Number Of Platelets In Patients Research Articles

Features of chronic heart failure on the background of thrombocytopenia in persons infected with the human immunodeficiency virus

The general context of the study is to review the characteristics of the course of chronic heart failure (CHF) in persons infected with the human immunodeficiency virus (HIV) as a part of a single-stage screening clinical trial.Aim: To determine the features of the CHF course in HIV-infected patients, depending on the presence of thrombocytopenia (TP).Material and Methods. In a multidisciplinary hospital, 240 patients with HIV infection were examined for four years, where a cohort of patients with CHF (160 people) was identified, further divided into groups of patients with TP (107 people) and without TP (53 people). Patients underwent the same amount of research, including echocardiography, non-invasive arteriography, and a detailed laboratory examination.Results. TP in patients with CHF and HIV infection is more common in smokers and people with alcohol dependence and is associated with hemodynamic disorders in the form of higher systolic and diastolic blood pressure, left atrium increase. With TP, the values of NT-proBNP in blood plasma are higher, and in the blood serum – tissue inhibitor of metalloproteinases-1, urea, bilirubin, while the concentration of sodium and potassium ions in the blood serum is lower. Anemia and leukopenia were more common in the group of patients with thrombocytopenia. Patients with TP are more adherent to taking protease inhibitors and non-steroidal anti-inflammatory drugs.Conclusion. The frequency of CHF detection in patients with HIV infection against the background of TP is almost 2 times higher and is more often accompanied by dilatation of the left atrium, higher blood pressure, an increase in TIMP-1 and a decrease in the concentration of potassium ions in the blood serum. The concentration of NT-proBNP in blood plasma has a close inverse correlation with the number of platelets in patients with CHF and TP, and the presence of severe TP ≤ 30 × 109 cells/l increases the chances of developing CHF with low left ventricular ejection fraction by 10.8 times. Smoking, possible alcohol dependence and adherence to taking protease inhibitors and NSAIDs are significant factors associated with the development of thrombocytopenia in HIV-infected patients with CHF.

Quantitative System Pharmacology Modeling of the Effect of Recombinant ADAMTS13 on Platelets in Patients with Congenital Thrombotic Thrombocytopenic Purpura

Background: Congenital thrombotic thrombocytopenic purpura (cTTP) is an ultra-rare, thrombotic microangiopathy due to an inherited deficiency of the von Willebrand factor (VWF)-cleaving protease, ADAMTS13.Accumulation of ultra-large VWF multimers increases blood clot formation in small blood vessels, leading to a low platelet count (thrombocytopenia). Treatment of cTTP consists of ADAMTS13 replacement with infusions of plasma-based therapies (PBT). Recombinant ADAMTS13 (rADAMTS13; Takeda Development Center Americas, Inc., Lexington, MA, USA) is in development for patients with cTTP.The ultra-rare nature of cTTP limits the sample size for a formally powered study to assess the impact of ADAMTS13 supplementation on efficacy outcome. This study was a proof-of-principle that model-based clinical trial simulations can be used to supplement clinical trial data in ultra-rare indications. Aims: To calibrate and validate a quantitative system pharmacology (QSP) model describing a mechanistic relationship between ADAMTS13, VWF and platelet numbers in patients with cTTP who received long-term prophylaxis with rADAMTS13 or PBT (fresh frozen or solvent/detergent treated plasma). In addition, to evaluate the treatment benefits of rADAMTS13 versus PBT using clinical trial simulations in virtual patients with cTTP as a supplement to the cTTP phase 3 study (NCT03393975) data. Methods: A QSP model was built using i) in vitro enzyme kinetic data and preclinical experimental data following administration of recombinant VWF and rADAMTS13 in ADAMTS13 knockout (KO) mice, and ii) data from the literature including platelet count and VWF activity from healthy human and untreated patients with cTTP. These data were used to calibrate the parameters corresponding to interactions between ADAMTS13 activity, VWF activity, and platelets. The model was validated against literature data from patients with cTTP treated with PBT and with data from a phase 2 study of patients with immune-mediated TTP treated with rADAMTS13 (NCT03922308). The model was then calibrated to longitudinal platelet data from the cTTP phase 3 study, in which patients with cTTP received prophylaxis for 6 months with PBT (exposure equivalent to ~10 IU/kg ADAMTS13 in the form of 10-20 mL/kg doses) and rADAMTS13 (40 IU/kg) either once every week (Q1W) or once every 2 weeks (Q2W) dosing intervals . The model was used to provide 1-year clinical trial simulations of isolated thrombocytopenia occurrences in a population of 1,000 virtual patients, phenotype-matched to patients in the cTTP phase 3 study. Results:Virtual clinical trial simulations using the QSP model suggested that rADAMTS13 administered over 1 year with a Q1W or Q2W schedule results in significantly fewer patients experiencing occurrences of thrombocytopenia (platelet count threshold of <150 x 10 9/L) compared to patients treated with PBT (hazard ratio [HR] relative to Q2W PBT: Q1W PBT, HR = 0.62; Q2W rADAMTS13, HR = 0.47; Q1W rADAMTS13, HR = 0.18). The treatment difference between rADAMTS13 and PBT was even more pronounced when a thrombocytopenia threshold of <100 x 10 9/L was used (Figure and Table). Simulations also predicted a prophylactic carry-over effect on platelets, with approximately 20 days taken to reach 90% of the long-term average platelet count when switching from rADAMTS13 to PBT or from PBT to rADAMTS13. A correlation analysis for a Q2W dosing of rADAMTS13 demonstrated that ADAMTS13 trough activity (C trough, Spearman ρ = 0.96) and time with ADAMTS13 activity above 10% (Spearman ρ = 0.93) showed stronger correlations with increased platelet count than the maximum plasma ADAMTS13 activity level (C max, Spearman ρ = 0.68). A correlation analysis for a Q1W dosing frequency produced similar results.Mean time above 10% ADAMTS13 activity was higher following rADAMTS13 versus PBT administration (5.2 vs 1.7 days). Conclusions: This model-based virtual clinical trial simulation predicts that dosing with rADAMTS13 would provide >50% reduction in the HR of thrombocytopenia occurrences over PBT. Patients may also have a higher level of protection for 2-3 weeks when switching from rADAMTS13 to PBT. These findings supplement the assessment of clinical benefit of rADAMTS13 in the cTTP phase 3 study and show that increased ADAMTS13 activity following rADAMTS13 administration is likely to reduce occurrences of thrombocytopenia in cTTP.

