Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings.

BACKGROUND: Non-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PURPOSE: This study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. METHODS: A potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. RESULTS: A natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. CONCLUSION: TMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

Elucidating the Mechanism of Action of the Attributed Immunomodulatory Role of Eltrombopag in Primary Immune Thrombocytopenia: An In Silico Approach.

Eltrombopag is a thrombopoietin receptor (MPL) agonist approved for the treatment of primary immune thrombocytopenia (ITP). Recent evidence shows that some patients may sustain platelet counts following eltrombopag discontinuation. The systemic immunomodulatory response that resolves ITP in some patients could result from an increase in platelet mass, caused either by the direct action of eltrombopag on megakaryocytes through MPL stimulation, or potential MPL-independent actions on other cell types. To uncover the possible mechanisms of action of eltrombopag, in silico analyses were performed, including a systems biology-based approach, a therapeutic performance mapping system, and structural analyses. Through manual curation of the available bibliography, 56 key proteins were identified and integrated into the ITP interactome analysis. Mathematical models (94.92% mean accuracy) were obtained to elucidate potential MPL-dependent pathways in non-megakaryocytic cell subtypes. In addition to the effects on megakaryocytes and platelet numbers, the results were consistent with MPL-mediated effects on other cells, which could involve interferon-gamma, transforming growth factor-beta, peroxisome proliferator-activated receptor-gamma, and forkhead box protein P3 pathways. Structural analyses indicated that effects on three apoptosis-related proteins (BCL2L1, BCL2, BAX) from the Bcl-2 family may be off-target effects of eltrombopag. In conclusion, this study proposes new hypotheses regarding the immunomodulatory functions of eltrombopag in patients with ITP.

Real-world use of thrombopoietin receptor agonists in older patients with primary immune thrombocytopenia.

The efficacy and safety of thrombopoietin receptor agonists (TRAs) in older patients with primary immune thrombocytopenia (ITP) are unknown. We investigated TRA response and switch, thrombotic/hemorrhagic risk, and sustained responses off-treatment (SROTs) in 384 patients with ITP aged ≥60 years. After 3 months, 82.5% and 74.3% of eltrombopag- and romiplostim-treated patients, respectively, achieved a response; 66.7% maintained the response (median follow-up, 2.7 years). Eighty-five (22.2%) patients switched to the alternative TRA; although no cross-toxicity was observed, 83.3% of resistant patients had a response after the switch. Thirty-four major thromboses (3 fatal) and 14 major hemorrhages (none fatal) occurred in 18 and 10 patients, respectively, while on TRAs and were associated with thrombosis history (subdistribution hazard ratio, 2.04, P = .05) and platelet count <20 × 109/L (subdistribution hazard ratio, 1.69; P = .04), respectively, at TRA start. A recurrent event occurred in 15.6% of patients surviving thrombosis, in all cases but 1 during persisting TRA treatment (incidence rate, 7.7 per 100 patient-years). All recurrences occurred in the absence of adequate antithrombotic secondary prophylaxis. Sixty-two (16.5%) responding patients discontinued TRAs; 53 (13.8%) patients maintained SROTs, which were associated with TRA discontinuation in complete response (P < .001). Very old age (≥75 years; 41.1%) was associated with the more frequent start of TRAs in the persistent/acute phase but not with response or thrombotic/hemorrhagic risk. TRAs are effective in older patients with ITP, with no fatal hemorrhages and with SROTs in a significant portion of patients. Caution is warranted in patients with a history of thrombosis, and a careful risk/benefit balance should be considered.

A simple HPLC assay for determining eltrombopag concentration in human serum.

Eltrombopag, a thrombopoietin receptor agonist, is used for the treatment of idiopathic thrombocytopenic purpura (ITP) and aplastic anemia. We developed a HPLC assay for the determination of serum eltrombopag concentration in ITP patients. An aliquot of a serum sample spiked with diclofenac as the internal standard (IS) was treated with acetonitrile to precipitate the proteins. Eltrombopag and the IS were separated on an octadecylsilyl silica-gel column using a mobile phase consisting of 10 mM 1-pentanesulfonic acid sodium salt, acetonitrile, and acetic acid. The detection wavelength was set at 265 nm. The calibration curve was linear at the concentration range of 0.15-12.5 µg/mL for eltrombopag (r = 0.9987). The recoveries of eltrombopag from the serum samples were greater than 95.9% with coefficients of variation (CVs) being less than 2.8%. The CVs for the intra-day and inter-day assays were 1.9-11.8% and 1.0-11.8%, respectively. This assay method could be used for therapeutic drug monitoring of eltrombopag in ITP patients.

