Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.

A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.

Thrombopoietin level predicts response to treatment with romiplostim in chemotherapy-induced thrombocytopenia.

Chemotherapy-induced thrombocytopenia (CIT) is a common complication of cancer treatment. Evidence has emerged supporting use of romiplostim to treat CIT but predicting clinical response to romiplostim is not possible. To determine utility of endogenous thrombopoietin (TPO) as a biomarker of romiplostim response, we performed an observational cohort study of patients with CIT and known baseline TPO levels receiving romiplostim. For weekly on-romiplostim platelet (Plt) count assessment, clinical response was defined as Plt ≥ 75 × 109 /L and ≥ 30 × 109 /L above pretreatment baseline. Overall, moderate, and superior classes of treatment response were defined based on fraction of Plt assessments meeting clinical response criteria (> 0, ≥ 0.6, and ≥ 0.8, respectively). Sixty-three patients with CIT were included; median age was 62 years, 41.3% were female, and median (IQR) romiplostim treatment duration was 14 (4-38) weeks. Median (IQR) TPO was lower in patients achieving moderate response to romiplostim vs those who did not, 234 (135-1085) pg/mL vs 665 (244-1970) pg/mL (p = .034) and lower still in patients achieving superior response vs those who did not, 212 (91-690) pg/mL versus 559 (173-1851) pg/mL (p = .023). Negative correlations were found between TPO level and baseline Plt and TPO level and response fraction. A positive correlation was found between TPO level and lowest effective romiplostim dose. In receiver operating characteristic (ROC) analysis, optimally discriminant TPO level thresholds (as defined by Youden's Index) were ≤ 457 pg/mL for moderate response and ≤ 260 pg/mL for superior response. In conclusion, TPO levels predict response to romiplostim in CIT, with lower levels predicting improved probability and depth of response.

Thrombopoietin Contributes to Enhanced Platelet Activation in Patients with Type 1 Diabetes Mellitus.

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM). Enhanced platelet reactivity is considered a main determinant of the increased atherothrombotic risk of diabetic patients. Thrombopoietin (THPO), a humoral growth factor able to stimulate megakaryocyte proliferation and differentiation, also modulates the response of mature platelets by enhancing both activation and binding to leukocytes in response to different agonists. Increased THPO levels have been reported in different clinical conditions characterized by a generalized pro-thrombotic state, from acute coronary syndromes to sepsis/septic shock, and associated with elevated indices of platelet activation. To investigate the potential contribution of elevated THPO levels in platelet activation in T1DM patients, we studied 28 T1DM patients and 28 healthy subjects. We measured plasma levels of THPO, as well as platelet-leukocyte binding, P-selectin, and THPO receptor (THPOR) platelet expression. The priming activity of plasma from diabetic patients or healthy subjects on platelet-leukocyte binding and the role of THPO on this effect was also studied in vitro. T1DM patients had higher circulating THPO levels and increased platelet-monocyte and platelet-granulocyte binding, as well as platelet P-selectin expression, compared to healthy subjects, whereas platelet expression of THPOR did not differ between the two groups. THPO concentrations correlated with platelet-leukocyte binding, as well as with fasting glucose and Hb1Ac. In vitro, plasma from diabetic patients, but not from healthy subjects, primed platelet-leukocyte binding and platelet P-selectin expression. Blocking THPO biological activity using a specific inhibitor prevented the priming effect induced by plasma from diabetic patients. In conclusion, augmented THPO may enhance platelet activation in patients with T1DM, potentially participating in increasing atherosclerotic risk.