Parameters of Iron Metabolism in Patients with Iron Deficiency Anemia and Erosive Lesions of the Gastroduodenal Zone

The most common disease in the world is erosive-ulcerative lesions of the gastroduodenal zone (EULGZ). These are chronic polyetiological, recurrent diseases with disruption of the neuro-endocrine and humoral mechanisms that regulate the secretory-trophic process in the gastroduodenal zone with the development of ulcers. In this disease, ulcers have different localization and clinical course, significantly reduce the quality of life, affect work capacity and personal life. Lack of timely diagnosis and treatment can have adverse consequences. The objective: to analyse the characteristics of clinical and metabolic disorders in iron deficiency anemia (IDA) and its combination with EULGZ in order to develop the methods for assessing the disease severity, optimizing diagnostics and treatment tactics. Materials and methods. The observation group included 26 patients (15 men and 11 women) with iron-deficiency anemia (IDA) on background of EULGZ with localization of ulcerative defects of the mucous membrane in the stomach or duodenal bulb. The diagnosis of IDA was verified on the basis of changes in the basic characteristics of clinical manifestations (signs of anemic hypoxia and sideropenic syndrome), specific changes in peripheral blood and indicators of iron metabolism. Patients with IDA and EULGZ, were examined by endoscopic methods, underwent ultrasound and, if necessary, radiological examination. The control group included 35 primary donors (16 women and 19 men), who underwent two laboratory tests for hepatitis B and C, as well as HIV infection/AIDS. All examined donors underwent a comprehensive examination of peripheral blood and determined biochemical indicators. The indicators of iron metabolism were determined in all participants: iron content in the blood serum (IS), total iron-binding capacity of the blood serum (TIBS). The index of unsaturated iron-binding capacity of blood serum was calculated as the difference between TIBS and IS. The coefficient of transferrin saturation with iron was determined as the ratio of the content of IS to TIBS. The content of transferrin was determined according to the TIBS indicator, ferritin – by the radiometric method. Results. Peripheral blood indicators in primary donors and morphometric changes in peripheral blood in patients with IDA were analyzed. The results of investigations of the indicators of iron exchange in blood serum are presented: iron concentration, total iron-binding capacity of serum and the degree of saturation of serum with iron. It was found that male donors have significantly higher levels of iron and ferritin in blood serum compared to female donors. The dynamic of changes in peripheral blood parameters in patients with IDA caused by long-term blood loss due to EULGZ during the treatment process is shown. Treatment of the EULGZ led to the significant changes of such indicators as the number of erythrocytes, hemoglobin concentration, erythrocyte indices towards normalization. The normalization of the reticulocyte count was observed on the 90th day of treatment. The detected changes in the number of platelets in patients with IDA due to EULGZ were observed in the absence of active bleeding from the digestive tract. Conclusions. As a result of the progression of IDA due to the erosive-ulcerative lesions of the gastroduodenal zone, the imbalance of iron metabolism deepens, serious disorders of iron metabolism develop, especially in the content of plasma ferritin. Patients with IDA caused by long-term blood loss as a result of EULGZ, should be prescribed a drug for oral use containing ferrous sulfate with a mucosa-protective effect at a dose of 2 mg/kg per day, followed by a decreased dose after normalization of the serum ferritin level.

