ALBERT

Description: Introduction Refractory anemia with ring sideroblasts (RARS), as a form of acquired primary sideroblastic anemia, represents about 11% of myelodysplastic syndromes (MDS) and is classified as a low risk MDS. It´s defined by the WHO as a pure dyserythropoietic disorder with presence of 〉15% ringed sideroblasts in the bone marrow. Abnormal expression of several genes of heme synthesis in this MDS subgroup and excessive accumulation of iron especially in mitochondria, the main place of reactive oxygen species (ROS) formation, contributes to the elevated oxidative stress. Oxidative stress is also known as one of the factors involved in the pathogenesis of MDS. High iron concentrations catalyse a Fenton reaction, where a hydroxyl radical is produced from hydrogen peroxide and causes an increase in ROS which may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. The aim of this study was to find a useful method for detection and identification of oxidatively modified proteins in plasma unique for RARS patients. Methods Carbonylated protein levels were determined spectrophotometrically using dinitrophenylhydrazine (DNPH) derivatization. Oxidatively modified proteins of plasma samples were derivatized with biotin hydrazide. The dialyzed biotin hydrazide labeled samples and negative controls were mixed with monomeric avidin resin. Captured carbonylated proteins were digested by trypsin and then identified by MS/MS mass spectrometry coupled to a nano-LC system. Mascot (Matrix Science, London, UK) was used for database searching (Swiss-Prot). Two unique peptides (with a higher Mascot score than the minimum for identification, P

Description: Introduction: Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoesis, associated with cytopenias and high risk of leukemic transformations with common morbidity. MDS are hematological malignancies of unclear etiology where oxidative/nitrative stress may contribute to the pathogenesis1. The posttranslational oxidative modifications of proteins and low molecular weight compounds are induced, revealing dysbalance of redox systems in vivo. Nitration of tyrosine either in free form or bound in proteins is important marker of nitric oxide synthase (NOS) activity shift in the presence of oxidative stress in favour of superoxide formation. The aim of this work was to assess whether 3-nitrotyrosine (3-NT) serum concentrations are enhanced also in MDS patients. Methods: Serum samples were obtained using blood of either MDS patients or healthy donors. All tested individuals agreed to the study at the time of blood collection. We proposed HPLC-MS/MS method to estimate 3-NT concentration in serum samples using QTRAP 4000 mass spectrometer (ABSciex, Prague, Czech Republic). Serum proteins were precipitated using ethanol, supernatants were evaporated, reconstituted in 0.1% HCOOH/2% methanol and injected onto HALO C18 microcolumn 100x0.5 mm (ABSciex, Prague, Czech Republic). Oxidative stress in MDS patients and controls was assessed by serum malondialdehyde concentrations measured by HPLC of 2-thiobarbituric acid MDA derivative using UV detection. Results: The sensitivity of method proposed for analysis of 3-NT in sera was sufficient for estimation of differences of 3-NT in patients and control samples. We have found enhanced concentrations of both MDA and 3-nitrotyrosine in serum of MDS patients as compared with healthy donors. Discussion: Enhanced MDA concentrations in MDS patients confirmed the presence of oxidative stress in MDS patients. The reactive oxygen species may oxidize tetrahydrobiopterin, important cofactor of NOS, resulting into nitric oxide synthase uncoupling with enhanced superoxide and consequently peroxynitrite production2. It is known that methylarginines, naturally occurring inhibitors of NOS, can profoundly increase superoxide generation from uncoupled NOS. Recently, we have found significantly enhanced concentration of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome3. The observed increased concentrations of 3-NT in MDS patients correspond with assumed enhanced peroxynitrite formation as compared with controls. 3-nitrotyrosine concentrations thus could serve as a new criterion of NOS changed activity in MDS patients. Literature: 1. Farquhar MJ, Bowen DT. Oxidative stress and the myelodysplastic syndromes. Int J Hematol. 2003;77:342-350. 2. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315-424. 3. Štikarová J, Suttnar J, Pimková K, Chrastinová-Mášová L, Čermák J, Dyr JE. Enhanced levels of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome. Journal of Hematology & Oncology. 2013;6:58. Disclosures No relevant conflicts of interest to declare.