Platelet enhancement by Carica papaya L. leaf fractions in cyclophosphamide induced thrombocytopenic rats is due to elevated expression of CD110 receptor on megakaryocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Carica papaya leaf juice/decoction has been in use in folk medicine in Srilanka, Malaysia and in few parts of India for enhancing the platelet counts in dengue. In Siddha medicine, a traditional form of medicine in India, papaya leaf juice has been used for increasing the platelet counts. Papaya leaf has been reported to enhance blood volume in ancient Ayurveda books in India. Carica papaya leaf is well known for its platelet enhancement activity. Although many preclinical and clinical studies have demonstrated the ability of papaya leaf juice for platelet enhancement, but the underlying mechanisms are still unclear. AIM OF THE STUDY: The study is aimed at identifying the key ingredients of papaya leaf extract and elucidate the mechanism (s) of action of the identified potent component in mitigating thrombocytopenia (Thp). MATERIALS AND METHODS: C. papaya leaf juice was subjected for sequential fractionation to identify the anti-thrombocytopenic phytochemicals. In vivo, stable thrombocytopenia was induced by subcutaneous injection of 70 mg/kg cyclophosphamide (Cyp). After induction, rats were treated with 200 and 400 mg/kg body weight papaya leaf juice and with identified fractions for 14 days. Serum thrombopoietin level was estimated using ELISA. CD110/cMpl, a receptor for thrombopoietin on platelets was measured by western blotting. RESULTS: Administration of cyclophosphamide for 6 days induced thrombocytopenia (210.4 ± 14.2 × 103 cells/µL) in rats. Treating thrombocytopenic rats with papaya leaf juice and butanol fraction for 14 days significantly increased the platelet count to 1073.50 ± 29.6 and 1189.80 ± 36.5 × 103 cells/µL, respectively. C.papaya extracts normalized the elevated bleeding and clotting time and decreased oxidative markers by increasing endogenous antioxidants. A marginal increase in the serum thrombopoietin (TPO) level was observed in Cyp treated group compared to normal and treatment groups. Low expression of CD110/cMpl receptor found in Cyp treated group was enhanced by C. papaya extracts (CPJ) and CPJ-BT. Furthermore, examination of the morphology of bone marrow megakaryocytes, histopathology of liver and kidneys revealed the ability of CPJ and fractions in mitigating Cyp-induced thrombocytopenia in rats. CONCLUSION: C. papaya leaf juice enhances the platelet count in chemotherapy-induced thrombocytopenia by increasing the expression of CD110 receptor on the megakaryocytes. Hence, activating CD110 receptor might be a viable strategy to increase the platelet production in individuals suffering from thrombocytopenia.

COVID-19 induces a hyperactive phenotype in circulating platelets.

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

Efficacy and safety of romiplostim in refractory aplastic anaemia: a Phase II/III, multicentre, open-label study.

A previous dose-finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open-label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1-4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5-52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 109 /l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66-95%]. Trilineage response was 39% (95% CI 22-58%) at week 53. The most common treatment-related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow-up. High-dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.

Increased B4GALT1 expression is associated with platelet surface galactosylation and thrombopoietin plasma levels in MPNs.

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies originating from hematopoietic stem cells, leading to an increase in mature blood cells in the peripheral blood. Sialylated derivatives of the glycan structure ß4-N-acetyllactosamine (Galß1,4GlcNAc or type-2 LacNAc, hereafter referred to as LacNAc) regulate platelet life span, hepatic thrombopoietin (TPO) production, and thrombopoiesis. We found increased TPO plasma levels in MPNs with high allele burden of the mutated clones. Remarkably, platelets isolated from MPNs had a significant increase in LacNAc expression that correlated with the high allele burden regardless of the underlying identified mutation. Megakaryocytes derived in vitro from these patients showed an increased expression of the B4GALT1 gene encoding ß-1,4-galactosyltransferase 1 (ß4GalT1). Consistently, megakaryocytes from MPN showed increased LacNAc expression relative to healthy controls, which was counteracted by the treatment with a Janus kinase 1/2 inhibitor. Altered expression of B4GALT1 in mutant megakaryocytes can lead to the production of platelets with aberrant galactosylation, which in turn promote hepatic TPO synthesis regardless of platelet mass. Our findings provide a new paradigm for understanding aberrant megakaryopoiesis in MPNs and identify ß4GalT1 as a potential actionable target for therapy.

Efficiently Restored Thrombopoietin Production by Ashwell-Morell Receptor and IL-6R Induced Janus Kinase 2/Signal Transducer and Activator of Transcription Signaling Early After Partial Hepatectomy.