Management of chronic liver disease-associated severe thrombocytopenia in Spain: a view from the experts

BACKGROUND: chronic liver disease (CLD) patients often present thrombocytopenia (TCP) and when severe, it may prevent them from undergoing necessary invasive procedures due to an increased bleeding risk. The lack of scientific evidence makes it impossible to determine key aspects of the current management and associated healthcare burden of these patients in Spain. PURPOSE: to gain insight into the current situation of patients with CLD-associated severe TCP undergoing invasive procedures in Spain, based on the experience of clinical experts. METHODS: national Delphi study involving 32 medical experts. RESULTS: the estimated prevalence of CLD-associated severe TCP is approximately 5,967, with an annual incidence of 1,148 new patients. Patients undergo a median of 1 (0-3) invasive procedures/year. Platelet transfusions (PTs) are the standard option to raise platelet counts and are associated with significant burden. The achievement of target platelet levels (≥ 50 X 109/l) after a transfusion is not routinely measured. The lack of effectiveness and short life span of transfused platelets can lead to procedure cancellations and bleeding events, which potentially affect patient outcomes. Adverse events occur in 1-25 % of patients, including mild (febrile and allergic reactions) and severe events (e.g., transfusion-related acute lung injury). Between 5-15 % of patients are unfit to receive PTs and approximately 3 % are treated off-label with thrombopoietin receptor agonists. CONCLUSIONS: this study provides a snapshot of the current situation in Spain, highlighting that the current management is poorly standardized and suboptimal in some cases. The results suggest the benefit of developing a consensus document to address some of these shortcomings and to advance in the search for alternatives to PTs

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Characteristics and outcomes of adults with cytomegalovirus-associated thrombocytopenia: a case series and literature review.

Cytomegalovirus (CMV) is a ubiquitous virus that infects people worldwide. CMV is known to trigger thrombocytopenia, but this association is probably underdiagnosed since CMV infection in healthy adults is usually either asymptomatic or causes only mild symptoms. A systematic literature review was carried out and yielded 23 publications that reported 25 patients. All haematology centres in Israel were searched for adult immunocompetent patients with CMV-associated thrombocytopenia, and five new cases were identified. The median age of the combined 30 patients was 33 years (range 18-80), 73% were men, 77% presented with CMV-related symptoms, 48% had enlarged spleens, 95% had atypical lymphocytes in peripheral blood and 68% had elevated transaminase levels. The response rate to first-line steroid-containing regimens was only 31%, whereas 11 patients who were treated with an anti-CMV agent had a response rate of 82%. Moreover, four patients received thrombopoietin receptor agonists (TPO-RA) to which three (75%) responded. Taken together, these distinctive features of a case with thrombocytopenia should alert to CMV infection as the source. While steroids were effective in less than one-third of the cases, both anti-CMV therapy and TPO-RA exhibited excellent efficacy, suggesting that those agents should be introduced earlier in the therapeutic course.

Platelet and immune characteristics of immune thrombocytopaenia patients non-responsive to therapy reveal severe immune dysregulation.

Multifactorial mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) might condition the ability of patients with ITP to respond to treatments. Examining their platelet and immune features, we aimed to detect singular characteristics of patients with ITP who do not respond to any treatment. We studied patients with chronic primary ITP who had been without treatment, or untreated (UT-ITP), for at least six months; included were responders to agonists of thrombopoietin receptors (TPO-RA), patients who showed no response to first- and second-line treatments (NR-ITP), and healthy controls. Platelets from NR-ITP patients exposed a reduced amount of sialic acid residues. Increased loss of platelet surface sialic acid residues was associated with increased platelet apoptosis. NR-ITP patients had an increased fraction of naive lymphocyte (L) B cells and a reduced LTreg (Lymphocyte T-regulator) subset. They also presented an anomalous monocyte and NK (Natural Killer) cells distribution. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls. In conclusion, our results indicate a severe deregulation of the immune system of NR-ITP. The inverse correlation between loss of sialic acid and LTreg count suggests a potential relationship between glycan composition on the platelet surface and immune response, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP.