Pre-Clinical Modeling of Concomitant Therapy with Azacytidine and Eltrombopag in MDS/AML

Hemorrhage-related morbidity and mortality frequently arises in patients with myelodysplastic syndromes (MDS). A major therapeutic goal in patients with MDS is the alleviation commonly occurring thrombocytopenia. Low platelet counts increase bleeding risk, limit tolerated doses and efficacy of standard therapies, and are associated with an increased risk of leukemic transformation. Preclinical and phase I/II studies on eltrombopag (EPAG), a megakaryopoiesis-stimulating thrombopoietin receptor (TPO-R) agonist, demonstrated safety and efficacy of the small molecule as monotherapy in MDS. However, a phase III study testing the ability of EPAG to increase platelet numbers in patients with intermediate or higher-risk MDS under azacytidine (AZA) therapy (NCT02158936) was prematurely terminated due to inferior performance of EPAG-treated vs. placebo-receiving patients. While the study did not find a difference in overall survival rates between the two groups, concerns pertaining to disease progression under concomitant treatment with EPAG and azacytidine have been raised.We assessed whether EPAG exerts antagonistic physiological activities in the context of anti-leukemia treatment with AZA exploiting a set of complementary pre-clinical ex vivo assays measuring aberrant cell proliferation, differentiation and self-renewal. MDS and AML patient-derived cell lines (n=4) and primary cell specimen (n=6) were treated with non-cytotoxic, clinically-relevant doses of EPAG and AZA in short-term liquid cultures, myeloid colony-forming assays, and long-term serial-replating assays. To elucidate functional effects of the combination treatment, we determined cell growth, apoptosis, differentiation and long-term colony initiation as a surrogate for self-renewal upon treatment. We also quantified cell surface presence of TPO-R, and assessed pathway activation downstream of the receptor using phosphoflow.Consistent with previous observations, continuous single agent treatment with either >0.3μM AZA, or 3-20mg/ml EPAG were found to be cytotoxic for leukemic cells. Simultaneous treatment with a non-cytotoxic functionally-relevant dosage of AZA (0.3μM) and EPAG at concentrations above 3mg/ml resulted in a striking decrease of cell viability compared to AZA single agent treatment in short-term cell line cultures. Lower-dose EPAG treatment (0.3 and 0.5mg/ml) along with AZA (0.3μM) did not lead to any significant changes in cell viability and growth in short term cultures (vs. single agent treated controls) of cell lines or primary cells. Apoptosis, cell surface differentiation marker analysis, and morphological assessment revealed no discernable differences upon combination treatment compared to AZA single agent treatment in cell lines. Characterization of aberrant self-renewal using serial replating colony assays revealed a significant decrease in the number of colony-initiating cells upon continuous single agent treatment with AZA, which was not diminished by combination treatment with EPAG. An AZA-resistant cell line also did not show altered long-term colony-forming cell numbers in the presence of both drugs. We found no evidence of increased leukemic self-renewal upon simultaneous treatment using primary cells in 5/6 specimen tested. One sample showed a transient increase in colony formation and average cell numbers per colony in the presence of both drugs, compared to AZA-treated controls, albeit the number of serial replatings was unchanged compared to AZA controls. We tested whether TPO-R/CD110-expressing aberrant cells would expand under EPAG single agent, or combination treatment with AZA, and found no significant changes in the frequency of CD110-positive cells compared to AZA single agent controls. TPO-R downstream signaling in primary mononuclear cells comprised a marked, yet transient impairment in STAT5 and AKT phosphorylation upon combination treatment, compared to single EPAG treatment.Togehter, our data show that simultaneous treatment of leukemic cells with non-cytotoxic, but clinically-relevant dosages of EPAG and AZA does not lead to a stimulation of leukemic cell growth, even in the presence of TPO-R, in several pre-clinical models. These results do not support a generalized mechanistic antagonism between the two drugs in leukemic cells. Further investigations are underway to understand the impact on non-leukemic hematopoiesis. DisclosuresSteidl:Bayer Healthcare: Consultancy; Novartis: Research Funding; Celgene: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding. Marques Ramos:Novartis Pharmaceuticals: Employment. Pallaud:Novartis Pharmaceuticals: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding.

