and its prognostic relevance is unknown. Methods: We analyzed platelet recovery after the first induction cycle in 291 consecutive AML patients receiving at least one cycle of intensive "3+7" based induction treatment. We defined excess platelet rebound (EPR) as a platelet increase above 500 G/L during hematologic recovery after the first induction cycle. Results: We observed EPR in 120 (41.2%) patients. EPR+ patients had lower platelets at diagnosis, higher marrow infiltration, more frequently NPM1 mutations, and were associated with ELN favorable risk. Absence of EPR correlated with complex karyotypes, ELN intermediate-I and adverse risk, and therapy-related AML. Overall survival was better in EPR+ patients than EPR- (median 125 versus 41 months; P=0.04), as was disease-free survival (Figure 1). By multivariate analysis, achievement of EPR+ was an independent parameter associated with favorable survival. Plasma thrombopoietin (TPO) levels at diagnosis indicated EPR+ (p&lt;0.0001), while GATA-1, GATA-2 and MPL mRNA expression did not differ between EPR+ and EPR- patients. Finally, transcription factors blocking early megakaryopoiesis were upregulated in EPR- patients, while NFE2 involved in late megakaryocyte differentiation was increased in EPR+ patients. Conclusion: This study suggests that excess platelet rebound (EPR+) defined as platelets exceeding 500 G/L is a common event identified in AML patients after the first induction cycle. EPR+ was predominantly observed in ELN favorable risk AML, and it was associated with significantly better overall and disease-free survival as compared to EPR- patients. In addition, achievement of EPR+ represented an independent prognostic parameter for improved survival in a multivariate analysis, and may contribute to prognostic assessment in AML patients undergoing intensive treatment with intention to cure. Disclosures No relevant conflicts of interest to declare.

A Systematic Review and Meta-Analysis of Safety and Efficacy for Pre-Procedural Use of Thrombopoietin Receptor Agonists in Hepatic Cirrhosis Patients

Introduction Thrombocytopenia is common in liver cirrhosis patients, which often complicates with patients' frequent issue with gastrointestinal bleeding and procedural needs. Based on biologic understanding of decreased thrombopoietin(TPO) level in liver cirrhosis patients, use of TPO agonists in liver cirrhosis have been actively studied. Over the period of time, new studies have come out about 2 FDA-approved TPO agonists, Avatrombopag and Lusutrombopag, for prophylaxis before procedure in liver cirrhosis patients with thrombocytopenia. In the past, there have raised concerns of increased risk of portal vein thrombosis and other thrombotic risks in other agents. In our study, we aimed to study the effectiveness and safety of TPO receptor agonists for pre-procedural use in patients with liver cirrhosis. Study design Study was conducted from August 2018 to July 2019. Previous studies were identified through database searching MEDLINE, CENTRAL, Clinicaltrial.gov and google search. Randomized clinical trials with active treatment arm with TPO receptor agonists in the use of liver cirrhosis patients, with intention of pre-procedural prophylaxis, and having placebo for direct comparisons were included. One of the major exclusions was TPO receptor agonists to increase platelet counts for anti-viral treatment in cirrhosis patients. 14 studies were identified. Studies were reviewed and eligibility was determined by two independent clinically trained researchers. 5 duplicated studies were removed, and in total of 7 studies were included for quantitative and qualitative analysis. Details of studies were collected by 2 independent researchers and compared. When there is a discrepancy, repeat review of the study was conducted. Studies were conducted or published from 2012 to 2018. 1 trial from Eltrombopag, 3 trials from Avatrombopag, and 3 trials from Lusutrombopag were included. Result Characteristics of included studies are summarized in table 1. Our studies found that 5.5(95% CI : 4.35-7.15) times higher risk ratio(RR) of reaching platelet target before procedure compared to placebo. Target platelet number goal was 50,000 to 80,000 depending on the study. Studies showed homogenous result with I-squared was 30.8% and q-statistics of p-value 0.193.(Figure 1) Subgroup analysis by FDA-approved medication of Avatrombopag and Lusutrombopag showed statistically significant higher risk ratio of 4.74(3.36-6.68) and of 5.52(3.65-8.34) each compared to placebo. Risk ratio for preventing platelet transfusion was not able to be calculated with heterogeneity of study with I-square higher than 90%. Lusutrombopag study showed significant benefits (RR 6.33, 95% CI 2.95-13.58) with heterogeneity inside the same medications, which might be explained with different doses in studies. No statistical significance in risk ratio in a study with Avatrombopag. Subgroup analysis limited to phase 3 studies showed risk ratio of preventing transfusion of 2.87(95% CI 2.15-3.82) but heterogeneity with q-statistics of p-value at 0.029. Relative risk for adverse event related to portal vein thrombosis was not statistically significant with RR of 0.99(95% CI : 0.35-2.85) in total of 1,229 patients.(Figure 2) Study result was homogenous result by I-square 0%, q-statistics of p-value 0.794. Other severe adverse events, major bleeding risk, overall thrombosis risk were not statistically significant. Only increased risk without statistical significance was reported in trail with Eltrombopag which was early terminated. Conclusion Our meta-analysis of pre-procedural use of TPO agonist in liver cirrhosis patients showed statistically significant benefit of reaching platelet count goal by 5.58 times with risk ratio, but no benefit of preventing transfusion. Compared to prior studies including use of TPO agonists for Interferon-Ribavirin treatment, meta-analysis limited to pre-procedural use did not show statistically significant increase in portal vein thrombosis. Serious adverse events including thrombosis events and bleeding risk were not statistically significant. Most studies described that portal-vein thrombosis events were often related to high platelet counts about 200,000 and longer use of TPO agonist. In current era with lesser use of Interferon and Ribavirin as an anti-viral therapy, TPO agonists use in setting of pre-procedure mostly with lower platelet targets can be safely used. Disclosures No relevant conflicts of interest to declare.