BACKGROUND AND AIMS: Thrombocytopenia has been described in most patients with acute and chronic liver failure. Decreased platelet production and decreased half-life of platelets might be a consequence of low levels of thrombopoietin (TPO) in these patients. Platelet production is tightly regulated to avoid bleeding complications after vessel injury and can be enhanced under elevated platelet destruction as observed in liver disease. Thrombopoietin (TPO) is the primary regulator of platelet biogenesis and supports proliferation and differentiation of megakaryocytes. APPROACH AND RESULTS: Recent work provided evidence for the control of TPO mRNA expression in liver and bone marrow (BM) by scanning circulating platelets. The Ashwell-Morell receptor (AMR) was identified to bind desialylated platelets to regulate hepatic thrombopoietin (TPO) production by Janus kinase (JAK2)/signal transducer and activator of transcription (STAT3) activation. Two-thirds partial hepatectomy (PHx) was performed in mice. Platelet activation and clearance by AMR/JAK2/STAT3 signaling and TPO production were analyzed at different time points after PHx. Here, we demonstrate that PHx in mice led to thrombocytopenia and platelet activation defects leading to bleeding complications, but unaltered arterial thrombosis, in these mice. Platelet counts were rapidly restored by up-regulation and crosstalk of the AMR and the IL-6 receptor (IL-6R) to induce JAK2-STAT3-TPO activation in the liver, accompanied by an increased number of megakaryocytes in spleen and BM before liver was completely regenerated. CONCLUSIONS: The AMR/IL-6R-STAT3-TPO signaling pathway is an acute-phase response to liver injury to reconstitute hemostasis. Bleeding complications were attributable to thrombocytopenia and platelet defects induced by elevated PGI2 , NO, and bile acid plasma levels early after PHx that might also be causative for the high mortality in patients with liver disease.

ß4GALT1 controls ß1 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. ß4GalT1 deficiency increases the number of fully differentiated MKs. However, the resulting lack of glycosylation enhances ß1 integrin signaling leading to dysplastic MKs with severely impaired demarcation system formation and thrombopoiesis. Platelets lacking ß4GalT1 adhere avidly to ß1 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, ß1 integrin deletion, specifically in MKs, restores thrombopoiesis. TPO and CXCL12 regulate ß4GalT1 in the MK lineage. Thus, our findings establish a non-redundant role for ß4GalT1 in the regulation of ß1 integrin function and signaling during thrombopoiesis. Defective thrombopoiesis and lack of ß4GalT1 further affect HSC homeostasis.

Plasma thrombopoietin levels as additional tool in clinical management of thrombocytopenic neonates.

Plasma thrombopoietin (Tpo) levels distinguish thrombocytopenia resulting from increased platelet destruction or decreased platelet production. We investigated whether measuring plasma Tpo levels in thrombocytopenic newborns is of diagnostic value to establish the underlying mechanism of thrombocytopenia.Tpo levels were measured with in-house developed ELISA in samples referred to our center because of thrombocytopenia noticed in the first 10 days of life. Clinical data were collected.Plasma Tpo levels <128 AU/ml were found in the majority (92%) of 121 newborns with immune-mediated thrombocytopenia (n = 104) and thrombocytopenia due to bacterial infections (n = 7); increased plasma Tpo levels (≥128 AU/ml) were found in thrombocytopenic newborns with severe asphyxia (n = 24). Highly increased plasma Tpo levels (>200 AU/ml) were found in thrombocytopenic neonates with congenital viral infections (n = 22) or amegakaryocytosis (n = 6). A plasma Tpo level <128 AU/ml excludes (negative predictive value 96%, 95% CI 90-99) severe asphyxia, congenital viral infections and amegakaryocytosis as the cause for thrombocytopenia in newborns.Increased plasma Tpo levels indicate that thrombocytopenia in newborns, as a result of various nonimmune disorders, is often caused by (temporary) bone marrow suppression/failure. Measurement of plasma Tpo levels provides the clinician with an additional tool to decide on the differential diagnosis, the necessity for subsequent diagnostics and treatment in neonates with thrombocytopenia.

