bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine
Authors: Majumder Rinku , Liang Xiaoe , Quinn-Allen Mary Ann , Kane William H. , Lentz Barry R. 
Published: October 2019

Abstract
Abstract 4344
Localization of blood coagulation enzymes and cofactors to the surface of procoagulant anionic phospholipid membranes is a central paradigm in hemostasis and thrombosis. The prototype for these complexes is the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. This enzyme complex consists of the enzyme factor Xa, the cofactor factor Va and calcium bound to a phospholipid surface. Formation of the prothrombinase complex results in a greater than 105-fold rate enhancement in the conversion of prothrombin to thrombin. Abnormal regulation of the prothrombinase complex has been demonstrated to be critical in the pathogenesis of thrombotic disorders.
Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. While reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va2 (Kd ∼6.5 mM) and to factor Xa (Kd ∼ 91 mM). C6PE and C6PS binding sites in factor Xa are specific, distinct and linked, as binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (Kdapp ∼40nM) of a partially active prothrombinase complex between factor Xa and factor Va2, compared to Kdapp for C6PS ∼2nM. We report here seven significant new observations:PE effects kcat more than KM. A kcat effect strongly implies an enzyme or cofactor conformational change, while a KM effect could be due to conformational changes or to altered binding of enzyme-cofactor and substrate to the membrane.PE promotes binding of factor Va to membranes.PE promotes assembly of a prothrombinase complex due its effects on factors Va and Xa independent of the presence of a membrane and thus not due to its effects on membrane packing.30% PE does not produce a measurable effect on factor Xa membrane binding.C6PE and C6PS bind synergistically to sites on factor Xa to alter its activity.C6PE binds to factor Va with greater affinity than does C6PS, but binding of these lipids to sites on this protein is not synergistic.Both PS and PE promote prothrombinase activity, but the magnitudes of these effects do not correlate with their effects on membrane interfacial packing.
These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.

Disclosures:
No relevant conflicts of interest to declare.
Clinical and Hematologic Manifestations in Patients with Diamond-Blackfan Anemia
Authors: Kim Soon Ki , Kang Hyoung Jin , Ahn Hyo Seop , Park Kyung Duk , Shin Hee Young , Kim Chul Soo 
Published: October 2019

Abstract
Abstract 4378

Background
Diamond-Blackfan anemia (DBA), characterized by impaired red cell production, is a rare condition that usually occurs symptomatic in early infancy. The purpose of this study was to assess the nationwide experiences of DBA that were encountered over a period of 20 years as a part of further molecular analyses.

Methods
Medical records of 56 patients diagnosed to have DBA were reviewed retrospectively from Nov. 1984 to July 31, 2010. Fifteen clinics including 13 university hospitals have been participated.

Results
Male to female ratio was 1.67:1 (males 35 cases, females 21). The median age of diagnosis was 4 months (range birth to 52 months of age), with the diagnosis of 74.1% before 1 year of age. From 2000 to 2009, 33 cases with DBA were registered, which showed the incidence of 6.6 cases per million. Excluding growth retardation, 21 cases (38.2%) showed congenital defects: thumb deformities (8 cases), ptosis (5), coarctation of aorta (5), ventricular septal defect (4), strabismus (3), cleft palate (2), mitral valve prolapsed (2). The mean hemoglobin (Hb) concentration was 5.1±1.9 g/dL, and median, 5.2 g/dL. The mean MCV was 93.4±11.6 fL, mean HbF 7.7±11.3%, mean reticulocytes 19,700/mm3 and mean adenosine deaminase 21.3. The mean cellularity of bone marrow was 75%, with the ME ratio of 20.4:1. Among 47 patients who had been treated with steroids, 23 patients (48.9%) did not need further steroids after remission. The mean duration of steroid use was 7 years and 2 months (maximum 22 years). Among 28 cases who received steroid, 9 patients had been controlled with the oral steroid of 5 mg or less per day. Eight patients with DBA that received hematopoietic transplantation have survived. Cancers developed in 2 cases (3.6%).

Conclusion
The incidence of DBA is similar with the data already published, but male predilection in our study. Although all patients responded to initial treatment with steroids, about a half needed further steroids after remission. It is necessary to collect the further data including management pathways from nationwide DBA registries, with the molecular analyses.