Hematologic recovery induced by eltrombopag in Japanese patients with aplastic anemia refractory or intolerant to immunosuppressive therapy.

Eltrombopag, an oral thrombopoietin-receptor agonist, stimulates hematopoiesis in patients with acquired aplastic anemia (AA) and has higher exposure in patients of East Asian origin. We evaluated the pharmacokinetics, efficacy, and safety of eltrombopag in Japanese patients with AA refractory or intolerant to immunosuppressive therapy (IST). Twenty-one patients (15 with non-severe AA, six with severe AA) with platelet counts < 30,000/µL received eltrombopag in a dose-escalation fashion (25, 50, 75, or 100 mg once daily) depending on individual platelet responses; the responders continued eltrombopag treatment beyond 6 months. The primary endpoint was hematologic response at 6 months, defined as improvements in blood counts or transfusion requirements. Ten (48%) patients achieved hematologic responses in at least one lineage at 6 months. Six patients achieved tri- and/or bi-lineage responses with continuation of eltrombopag treatment, with two patients no longer requiring eltrombopag treatment. The most common adverse events were nasopharyngitis and abnormal hepatic function, with the majority being grade 1 or 2. Cytogenetic abnormalities were observed in three patients; however, no progression to myelodysplastic syndrome/other malignancy was observed. Eltrombopag can safely restore multi-lineage hematopoiesis in Japanese patients with AA refractory or intolerant to IST.Clinical Trial registration NCT02148133.

Glucocorticoids promote response to thrombopoietin-receptor agonists in refractory ITP: a case series.

A proportion of patients with immune thrombocytopenic purpura are refractory to multiple therapies including thrombopoietin-receptor agonists (TPO-RA). We report 10 patients who did not respond to a TPO-RA until the addition of a glucocorticoid. These patients were previously treated with a median of 6 therapies. One patient elected to discontinue both medications despite persistent thrombocytopenia. The remaining 9 patients continued on the combination of prednisone (doses 5 mg every other day to 10 mg daily) and a TPO-RA. Combination therapy with low dose glucocorticoid and a TPO-RA may be an option for patients unresponsive to a TPO-RA alone.

[Successful management of primary immune thrombocytopenia with romiplostim during open heart surgery in a hemodialysis patient].

A 66-year-old male undergoing maintenance hemodialysis presented with mild thrombocytopenia. He also had aortic valve stenosis and required aortic valve replacement. In addition, he required anticoagulation therapy with warfarin because of chronic subclavian artery occlusion. He was eventually diagnosed with immune thrombocytopenic purpura (ITP), although there were no bleeding tendencies. The patient was preoperatively treated with thrombopoietin receptor agonist (romiplostim®) because of the risk of bleeding complication during cardiac surgery. The platelet count rapidly increased with low-dose romiplostim, and no thrombotic complication occurred. During surgery, no significant bleeding complications were observed. This report suggests that romiplostim is a useful treatment option for the management of bleeding complication during cardiac surgery in a hemodialysis patient with ITP.

Lusutrombopag for the Treatment of Thrombocytopenia in Patients With Chronic Liver Disease Undergoing Invasive Procedures (L-PLUS 2).

Thrombocytopenia may be associated with increased bleeding risk impacting timing and outcome of invasive procedures in patients with chronic liver disease (CLD). Lusutrombopag, a small-molecule, thrombopoietin (TPO) receptor agonist, was evaluated as a treatment to raise platelet counts (PCs) in patients with thrombocytopenia and CLD undergoing invasive procedures. L-PLUS 2 was a global, phase 3, randomized, double-blind, placebo-controlled study. Adults with CLD and baseline PCs < 50 × 109 /L were randomized to receive once-daily lusutrombopag 3 mg or placebo ≤ 7 days before an invasive procedure scheduled 2-7 days after the last dose. The primary endpoint was avoidance of preprocedure platelet transfusion and avoidance of rescue therapy for bleeding. A key secondary endpoint was number of days PCs were ≥ 50 × 109 /L throughout the study. Safety analysis was performed on patients who received at least one dose of study drug. This study occurred between June 15, 2015, and April 19, 2017, with a total of 215 randomized patients (lusutrombopag, 108; placebo, 107); 64.8% (70/108) of patients in the lusutrombopag group versus 29.0% (31/107) in the placebo group met the primary endpoint (P < 0.0001; difference of proportion 95% confidence interval [CI], 36.7 [24.9, 48.5]). The median duration of PCs ≥ 50 × 109 /L was 19.2 days with lusutrombopag (without platelet transfusion) compared with 0.0 in the placebo group (with platelet transfusion) (P = 0.0001). Most adverse events were mild or moderate in severity, and rates were similar in the lusutrombopag and placebo groups (47.7% and 48.6%, respectively). Conclusion: Lusutrombopag was superior to placebo for reducing the need for platelet transfusions and achieved durable PC response in patients with thrombocytopenia and CLD undergoing invasive procedures, with a safety profile similar to placebo.