Full spectrum of clonal haematopoiesis-driver mutations in chronic heart failure and their associations with mortality.

AimsSomatic mutations in haematopoietic stem cells can lead to the clonal expansion of mutated blood cells, known as clonal haematopoiesis (CH). Mutations in the most prevalent driver genes DNMT3A and TET2 with a variant allele frequency (VAF) ≥ 2% have been associated with atherosclerosis and chronic heart failure of ischemic origin (CHF). However, the effects of mutations in other driver genes for CH with low VAF (<2%) on CHF are still unknown.Methods and resultsTherefore, we analysed mononuclear bone marrow and blood cells from 399 CHF patients by deep error‐corrected targeted sequencing of 56 genes and associated mutations with the long‐term mortality in these patients (3.95 years median follow‐up). We detected 1113 mutations with a VAF ≥ 0.5% in 347 of 399 patients, and only 13% had no detectable CH. Despite a high prevalence of mutations in the most frequently mutated genes DNMT3A (165 patients) and TET2 (107 patients), mutations in CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2 were associated with increased death compared with the average death rate of all patients. To avoid confounding effects, we excluded patients with DNMT3A‐related, TET2‐related, and other clonal haematopoiesis of indeterminate potential (CHIP)‐related mutations with a VAF ≥ 2% for further analyses. Kaplan–Meier survival analyses revealed a significantly higher mortality in patients with mutations in either of the seven genes (53 patients), combined as the CH‐risk gene set for CHF. Baseline patient characteristics showed no significant differences in any parameter including patient age, confounding diseases, severity of CHF, or blood cell parameters except for a reduced number of platelets in patients with mutations in the risk gene set in comparison with patients without. However, carrying a mutation in any of the risk genes remained significant after multivariate cox regression analysis (hazard ratio, 3.1; 95% confidence interval, 1.8–5.4; P < 0.001), whereas platelet numbers did not.ConclusionsSomatic mutations with low VAF in a distinct set of genes, namely, in CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2, are significantly associated with mortality in CHF, independently of the most prevalent CHIP‐mutations in DNMT3A and TET2. Mutations in these genes are prevalent in young CHF patients and comprise an independent risk factor for the outcome of CHF, potentially providing a novel tool for risk assessment in CHF.

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&lt;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&lt;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&amp;lt;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.