The Ribosomal Biogenesis Inhibitor CX-5461 Is an Anti-Cancer Therapeutic That Increases Platelet Count in Mice and in Humans

Background: The ribosomal biogenesis inhibitor CX-5461 is a first-in-class selective inhibitor of ribosomal RNA gene transcription with single-agent antitumor activity against advanced hematologic cancers and synergistic activity when combined with front-line and emerging agents (Devlin et al. 2015, Sornkom et al. 2018, Maclachlan et al. 2018). It has predictable pharmacokinetics and a safety profile allowing prolonged dosing (Khot et al, Cancer Discov 2019). Unexpectedly, CX-5461 treatment increases platelet count by ~50% in tumour diseased mice; an effect that was maintained for up to 6 months of treatment. Aims: We investigated mechanisms that may contribute to increased platelet counts following CX-5461 treatment in a liver carcinoma mouse model or disease-free mice and evaluated blood test data from patients with haematological malignancies treated with CX-5461. Methods: We evaluated blood cell numbers in blood from vehicle- or CX-5461 treated mice with liver carcinoma, and blood cell numbers, bone marrow megakaryocytes, platelet receptors, immature platelets, platelet function, thrombopoietin (TPO) and inflammatory cytokine levels in disease-free C57BL/6 KaLwRij mice treated with 35 mg/kg CX-5461 or vehicle (n=15). Human platelet receptors and function were assessed in CX-5461-treated platelet-rich plasma by flow cytometry, platelet spreading assays and light transmission aggregometry. We evaluated temporal platelet count, and the platelet activation marker soluble glycoprotein (GP) VI in patient plasma samples who had received a single dose of 25-250 mg/m2 CX-5461 (n=16) with 1-way ANOVA. Results: In a model of liver carcinoma, mice with disease displayed significantly elevated (2.3-fold) platelet counts after 3 months treatment with biweekly injections of 35 mg/kg CX-5461 compared with vehicle-treated diseased mice (p = 0.0013). Disease-free mice treated for 7 or 14 days (3 or 6 x 35 mg/kg doses) with CX-5461 showed ~60% increase in platelet count; in comparison white and red cells were mildly suppressed. The CX-5461-mediated platelet increase at d7 (p&lt;0.0005) was reversible within 1 week. At d14 (p&lt;0.01, 1.7-fold increase in platelet count) a significant increase in megakaryocytes (p&lt;0.05) and immature platelets (p&lt;0.01) was observed. CX-5461 treatment had no effect on plasma TPO, platelet lifespan or platelet GPVI or GPIbα levels. Integrin αIIb was significantly elevated. Inflammatory cytokines interleukin (IL)-6 (p&lt;0.05) and TNFα (p&lt;0.01) increased at d7 in plasma. After pre-treatment for 30 min with CX-5461 ex vivo, human platelet function (aggregation and platelet spreading on collagen and fibrinogen) and receptor levels were normal. In 8/16 patients receiving CX-5461, increases of up to 34% in platelet counts were measured at day 15 of CX-5461 treatment, with no change in sGPVI. Conclusions: CX-5461 treatment of mice for two weeks increased platelet counts, megakaryocyte number and immature platelet fraction by an unknown mechanism however IL-6 has been reported by others to enhance megakaryopoeisis. Brief CX-5461 treatment in a group of patients with advanced haematological malignancy resulted in small increases in platelet count with retained platelet activation and treatment ex-vivo demonstrated platelet function to be unaffected. Future work will explore the link between CX-5461 treatment and megakaryopoiesis and thrombopoiesis, including the potential for CX-5461 to ameliorate drug-induced thrombocytopenia. Disclosures Harrison: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hannan:Pimera: Membership on an entity's Board of Directors or advisory committees, Research Funding.