Description: Fibrinogen adsorption on a surface results in the modification of its functional characteristics. Our previous studies revealed that fibrinogen adsorbs onto surfaces essentially in 2 different orientations depending on its concentration in the solution: “side-on” at low concentrations and “end-on” at high concentrations. In the present study, we analyzed the thrombin-mediated release of fibrinopeptides A and B (FpA and FpB) from fibrinogen adsorbed in these orientations, as well as from surface-bound fibrinogen-fibrin complexes prepared by converting fibrinogen adsorbed in either orientation into fibrin and subsequently adding fibrinogen. The release of fibrinopeptides from surface-adsorbed fibrinogen and from surface-bound fibrinogen-fibrin complexes differed significantly compared with that from fibrinogen in solution. The release of FpB occurred without the delay (lag phase) characteristic of its release from fibrinogen in solution. The amount of FpB released from end-on adsorbed fibrinogen and from adsorbed fibrinogen-fibrin complexes was much higher than that of FpA. FpB is known as a potent chemoattractant, so its preferential release suggests a physiological purpose in the attraction of cells to the site of injury. The N-terminal portions of fibrin β chains including residues Bβ15-42, which are exposed after cleavage of FpB, have been implicated in many processes, including angiogenesis and inflammation.

Description: Introduction Acute myeloid leukemia (AML) represents an aggressive bone marrow malignancy with diverse genetic abnormalities. The tumor cells, to support their own growth and proliferation, may alter metabolism 1) to accelerated glycolysis to provide energy/biosynthetic precursors and 2) to produce intermediates by active TCA cycle for synthesis of essential biomolecules [1]. Mutations of isocitrate dehydrogenase (IDH1/2) as frequent mutations (affecting approximately 20% of AML patients) cause the reduction of α-ketoglutarate to D-2-hydroxyglutarate instead of oxidative decarboxylation of isocitrate to α-ketoglutarate. IDH2 mutations occur almost exclusively in hematopoietic tumors. Whereas IDH2R140 mutation is frequently accompanied by normal cytogenetics and NPM1 mutations, IDH2R172 is frequently the only mutation detected in AML. In addition, IDH2R172 confers a poor prognosis in patients with AML [2, 3]. The aim of this study was to characterize and uncover the differences in metabolism of AML patients with or without IDH2 mutations in diagnosis (the initial stage), in remission (after chemotherapy treatment), and before transplantation (after conditioning). Methods Serum metabolomics profiles were generated with samples obtained at diagnosis, in remission, and before transplantation from patients (n=20) treated in the Institute of Hematology and Blood Transfusion, Prague, Czech Republic. Patients were assigned in IDH2WT (n=7) and IDH2R140/ IDH2R172 (n=7/6) groups. Targeted metabolomic profiling of 19 metabolites related mainly to TCA cycle and glycolysis was performed by LC-MS/MS. Results Using non-parametric randomized block analysis of variance (Friedman test) we found significant differences in levels of 13 metabolites among treatment periods for all AML patients. Moreover, when comparing each treatment periods for all patients, we found significantly decreasing levels of 12 metabolites between the initial stage and the period before transplantation. If we considered the division of patients into groups according to IDH2 mutations, we found significantly different levels of 4 metabolites among treatment periods for samples of patients with IDH2R140,namely 2 metabolites of TCA cycle: isocitrate, succinate; and 3-hydroxybutyrate and urate. We obtained significantly different levels in the same number of other metabolites for IDH2WT. In samples of patients with IDH2R172 we found significantly different levels of 8 metabolites among treatment periods, namely 5 metabolites of TCA cycle: citrate, 2-hydroxyglutarate, succinate, fumarate, malate; 2 metabolites of glycolysis: 3-phosphoglycerate, phosphoenolpyruvate; and pyroglutamate of glutathione metabolism. Furthermore, using Nemenyi post-hoc test we ascertained significantly decreasing levels of named metabolites in IDH2R140 and IDH2R172 samples before transplantation with respect to the initial stages of AML (diagnosis). Conclusion Overall, this study identified significant changes in several metabolites of TCA cycle and glycolysis between initial and final treatment periods and also between remission and period before transplantation in AML patients. With respect to IDH2 mutations we found more significant changes in metabolites specifically in TCA cycle for IDH2R172 patients relative to IDH2R140. The results of our preliminary targeted metabolomic profiling of AML patients with IDH2 mutations are corresponding with the already described differences in morphological and genetic patterns in the patients and, moreover, support the classification of IDH2R172 as separate AML subtype. Metabolomic profiling thus seems to be a new valuable tool providing additional important information. Acknowledgment This work was supported by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports), by the project of the Ministry of Health, Czech Republic, 00023736, by Grant from the Academy of Sciences, Czech Republic, P205/12/G118, and by ERDF OPPK CZ.2.16/3.1.00/24001. [1] DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. Cell Metab. 2008;7(1):11-20. [2] Rakheja D, Konoplev S, Medeiros LJ, Chen W. Hum Pathol. 2012;43(10):1541-51. [3] Meggendorfer M, Cappelli LV, Walter W, Haferlach C, Kern W, Falini B, Haferlach T. Leukemia. 2018;32(5):1249-1253. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The protein microarrays are becoming the leading technology in proteomic research area. They enable to implement both features of proteins that can be altered in disease as quantitative proteomics (levels in biological samples) as well as functional proteomics (determination of their selective interactions with other biomolecules). Protein microarray techniques such as sandwich immunoassays, antigen capture immunoassay and direct immunoassays use labeling and antibodies. However, these labels can interfere with the analyte binding site and thus affect protein activity. Using antibodies requires a prior knowledge of the studied proteins which is in the case of a heterogeneous disease with little knowledge in its proteomic field a huge disadvantage. Surface plasmon resonance (SPR) is a label-free and direct method allowing quantification as well as monitoring of protein-protein interactions simultaneously and in real time. Myelodysplastic syndrome (MDS) is a heterogeneous group of hematological malignancies. It affects pluripotent hematopoietic stem cell and is manifested by variety of clinical symptoms according to predominant involvement of development lineage. The high risk of MDS to transform into acute myeloid leukemia makes it a suitable model for study of biological processes leading to leukemia development. In this work, we use SPR imaging for simultaneous screening of blood plasma of MDS patients followed by mass spectrometry (MS) for identification of interacting partners and analysis of protein network properties. Proteins, whose levels are either elevated during MDS disease or interaction with their receptors/ligands is a part of signaling pathway, were employed. Method SPR imaging system with polarization contrast and internal referencing was combined with dispersionless microfluidics for parallel screening of blood plasma samples. Proteins involved in pathogenesis of MDS and their physiological counterparts were immobilized under flow to create 6x6 sensing spots. Specifically, these include integrin αMβ2 (LFA1), intercellular adhesion molecule 1 (ICAM1), integrin α4β1 (VLA4), vascular cell adhesion protein 1 (VCAM1) and cytotoxic T-lymphocyte protein 4 (CTLA4). The sensor surface functionalization was optimized with respect to its ability to provide a low-fouling sensing surface with biologically active receptors. Plasma samples of controls and MDS patients were flowed along the functionalized surface and differences in individual interactions were evaluated. Selected interacting partners were further identified using 2D-HPLC/ESI-MS/MS. Identified proteins were analyzed by String Networks and Power Graph Analysis. Results and Conclusion Significant differences in the protein profiles among different MDS groups of patients as well as relative to control healthy donors were observed using SPR imaging; tens of interacting proteins were identified by mass spectrometry. Protein interaction networks were explored through clustering of proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. Identified proteins are involved in several processes; regulation of immune system, ubiquitinylation and protein degradation, cell signaling, hemostasis, protein synthesis, cell adhesion, metastasis, and inhibition of blood coagulation. Using of Power Graphs, a novel representation of (protein) networks, provided valuable insight into the existence of protein complexes, their internal organization, and their relationships. Interaction networks also indicated possible pathways involved in MDS pathogenesis (especially Src tyrosine kinases). The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aα chain - fibrinopeptides A and the Bβ chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined two unrelated boys aged 4 (boy 1) and 14 (boy 2) for susp. dysfibrinogenemia. Routine coagulation tests revealed prolonged thrombin and reptilase time in both boys and also showed decreased functional fibrinogen levels in both kids (Tab. 1). Boy 1 presented with bleeding manifestation – easy bruising, epistaxis, and bleeding after tooth extraction. His 26-year-old mother presented with similar coagulation findings and with mild bleeding complications. Boy 2 was asymptomatic. Fibrin polymerization experiments carried out on plasma samples showed prolonged lag time and significantly reduced final turbidity in both cases. Measurement of kinetics of fibrinopeptide release showed a decreased amount of the released fibrinopeptide A in both patients. DNA sequencing of boy 1 revealed a point mutation in exon 2 of the FGA gene at the position 3456 G/A, which causes the substitution of Aα 16 Arg to His (fibrinogen Praha V). The boy was found to be heterozygous for the mutation as well as his mother. DNA analysis of boy 2 revealed the same point mutation in the FGAgene, causing the same substitution of Aα 16 Arg to His (fibrinogen Kralupy nad Vltavou). Conclusion In this study we report two cases of congenital defects in the fibrinogen Aα Arg16-Gly17 bond found during childhood. It has been described earlier that the replacement of Aα 16 arginine by histidine decelerates thrombin catalyzed fibrinopeptide A release. Although both kids presented with the same genetic defect and with similar coagulation results, they have different clinical manifestation of the disease. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Table 1: Routine coagulation test results Boy 1 Boy 1's mother Boy 2 Normal APTT 40.1 s 34.6 s 35.3 s 27.5 - 36.1 s Prothrombin time 17.5 s 14.4 s 16.9 s 11.7 - 15.1 s Thrombin time 40.9 s 36.2 s 44.8 s 17.6 - 21.6 s Reptilase time 58.3 s 51.6 s 55.7 s 16.0 - 20.0 s Fibrinogen (Clauss) 0.51 g/l 1.12 g/l 0.58 g/l 2.00 - 4.20 g/l Fibrinogen (Immuno) 2.47 g/l 2.68 g/l 2.31 g/l 2.00 - 4.20 g/l Age 4 26 14 - Disclosures No relevant conflicts of interest to declare.