Problems of safety and quality of donor blood components

Objective. Analysis of issues related to the safety and quality of donated blood and its components.&#x0D; Materials and methods. Many years of experience of hematologists, immunologists, isoserologists, morphologists, biochemists in studying the composition, morpho-functional properties of cells and blood plasma, the use of donor blood and its components in clinical practice have made it possible to reconsider method of chemotherapy “multilateral action”.&#x0D; Results and discussion. One of the main axioms of modern transfusion medicine: chemotherapy should be performed strictly according to the indications and those blood components that are needed to ensure the viability of the body. The development of transfusiology has proved, with few exceptions, the inexpediency and even harmfulness of the use of whole donor blood. When using blood and its components, it is necessary to clearly consider extremely important point: the use will be in a planned manner, or in special circumstances. Blood components and blood plasma preparations have unique medicinal properties and there is currently no alternative to them. Each country is recommended to switch to self-sufficiency of blood components and their derivatives, to organize their own production structures that would meet the country’s domestic needs in blood components and preparations. The main components of the ideology of component chemotherapy are: recovery of blood component deficiency is not achieved on a “drop by drop” basis; no need to completely replace the existing deficiency of a cellular or protein component. Transfusions of blood components should be treated as a responsible invasive medical procedure – an operation that can have both immediate and long-term complications and consequences. Unreasonable transfusions of whole canned blood, especially after long periods of storage, are not only ineffective, but often pose a danger. In canned blood, during storage, complex biochemical metabolic processes take place both in cells and in plasma, which ultimately reduce the quality of both the blood itself and the morpho-functional properties of its individual components. Thus, 8-day storage of erythrocytes is the threshold after which erythrocytes begin to adversely affect the patient. Morpho-functional properties of blood components are directly dependent on the shelf life and distance of transportation. During the storage of blood and erythrocyte mass, the level of ATP decreases, on which the elasticity of the erythrocyte membrane depends. The magnitude of the negative electrical charge of the surface membrane of blood cells decreases. The number of prehemolytic forms of erythrocytes and cells that are not capable of reverse transformation increases. Within 1-4 days, leukocytes die, bacteria are released, so after this period, the greatest number of complications and reactions. Leukocytes and platelets form microaggregates at an early stage of storage, which can cause microembolism and distress syndrome. Microclots are formed, which include lysed blood cells and fibrin. The number of microclots increases every day, reaching on day 21 to 100 thousand/ml, so when transfusing it is advisable to use antiplatelet filters, rather than leukocyte. The use of bed leukofilters after a long period of storage of erythromass is not advisable, because there are no leukocytes. In addition, up to 2 % of erythrocytes are lost. The pH decreases, the content of 2,3-DFG, which is responsible for oxygen transport function (decreases by 50 % on the third day), hemolysis increases (up to 200 mg% of free hemoglobin). The concentration of potassium and ammonium ions increases. The recommended threshold for erythrocyte concentrate transfusions is a hemoglobin level of less than 70 g/l in adults and most children. It is important to use fresh-frozen plasma, erythrocyte concentrate and platelets obtained from one donor. Modern blood separators technically provide such an opportunity. Conditionally acceptable number of platelets in patients with injuries is more than 50×109/l cells, and in patients with combined brain injury is 100×109/l. Platelet concentrate obtained by the manual method from 4-5 donors leads to the development of refractoriness. With increasing shelf life of platelets, the functional properties of cells deteriorate significantly. All attempts to create the so-called artificial blood in the 19th century ended at the level of scientific developments. Synthetic and bioengineered cellular components of blood, hematopoietic factors, as well as hematopoietic stem cells are considered promising in the future. One of the possible ways to solve the problem of long-term storage of blood components, especially liquid groups, is cryopreservation of individual cells (erythrocytes) at moderately low (-20; -40; -80 ºС) and ultra-low (-165-196 ºС) temperatures, followed by deglycerization (washing), the use of special solutions for resuspension. With the development of low-temperature electric refrigeration equipment in Ukraine, this has become a reality.&#x0D; Conclusions. Practice has shown that both the blood itself and its components can neither be produced nor extracted as minerals, it can only be shared. All of the above indicates that blood donation is and will remain the main source of cellular components of the blood for at least the next decades.

Comparison of platelet count reduction in patients with essential thrombocythaemia treated with hydroxyurea and thromboreductin. Single centre experience (RCD code: VIII)

Essential thrombocythemia is one of the Ph-negative myeloproliferative neoplasms treated with hydroxyurea. An alternative strategy may be a therapy with thromboreductin. The aim of the study was to compare the effectiveness of hydroxyurea and thromboreductin treatment, defined by a decrease in the platelet count. The study group consisted of 154 patients with essential thrombocythemia diag‐ nosed and treated at the Outpatient Clinic of Hematology in Krakow, Poland between 1995 and 2016. Patients were included in the study at the start of cytoreductive treatment. 102 patients was treated with hydroxyurea and 52 patients treated with thromboreducin. We set the limit values for the number of platelets on levels : &lt;800 x 10 9 /L, &lt;600 x 10 9 /L, &lt;450 x 10 9 /L and &lt;350 x 10 9 /L. Afterwards, the analysis of the time required to achieve each point was performed. A comparison of hydroxyurea and thromboreductin groups showed that the number of platelets at the beginning of therapy was significantly lower in patients treated with hydroxyurea. Platelets value in the last control was significantly lower in patients treated with thromboreductin than hydroxyurea. The change in total platelet count over the time was significantly higher in the thromboreductin group. Patients treated with thromboreductin had a faster platelets reduction lower than 450 x10 9 /L. Tromboreductin is effective in reducing the number of platelets in patients with resistant essential thrombocythemia or intolerant of hydroxyurea regardless of age. JRCD 2018; 3 (8): 266–270

Types of bcr-abl and their correlations with the blood count in chronic myeloid leukemia (CML) in Togo