Determinants of aspirin resistance in patients with type 2 diabetes.

BACKGROUND: Cardiovascular disease is a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM). Numerous patients with T2DM show resistance to aspirin treatment, which may explain the higher rate of major adverse cardiovascular events observed compared with non-diabetes patients, and it has recently been shown that aspirin resistance is mainly related to accelerated platelet turnover with persistent high platelet reactivity (HPR) 24h after last aspirin intake. The mechanism behind HPR is unknown. The aim of this study was to investigate the precise rate and mechanisms associated with HPR in a population of T2DM patients treated with aspirin. METHODS: Included were 116 consecutive stable T2DM patients who had attended our hospital for their yearly check-up. HPR was assessed 24h after aspirin intake using light transmission aggregometry (LTA) with arachidonic acid (AA) and serum thromboxane B2 (TXB2) measurement. Its relationship with diabetes status, insulin resistance, inflammatory markers and coronary artery disease (CAD) severity, using calcium scores, were investigated. RESULTS: Using LTA, HPR was found in 27 (23%) patients. There was no significant difference in mean age, gender ratio or cardiovascular risk factors in patients with or without HPR. HPR was significantly related to duration of diabetes and higher fasting glucose levels (but not consistently with HbA1c), and strongly related to all markers of insulin resistance, especially waist circumference, HOMA-IR, QUICKI and leptin. There was no association between HPR and thrombopoietin or inflammatory markers (IL-6, IL-10, indoleamine 2,3-dioxygenase activity, TNF-α, C-reactive protein), whereas HPR was associated with more severe CAD. Similar results were found with TXB2. CONCLUSION: Our results reveal that 'aspirin resistance' is frequently found in T2DM, and is strongly related to insulin resistance and severity of CAD, but weakly related to HbA1c and not at all to inflammatory parameters. This may help to identify those T2DM patients who might benefit from alternative antiplatelet treatments such as twice-daily aspirin and thienopyridines.

Thrombomodulin and Thrombopoietin, Two Biomarkers of Hemostasis, Are Positively Associated with Adherence to the World Cancer Research Fund/American Institute for Cancer Research Recommendations for Cancer Prevention in a Population-Based Cross-Sectional Study.

A pro-coagulative state is related to increased risk of cardiovascular diseases but also certain cancers. Since experimental and smaller human studies suggest that diet, physical activity, and body weight may all affect coagulation, we evaluated associations between these lifestyle factors and hemostatic biomarkers in a population-based study. Cross-sectional baseline data from 2267 randomly selected participants of EPIC-Heidelberg (age range 35-65 years) was used. Fibrinogen, glycoprotein IIb/IIIa, P-selectin, thrombomodulin (TM), and thrombopoietin (TPO) were measured in baseline plasma samples. A score reflecting adherence to the World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) recommendations for cancer prevention was created. Associations between the WCRF/AICR score as well as its individual components and hemostatic biomarkers were analyzed by linear regression models. Multivariable-adjusted geometric means (95% confidence intervals) of TM and TPO were higher with greater adherence to the WCRF/AICR recommendations (TM, lowest vs. highest score category: 2.90 (2.7,3.1) vs. 3.10 (2.9,3.3) ng/mL, plinear trend = 0.0001; TPO: 328 (302,356) vs. 348 (321,378) pg/mL, plinear trend = 0.0007). These associations were driven by lower alcohol and meat consumption among persons with higher WCRF/AICR scores. Our results indicate that lifestyle factors favorably affect TM and TPO, two hemostatic factors implicated in chronic disease development.

Thrombopoietin Contributes to Extreme Thrombocytosis After Pediatric Pancreatectomy With Islet Autotransplantation.