Description: Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aa, Bb, and g) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aa chain - fibrinopeptides A and the Bb chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined four unrelated children suspected dysfibrinogenemia. The first patient was a 5-yr old boy with prolonged thrombin time and low functional fibrinogen level. He presented with easy bruising and epistaxis. Genetic analysis revealed a heterozygous substitution Aalpha R16H. The second patient was a 13-yr old girl with prolonged thrombin and reptilase times and low functional fibrinogen level. She presented with menorrhagia. Genetic analysis revealed a heterozygous substitution Aalpha G17V. The third patient was a 12-yr old asymptomatic boy. Genetic analysis revealed a heterozygous substitution Aalpha R16H. Kinetics of fibrinopeptide release was impaired in all these cases. The fourth patient was a 3-yr old girl with low functional fibrinogen level. She presented with easy bruising. Genetic analysis revealed a heterozygous substitution Aalpha K448N. All patients presented with impaired fibrin polymerization. Conclusion All patients were found to be heterozygous for point mutations in FGA gene. Three mutations were found in the site of fibrinopeptide cleavage and one in the alphaC-domain. Mutations had different clinical manifestations from asymptomatic to bleeding. Mutations Aalpha Arg16His and Aalpha Gly17Val are among the most common fibrinogen mutations and both decelerate fibrinopeptide A cleavage from mutant fibrinogens. We describe here a novel previously unreported mutation Aalpha Lys448Asn affecting fibrin formation. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Disclosures No relevant conflicts of interest to declare.

Description: Fibrinogen is a 340 kDA glycoprotein that plays a vital role in the hemostasis. The three pairs of polypeptide chains (Aα, Bβ and γ) form a heterodimer structure of fibrinogen, each chain is encoded by a distinct gene (FGA, FGB and FGG). Congenital dysfibrinogenemia is a rare qualitative disorder characterized by structural alterations in fibrinogen molecule resulting in its defective function. The clinical manifestation of majority cases is asymptomatic (50%); about 30% are associated with bleeding tendencies and only some cases are thrombotic (20%). In this study, we evaluated 25 patients with heterozygous missense mutations in exon 2 of FGA gene from 13 unrelated families, all with low levels of functional fibrinogen and prolonged clotting time. All mutations were located around thrombin cleavage site at N-terminus of a fibrinogen Aα chain. Mutation Aα G13E was found in 6 families (13 patients), Aα R16H in 4 families (6 patients), Aα R16C in 1 family (4 patients), and Aα R19G in 1 family (2 patients). Correlations between the genotype and phenotype were studied by fibrin polymerization, fibrinolysis, kinetics of fibrinopeptides release, and electron and confocal microscopy. The viscoelastic properties of fibrin clots were measured. All mutations caused a delay or absence of fibrinopeptide A release and prolonged fibrin polymerization. Clinical phenotypes of mutations were, unexpectedly and importantly, highly heterogeneous. Out of 25 patients 17 were asymptomatic, 7 had the bleeding tendencies and 1 patient had thrombosis. One woman with Aα G13E had 2 spontaneous abortions and 3 pregnant women with Aα G13E, Aα R16C and Aα R19G mutations suffered for hemorrhages or postpartum bleeding. Eventually, all had successful delivery under provided care with a hematologist. Small surgeries were performed to patients with Aα R16H supplied with fibrinogen concentrates and antifibrinolytics. Our observations show that the patients should be carefully treated based both on the results of the laboratory analyzes and personal and family history. This work was supported by the project of the Ministry of Health, Czech Republic, for conceptual development of research organization 00023736, by Grant from the Academy of Sciences, Czech Republic nr. P205/12/G118, and by ERDF OPPK CZ.2.16/3.1.00/24001. Disclosures No relevant conflicts of interest to declare.