Décrire les différents types de transcrits de fusion moléculaire bcr-abl afin d'évaluer leur fréquence et étudier leurs influences sur les paramètres de l'hémogramme au diagnostic. Il s'agit d'une étude prospective de 34 patients porteurs d'une leucémie myéloïde chronique au Togo. La recherche des transcrits de fusion a été réalisée au laboratoire d'hématologie biologique de l'hôpital Henri Mondor à Créteil (France). L'âge moyen des patients était de 42,32±14,87 ans avec des extrêmes de 9 et 65ans. On notait une prédominance masculine avec un sex- ratio de 1,61 (21 hommes et 13 femmes). L'étude moléculaire révélait deux types de transcrits b3-a2 et b2-a2. Dix-neuf patients (55,88%) avaient le transcrit b3-a2, 13 (38,24%) le transcrit b2-a2 (32,10 %) et deux patients possédaient les deux types de transcrits b3-a2 et b2-a2 (5,88%). Au diagnostic, le taux d'hémoglobine moyen, le nombre moyen de globules blancs, le chiffre moyen des plaquettes chez les patients porteurs du transcrit b3-a2 étaient respectivement de 99,2g/l ; 207,63G/l et 451,28G/l. Pour les patients porteurs du transcrit b2-a2 les valeurs sont 104,6g/l, 114,32G/l et 486,11G/l. Les patients ayant les deux types de transcrit de fusion avaient respectivement 67g/l, 867G/l et 780G/l. Les paramètres de l'hémogramme sont significativement plus perturbés chez les patients porteurs des deux types de transcrits b3-a2 et b2-a2.

An investigation of the effects of the mean platelet volume, platelet distribution width, platelet/lymphocyte ratio, and platelet counts on mortality in patents with sepsis who applied to the emergency department.

The aim of this study is to examine the role of the mean platelet volume (MPV), platelet distribution width (PDW), platelet/lymphocyte ratio (PLR), and platelet values for predicting mortality in patients with sepsis. This is a retrospective study, involving patients 18 years and above who were diagnosed with sepsis. Blood samples were analyzed for platelets characteristics (counts, MPV, PDW, and PLR). The patients were separated into two groups namely the survivors and deceased. The two groups' MPV, PDW, PLR, and platelet counts which were considered to have an effect on mortality, were compared. Three hundred and thirty patients who were diagnosed with sepsis in our emergency department and complying with the study participation criteria were studied retrospectively. Comparison of the MPV, PDW, PLR, and platelet counts of the deceased and survivors showed that the MPV, PDW, and PLR were higher in the deceased while the platelet counts were higher in the survivors. The low number of platelets in patients with sepsis at the moment of application and the high PDW and PLR values are valuable for predicting a high mortality.

Особливості змін показників системи гемостазу та рівня с-реактивного протеїну у хворих на гострий холецистит

Introduction. Acute cholecystitis, which is requently complicated by the bile ducts mechanical obstruction, is characterized by a variety of symptoms – from local inflammation to significant changes in hepatocytes and cholestatic intoxication. These symptoms often manifest violation of the liver functions, which is involved in the synthesis of most proteins, as the system of hemostasis, as well as the so-called «acute phase proteins».The aim of the study – to determine and conduct comparative analysis of hemostatic parameters and level of C-reactive protein (CRP) in patients with acute calculous cholecystitis (ACC).Methods of the research. 67 people were examined: 20 of them – healthy, 25 – patients with ACC without complications (1 group) and 22 – patients with ACC complicated with obstructive jaundice (2 group ). The study of hemostasis was performed by determining platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration, the content of soluble fibrin-monomeric complexes (SFMC), antithrombin III activity (ATIII) and Hagemann-dependent fibrinolysis. Determination of CRP level in blood serum was also performed.Results and Discussion. We found a significant elongation of the PT, APTT, increased concentration of fibrinogen, SFMC content, Hagemann-dependent fibrinolysis and the level of CRP in patients with ACC in comparison with the control group (p&lt;0.05, p&lt;0.001). In addition, a significant decrease in ATIII level and reduction of the number of platelets in patients with ACC in comparison with the practically healthy people (p&lt;0.05) was found.Conclusions. The basis of the identified changes in parameters of hemostasis system most likely lies in hepatocellular insufficiency as the cause of the reduced synthesis of many components of haemostasis on the background of inflammatory liver damage. Disorders of primary and secondary hemostasis with insufficient anticoagulants activity can lead to multiple lesions in hemodynamics and microcirculation. The increase in the content of SFMC in plasma suggests the presence of DIC syndrome. A prolongation of PT and APTT will characterize DIC syndrome as the use factors of a phase hypocoagulation.A significant increase in the level of CRP in patients with ACC complicated with obstructive jaundice indicates its involvement in the progress of the inflammatory process.

THE ROLE OF PLATELETS IN FORMATION OF NEUTROPHIL EXTRACELLULAR TRAPS IN PATIENTS WITH IMMUNOCOMPLEX PATHOLOGY

The article presents the results of the assessment of the role of platelets in formation of extracellular traps by neutrophils. We have detected the ability of platelets to oppress the formation of extracellular traps by neutrophils in vitro cultures in patients with rheumatoid arthritis (RA) (n = 42) and systemic lupus erythematosus (SLE) (n = 24), but not in patients with hemorrhagic vasculitis (GW) (n = 15). The study has revealed the interrelation of NETosis and rheumatoid factor in patients with RA and SLE, as well as NET formation and number of platelets in patients with GW.