OBJECTIVE: This study aims to explore the role of thrombopoietin (TPO) production in extreme thrombocytosis that is often observed after pancreatectomy with islet autotransplantation (IAT) and the effectiveness of hydroxyurea in thrombocytosis management. METHODS: Retrospective chart review was performed for all patients who underwent pancreatectomy with IAT at our institution between April 1, 2015, and December 31, 2016. Data evaluated included demographics, platelet counts, TPO levels, and thrombocytosis management strategies. RESULTS: Twelve total and 1 subtotal pancreatectomy with IAT cases were reviewed. All operations included splenectomy. No major surgical or thrombotic complications occurred. Thrombopoietin levels, normal preoperatively, rose significantly (median, 219 pg/mL) soon after surgery, peaking on median postoperative day 3. Platelet counts, also normal preoperatively, increased within a week of surgery, with 92% over 1000 K/µL (median peak platelet count, 1403 K/µL). Platelet counts and TPO levels dropped after hydroxyurea initiation in most patients. CONCLUSIONS: After pancreatectomy with IAT, patients experienced marked TPO rise and subsequent thrombocytosis, and both decreased significantly after hydroxyurea initiation. These data suggest that TPO elevation and associated increased platelet production may be one driver of early extreme post-total pancreatectomy with islet autotransplantation thrombocytosis, and this process may be modulated by hydroxyurea.

Diagnosing Immune Thrombocytopenia.

Although the detection of a characteristic autoantibody can prove immune thrombocytopenia (ITP), this diagnosis is often based on the exclusion of other causes of thrombocytopenia. Direct glycoprotein (GP)-specific tests have the property required to demonstrate such a characteristic autoantibody. In contrast, platelet-associated immunoglobulin G or antibody detection in plasma or serum is an insufficient diagnostic test. Moreover, data for commercial capture assays are sparse and their use is currently not recommended. A significant drawback of direct GP-specific tests is their low sensitivity, and a negative test result has no relevance. It is therefore also useful to establish a diagnosis of (primarily) hyperdestructive thrombocytopenia. A full blood count together with the immature platelet fraction has an excellent positive predictive value for ITP. Plasma glycocalicin has no apparent diagnostic value in identifying ITP patients, and conflicting data for TPO preclude its use for diagnostic purposes.

Repeated arctigenin treatment produces antidepressant- and anxiolytic-like effects in mice.

Depression is the root of various diseases. It is one of the most debilitating conditions globally. Antidepressant drugs are usually the first-line of depression treatment. Arctigenin (ARC), one of active ingredient of Arctium lappa L, has been found to exert neuroprotective, anti-decrepitude, and anti-inflammatory activities. Thus, the aim of this study was to investigate the potential antidepressant- and anxiolytic-like effects of ARC using acute and chronic mild stress (CMS) mice model. ICR mice model received acute stress or chronic mild stress assessed by open field test (OFT), novelty suppressed feeding (NSF), sucrose preference test (SPT), forced-swimming test (FST), and tail suspension test (TST). After the final test, blood was collected to detect the serum levels of angiogenin (ANG), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay (ELISA). The behavioral results showed that repeated ARC (10, 30 mg/kg) administration significantly relieved the antidepressant- and anxiolytic-like effects. And repeated ARC administration at the dose of 10 and 30 mg/kg could significantly block depressive- and anxiety-like behaviors caused by CMS. Finally, ELISA results showed that ARC administration increased the serum levels of angiogenin (ANG), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF). Results showed that chronic ARC administration produces antidepressant- and anxiolytic-like effects, which provides direct evidence for the first time that ARC may be a novel strategy for the treatment of depression and even stress-related disorders. The present data supports further exploration for developing ARC administration as a novel therapeutic strategy for depression and even stress-related disorders.

Morphological and optical properties of human immature platelet-enriched population produced in immunodeficient mice.