Iron chelating properties of Eltrombopag: Investigating its role in thalassemia-induced osteoporosis.

Chronic blood transfusions are responsible to cause iron overload, which leads to several complications to end organs and osteoporosis. Iron chelation is needed to remove iron excess and to contain bone-mass loss. Deferasirox is the most recent oral iron chelator that prevents transfusion related iron overload complications. Recently Eltrombopag (ELT) iron chelating properties are emerging. ELT is an agonist at Thrombopoietin receptor, used in treatment of thrombocytopenia. We tested ELT and Deferasirox in iron overloaded osteoclasts from thalassemic patients and donors measuring intracellular iron, TRAP expression and osteoclast activity. We confirmed ELT iron chelation capacity also in bone tissue and a synergic effect when used with Deferasirox. Moreover, having demonstrated its effects on osteoclast activity, we suggest for the first time that ELT could ameliorate bone tissue's health reducing bone mass loss.

Aptamer-based proteomics of serum and plasma in acquired aplastic anemia.

Single-stranded oligonucleotides containing deoxyuridine are aptamers (SOMAmers) that can bind proteins with high specificity and affinity and slow dissociation rates. SOMAscan, an aptamer-based proteomic technology, allows measurement of more than 1,300 proteins simultaneously for the identification of new disease biomarkers. The aim of the present study was to identify new serum and plasma protein markers for diagnosis of acquired aplastic anemia (AA) and response to immunosuppressive therapies (IST). SOMAscan was used to screen 1,141 serum proteins in 28 AA patients before and after therapy and 1,317 plasma proteins in seven SAA patients treated with standard IST and a thrombopoietin receptor agonist. From our analysis, 19 serum and 28 plasma proteins were identified as possible candidate diagnostic and prognostic markers. A custom immunobead-based multiplex assay with five selected serum proteins (BMP-10, CCL17, DKK1, HGF, and SELL) was used for validation in a verification set (n = 65) of samples obtained before and after IST and in a blinded validation cohort at baseline (n = 16). After technical validation, four biomarkers were employed to predict diagnosis (accuracy, 88%) and long-term response to IST (accuracy, 79%). In conclusion, SOMAscan is a powerful tool for the identification of new biomarkers. We propose further larger studies to validate new candidate serum and plasma diagnostic and prognostic markers of AA.

Risk of thrombosis with anti-phospholipid syndrome in systemic lupus erythematosus treated with thrombopoietin-receptor agonists.

OBJECTIVES: The use of thrombopoietin-receptor agonists (TPO-RAs) has increased as a second-line therapy in ITP, but the efficacy and safety of such drugs has not been evaluated in SLE-associated ITP. METHODS: This was a multicentre retrospective cohort study from 2009 to 2016. Participating centres (n = 11) were secondary- or tertiary-care hospitals belonging to the French national network for adult ITP. RESULTS: We included 18 patients with SLE-ITP treated with TPO-RAs; 10 (55%) had aPL, 5 (27%) showing definite APS. Except for one patient, all (94%) achieved response with TPO-RAs overall. After a median follow-up of 14.7 months with TPO-RAs, four arterial thrombosis events (including one catastrophic APS) occurred in four patients. Two venous thrombosis events occurred in a patient without APS or aPLs. CONCLUSION: Our results suggest that aPLs should be systematically screened before TPO-RA initiation in patients with SLE. With aPL positivity, alternative therapy should be discussed (if possible), especially in patients with definite APS or suboptimal adherence to anti-coagulation therapy.