Platelet-Like-Particles Sheared From Myosin-II-Inhibited Megakaryocytes Highlights The Elevated Thrombocrit Of May-Hegglin Anomaly

Megakaryocytes (MKs) in the marrow extend projections into blood flow and generate platelets under shear. Understanding MK differentiation and platelet production is of broad clinical importance and extends a need to augment platelet numbers in patients. Reversible but sustained inhibition of non-muscle myosin-II (NMM-II) with the drug blebbistatin increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation under shear. Using a cone and plate rheometer to apply fluid shear to drug-treated MKs in bulk, platelet-like-particles (PLPs) that are collagen-I responsive can be generated with intermediate shear. The MKs naturally down-regulate NMM-IIA activity through phosphorylation of S1943, but this site proves shear sensitive, consistent with results for human platelets. Using micropipette aspiration of MKs, inhibition of NMM-IIA is found necessary to generate CD41+ fragments that approximate the size of human platelets. Localization of NMM-IIA to the fragments is modulated by S1943 as seen by unique distribution patterns resulting from specific S1943 mutations that can be abrogated by addition of blebbistatin. The approach is extended to clinically relevant mutations associated with May-Hegglin anomaly (MHA) co-expressed with wild type protein to mimic heterozygotes. As with blebbistatin inhibition of myosin, May-Hegglin mutants result in a higher frequency of fragmentation during micropipette aspiration, indicating a dominant negative effect. Immunofluorescence documents abnormal myosin aggregation in cells transfected with May-Hegglin myosin mutations compared to wild type constructs. Finally, peripheral blood from a patient with a D1414N May-Hegglin mutation is cultured to produce megakaryocytes used to support both the micropipette and immunofluorescence results. These findings reveal a phospho-switch in NMM-II, from inactive to active in the terminal stages of platelet-poiesis, and that proper myosin activity is critical to fragment size and number. Disruption of normal activity enhances fragment generation suggesting a novel mechanism in MHA: in particular, MHA thrombocytopenia results in an increased thrombocrit due to abnormally large platelets, which overcompensates for the reduction in platelet number. Disclosures:No relevant conflicts of interest to declare.

Eltrombopag Increases Platelet Numbers in Thrombocytopenic Patients With HCV Infection and Cirrhosis, Allowing for Effective Antiviral Therapy

Thrombocytopenia is common among patients with hepatitis C virus (HCV) infection and advanced fibrosis or cirrhosis, limiting initiation and dose of peginterferon-alfa (PEG) and ribavirin (RBV) therapy. The phase 3 randomized, controlled studies, Eltrombopag to Initiate and Maintain Interferon Antiviral Treatment to Benefit Subjects with Hepatitis C-Related Liver Disease (ENABLE)-1 and ENABLE-2, investigated the ability of eltrombopag to increase the number of platelets in patients, thereby allowing them to receive initiation or maintenance therapy with PEG and RBV. Patients with HCV infection and thrombocytopenia (platelet count <75,000/μL) who participated in ENABLE-1 (n = 715) or ENABLE-2 (n = 805), from approximately 150 centers in 23 countries, received open-label eltrombopag (25-100 mg/day) for 9 weeks or fewer. Patients whose platelet counts reached the predefined minimal threshold for the initiation of PEG and RBV therapy (95% from ENABLE-1 and 94% from ENABLE-2) entered the antiviral treatment phase, and were assigned randomly (2:1) to groups that received eltrombopag or placebo along with antiviral therapy (24 or 48 weeks, depending on HCV genotype). The primary end point was sustained virologic response (SVR) 24 weeks after completion of antiviral therapy. More patients who received eltrombopag than placebo achieved SVRs (ENABLE-1: eltrombopag, 23%; placebo, 14%; P = .0064; ENABLE-2: eltrombopag, 19%; placebo, 13%; P = .0202). PEG was administered at higher doses, with fewer dose reductions, in the eltrombopag groups of each study compared with the placebo groups. More patients who received eltrombopag than placebo maintained platelet counts of 50,000/μL or higher throughout antiviral treatment (ENABLE-1, 69% vs 15%; ENABLE-2, 81% vs 23%). Adverse events were similar between groups, with the exception of hepatic decompensation (both studies: eltrombopag, 10%; placebo, 5%) and thromboembolic events, which were more common in the eltrombopag group of ENABLE-2. Eltrombopag increases platelet numbers in thrombocytopenic patients with HCV and advanced fibrosis and cirrhosis, allowing otherwise ineligible or marginal patients to begin and maintain antiviral therapy, leading to significantly increased rates of SVR. Clinical trial no: NCT00516321, NCT00529568.