Ultrastructure analysis of immature platelets is difficult because of the lack of a suitable marker and their relatively low concentration in total platelets. We investigated the morphological and optical properties of human immature platelets produced and enriched in immunodeficient mice via human CD34-positive cell administration. Immunodeficient mice were injected with human CD34-positive cells and administered eltrombopag orally for 14 days (eltro-mice). Some of these mice were maintained for 2-3 months (steady-state-mice). Platelets were double-stained with a human CD41 antibody and a nuclear staining dye (Sysmex hematology analyzer XN series reagent), and then analyzed by flowcytometry FCM to identify human immature platelets. Human CD41-positive cells were isolated from citrated blood by magnetic cell sorting with human CD41 antibody, and examined using electron microscopy. Flow cytometric analysis with the XN reagent demonstrated that peripheral blood from eltro-mice had a higher percentage of immature platelet fraction in human platelets than that from steady-state-mice. The geometric mean of XN reagent fluorescence for human platelets, divided with that for mouse platelets, revealed that the ratios in eltro-mice were significantly higher than those in steady-state-mice, thus indicating that immature platelets were highly enriched in eltro-mice. Scanning and transmission electron microscopy revealed that human citrated platelets isolated from eltro-mice tended to be larger (n = 15, p = 0.276) and contained more mitochondria than those isolated from steady-state-mice (n = 10, p = 0.0002). Therefore, an increased number of mitochondria, rather than platelet size, is a distinctive feature of immature platelets.

Epidemiological, clinical and genetic characterization of aplastic anemia patients in Pakistan.

Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.

Thrombopoietin level predicts response to treatment with eltrombopag and romiplostim in immune thrombocytopenia.

Thrombopoietin receptor agonists (TPO-RAs) are used to treat immune thrombocytopenia (ITP), but predicting clinical response to TPO-RAs before initiation is not possible. To determine whether endogenous TPO levels predict treatment response to TPO-RAs we performed a retrospective analysis of ITP patients with known baseline TPO levels who received TPO-RAs. Data was collected for ITP patients with a baseline TPO level treated with eltrombopag or romiplostim. Multiple logistic regression was used to model the probability of 3 classes of treatment response (overall, moderate, and superior) based on TPO level; receiver operating characteristic (ROC) analysis was performed to identify optimal TPO thresholds for response; correlations between TPO level and various response characteristics were analyzed. A total of 67 patients (37 receiving eltrombopag and 46 receiving romiplostim) were included. Logistic regression models demonstrated a significant predictive relation between TPO level and probability of all classes of response; per 10 pg/mL TPO increase, odds ratio for overall response to eltrombopag was 0.524 (95% CI 0.327, 0.837) and romiplostim was 0.905 (95% CI, 0.844, 0.970). TPO level was inversely correlated with all classes of response; for overall response, r = -0.719 (P < .001) for eltrombopag and r = -0.584 (P < .001) for romiplostim. ROC analysis identified TPO thresholds of ≤136 pg/mL (eltrombopag) and ≤209 pg/mL (romiplostim) as optimally discriminating between responders and nonresponders. Most non-responders had high TPO levels but did respond after addition of low-dose prednisone. In conclusion, TPO levels predict response to eltrombopag and romiplostim in ITP patients, with lower levels predicting improved probability and magnitude of response.

Icaritin Provokes Serum Thrombopoietin and Downregulates Thrombopoietin/MPL of the Bone Marrow in a Mouse Model of Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is a common acquired autoimmune disease, and thrombopoietin (TPO) is an important cytokine that regulates the production of megakaryocytes and platelets. We have identified a biologically active component, icaritin, from a Chinese herba epimedii extract. Icaritin promotes platelet production and regulates T cell polarization, but its mechanism is not clear. In this study, the BALB/c mouse model of ITP was established by injection of an antiplatelet antibody every other day for seven total times. The antiplatelet sera were derived from guinea pigs immunized with the platelets of BALB/c mice. Mice with ITP were treated with icaritin at low, moderate, or high doses of 4.73, 9.45, and 18.90 mg/kg, respectively, for fourteen consecutive days. The present study shows that icaritin can significantly increase peripheral blood platelet counts and thrombocytocrit, increase the TPO level in serum, attenuate splenomegaly, and reduce the abnormal proliferation of megakaryocytes in the spleen and bone marrow. Icaritin can also downregulate the expression of bone marrow TPO, myeloproliferative leukemia virus oncogene (MPL), and p-Stat3. Our results suggest that icaritin can significantly improve the health of mice with ITP via possible downregulation of p-Stat3 expression in the JAK2/Stat3 phosphorylation signaling pathway and regulation of bone marrow TPO/MPL metabolism.