Phenotypic Screening for Inhibitors of a Mutant Thrombopoietin Receptor.

An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.

Thrombocytopenia after liver transplantation: Should we care?

Transient thrombocytopenia is a common phenomenon after liver transplantation. After liver transplantation (LT), platelet count decreases and reaches a nadir on postoperative days 3-5, with an average reduction in platelet counts of 60%; platelet count recovers to preoperative levels approximately two weeks after LT. The putative mechanisms include haemodilution, decreased platelet production, increased sequestration, medications, infections, thrombosis, or combination of these processes. However, the precise mechanisms remain unclear. The role of platelets in liver transplantation has been highlighted in recent years, and particular attention has been given to their effects beyond hemostasis and thrombosis. Previous studies have demonstrated that perioperative thrombocytopenia causes poor graft regeneration, increases the incidence of postoperative morbidity, and deteriorates the graft and decreases patient survival in both the short and long term after liver transplantation. Platelet therapies to increase perioperative platelet counts, such as thrombopoietin, thrombopoietin receptor agonist, platelet transfusion, splenectomy, and intravenous immunoglobulin treatment might have a potential for improving graft survival, however clinical trials are lacking. Further studies are warranted to detect direct evidence on whether thrombocytopenia is the cause or result of poor-graft function and postoperative complications, and to determine who needs platelet therapies in order to prevent postoperative complications and thus improve post-transplant outcomes.

Splenectomy for immune thrombocytopenia: down but not out.

Splenectomy is an effective therapy for steroid-refractory or dependent immune thrombocytopenia (ITP). With the advent of medical alternatives such as rituximab and thrombopoietin receptor antagonists, the use of splenectomy has declined and is generally reserved for patients that fail multiple medical therapies. Splenectomy removes the primary site of platelet clearance and autoantibody production and offers the highest rate of durable response (50% to 70%) compared with other ITP therapies. However, there are no reliable predictors of splenectomy response, and long-term risks of infection and cardiovascular complications must be considered. Because the long-term efficacy of different second-line medical therapies for ITP have not been directly compared, treatment decisions must be made without supportive evidence. Splenectomy continues to be a reasonable treatment option for many patients, including those with an active lifestyle who desire freedom from medication and monitoring, and patients with fulminant ITP that does not respond well to medical therapy. We try to avoid splenectomy within the first 12 months after ITP diagnosis for most patients to allow for spontaneous or therapy-induced remissions, particularly in older patients who have increased surgical morbidity and lower rates of response, and in young children. Treatment decisions must be individualized based on patients' comorbidities, lifestyles, and preferences. Future research should focus on comparing long-term outcomes of patients treated with different second-line therapies and on developing personalized medicine approaches to identify subsets of patients most likely to respond to splenectomy or other therapeutic approaches.

Thrombopoietin Receptor Agonist Mitigates Hematopoietic Radiation Syndrome and Improves Survival after Whole-Body Ionizing Irradiation Followed by Wound Trauma.

Ionizing radiation combined with trauma tissue injury (combined injury, CI) results in greater mortality and H-ARS than radiation alone (radiation injury, RI), which includes thrombocytopenia. The aim of this study was to determine whether increases in numbers of thrombocytes would improve survival and mitigate H-ARS after CI. We observed in mice that WBC and platelets remained very low in surviving RI animals that were given 9.5 Gy 60Co-γ-photon radiation, whereas only lymphocytes and basophils remained low in surviving CI mice that were irradiated and then given skin wounds. Numbers of RBC and platelets, hemoglobin concentrations, and hematocrit values remained low in surviving RI and CI mice. CI induced 30-day mortality higher than RI. Radiation delayed wound healing by approximately 14 days. Treatment with a thrombopoietin receptor agonist, Alxn4100TPO, after CI improved survival, mitigated body-weight loss, and reduced water consumption. Though this therapy delayed wound-healing rate more than in vehicle groups, it greatly increased numbers of platelets in sham, wounded, RI, and CI mice; it significantly mitigated decreases in WBC, spleen weights, and splenocytes in CI mice and decreases in RBC, hemoglobin, hematocrit values, and splenocytes and splenomegaly in RI mice. The results suggest that Alxn4100TPO is effective in mitigating CI.