Does TP53 mutation promote ovarian cancer metastasis to omentum by regulating lipid metabolism?

TP53 (Tumor Protein 53, previously known as p53) is probably the best known of all tumor suppressor genes, and is mutated in nearly all (96%) high-grade serous ovarian cancer (HGS-OvCa), which is the most common histopathological type of epithelial ovarian cancer (EOC). Recently, TP53 is found to involve in regulating cell metabolic pathways besides its classical tumor suppressive functions. In addition, emerging evidence suggests that mutant TP53 is associated with cancer metastasis. Through summarizing and comparing the roles of wild-type TP53 and mutant TP53 in the progression of various types of cancer, we hypothesize that mutant TP53 in HGS-OvCa cells interacts with sterol regulatory element-binding proteins (SREBPs) and guanidinoacetate N-methyltransferase (GAMT), leading to increased gene expression of key enzymes involved in fatty acids (FAs) and cholesterol biosynthesis and the inhibition of fatty acid oxidation (FAO), thus promotes lipid anabolism to accelerate tumor growth and progression. Elevated platelet number in patients’ tumor microenvironment results in increased TGF-β production. Then, TGF-β acts in concert with mutant TP53 to promote HGS-OvCa metastasis by assembling a mutant-TP53/p63/Smads protein complex, in which p63’s functions as metastasis suppressor are antagonized, and by enhancing the activities of the Slug/Snail and Twist families to drive induce EMT-like transition. Then adipocyte-derived IL-8 facilitates the metastasis of transformative cancer cells to abdominal adipose tissue (e.g., omentum). Once metastasis is established, mutant TP53 together with adipocyte-derived IL-8 upregulates Fatty acid-binding protein 4 (FABP4) expression and then promotes FAs absorption from adipocytes to support rapid tumor growth in adipocyte-rich metastatic environments. In summary, these indicate that mutant TP53 may play determinant roles in the progression of HGS-OvCa.

Shear-Optimized Platelet-Like-Particles from High Ploidy MKS: From Segregation to Composition and Activation

Abstract Abstract 3456 Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet' projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro. To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures. Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures: No relevant conflicts of interest to declare.

Shear-Optimized Platelet-Like-Particles From High Ploidy Mks: From Segregation to Composition and Activation

Abstract 3456Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet' projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro.To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures.Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures:No relevant conflicts of interest to declare.

Frequent downregulation or loss of CD79a expression in plasma cell myelomas: Potential clue for diagnosis

Plasma cell myeloma is a frequent hematogeneous disorder that occurs mainly in older people. Not only bone marrow smears but also clots and/or biopsied specimens are often taken for confirmation of pathological diagnosis. Some specimens show sheet-like plasma cell proliferation associated with immunoglobulin monotype on immunohistology, which readily leads to diagnosis, but many samples do not clearly show light-chain restriction. The aim of the present study was therefore to examine CD79a expression because some samples had reduced expression or none at all. The immunoreactivity of CD79a was categorized into three groups: positive, weakly positive and negative, compared with scattering non-neoplastic plasma cells in the same specimen. Out of 100 specimens of plasma cell myeloma, 48% were positive for CD79a, 15% were weakly positive, and 37% were negative. In contrast, overexpression of cyclinD1 was detected in 26% of examined samples. CD79a-negative cases had a significantly lower percentage of positive staining for cyclinD1 than CD79a-positive or weakly positive cases. Clinicopathological data showed that CD79a-negative expression was associated with decreased platelet numbers in patients. The present study indicates that downregulation or loss of CD79a and/or overexpression of cyclin D1, observed in 59% of neoplastic plasma cell samples, could provide a strong diagnostic clue without regard to the results of immunoglobulin light-chain restriction.

Does tirofiban prevent platelet loss in patients after cardiogenic shock during continuous renal replacement therapy?

Link and colleagues present a pilot study investigating platelet function and platelet numbers in patients with cardiogenic shock and acute kidney failure undergoing continuous venovenous haemodialysis. Their data indicate a significantly reduced platelet loss with combined therapy of unfractionated heparin plus tirofiban, the glycoprotein IIb/IIIa antagonist, compared with unfractionated heparin therapy alone. Owing to the small sample size, however, the potential impact of additional treatment variables (antiplatelet agents, intraaortic counterpulsation) could not be clarified. A substantially larger, adequately powered study is therefore called for to establish the potential clinical relevance of these findings.