Disclosures:
No relevant conflicts of interest to declare.
Survival Of AML Patients Relapsing After Allogeneic Stem Cell Transplantation: A Center For International Blood and Marrow Transplant Research Study
Authors: Bejanyan Nelli , Weisdorf Daniel J. , Wang Hailin , Logan Brent R. , Devine Steven M. , de Lima Marcos , Bunjes Donald W. , Zhang Mei-Jie 
Published: October 2019

Abstract

Despite the curative potential of allogeneic hematopoietic cell transplantation (alloHCT), a large number of patients with acute myeloid leukemia (AML) experience leukemia relapse after HCT. Although subsequent remissions can be achieved with salvage therapy, they are usually short lived. We evaluated survival of AML patients relapsing after alloHCT to identify prognostic factors for survival after post-transplant relapse. 1788 pediatric and adult AML CR1 & CR2 patients relapsed post-transplant (1990-2010). The median age at HCT was 30 years (yr; range, <1-76). De novo leukemia occurred in 81% and intermediate risk cytogenetics in 45%. 1374 patients (77%) underwent myeloablative (MA) and 936 patients (52%) related donor HCT. Active GVHD prior to relapse occurred in 727 subjects (41%). Median time from transplant to relapse was 7 months (mo; range, 1-177). At relapse, 357 patients (20%) received supportive care, whereas 1231 patients (69%) received intensive therapy, including chemotherapy (CT) alone (n=660), donor lymphocyte infusion (DLI)±CT (n=202; DLI+CT=51) and 2nd alloHCT±CT and/or DLI (n=369; 49% 2nd alloHCT were myeloablative). Among evaluable patients (n=846) who received intensive therapy, CR rate was 29% (n=246) [15.6% for CT alone (n=443), 37.4% for DLI±CT (n=139) and 47.3% for 2nd alloHCT±CT±DLI (n=264)]. After a median follow up of 39 mo (range, <1-193) after relapse, 229 patients (13%) survived with 5-yr overall survival of 3% (95% CI 2-5%) for patients relapsing within 6mo, 11% (95% CI 8-14%; p<0.0001 comparing to <6 mo) for 6mo-2yr, 21% (95% CI 14-28%; p<0.0001 comparing to <6 mo) for 2-3yr and 31% (95% CI 23-39%; p<0.0001 comparing to <6 mo) for ≥3yr groups. Persisting AML (n=1271, 71%) was the most common cause of death. In multivariable analysis, longer time from alloHCT to relapse (RR 0.55 6mo-2yr, 0.39 2-3yr and 0.28 ≥3yr; p<0.0001) and reduced intensity conditioning (RIC) (RR=0.77; 95% CI 0.66-0.88, p=0.0002) were associated with significantly lower mortality. In contrast, age over 40 yr (RR=1.42, 95% CI 1.24-1.64; p<0.0001), active GVHD at relapse (RR=1.25, 95% CI 1.13-1.39; p<0.0001), adverse cytogenetics (RR=1.37, 95% CI 1.09-1.71; p=0.0062), mismatched URD (RR=1.61, 95% CI 1.22-2.13; p=0.0008) or cord blood (RR=1.23, 95% CI 1.06-1.42; p=0.0078) HCT had inferior survival. We confirm poor survival of AML patients relapsing after alloHCT. Yet patients with relapse beyond 6 mo have better survival and may benefit from intensive therapy, DLI or 2nd alloHCT.

Disclosures:
No relevant conflicts of interest to declare.
Free Thiol Groups In Von Willebrand Factor (VWF) Are Required For Its Proper Function Under Physiological Flow Conditions
Authors: Solecka Barbara , Fuchs Birte , Weise Christoph , Kannicht Christoph 
Published: October 2019

Abstract

Background
The multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested.

Aims
Objective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation.

Methods
Free and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry.

Results
Blocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified.

Conclusion
Blocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment.

Disclosures:
Solecka: Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.
Recombinant Human Thrombopoietin Promotes The Recovery Of Megakaryocyte Lineage In Patients With Severe Aplastic Anemia Receiving Immunosuppressive Therapy
Authors: Shao Zonghong , Dong Qi'e , Fu Rong 
Published: October 2019

Abstract

Objective
To assess the effectiveness of recombinant human thrombopoietin (rhTPO) in severe aplastic anemia (SAA) patients receiving immunosuppressive therapy (IST).

Methods
Eighty SAA patients receiving IST during a period from January 2007 to December 2011 were included in this retrospective analysis. Thirty-two subjects also received rhTPO treatment (15,000 U, three times a week, subcutaneously). The remaining 48 patients did not receive rhTPO treatment. The choice of using (or not using) rhTPO was based on physician discretion, and more importantly, patient will (partly based on financial capability to afford the medication). rhTPO was discontinued when platelet count returned to normal range. Hematologic response, bone marrow recovery, transfusion interval (platelet or red-cells), and the time to transfusion-free status were compared.

Result
At 6th months after the treatment, hematologic response along at least one lineage was achieved in 65.6% of the subjects receiving rhTPO vs. 41.7% in those who did not receive rhTPO (p=0.04). Response rate of megakaryocyte and erythroid lineage at 3rd months was also higher in subjects receiving rhTPO than in those who did not (43.8% vs. 10.4%, p=0.001; 50.0% vs. 20.8%, p=0.006). The mean number of megakaryocyte per bone marrow slide was higher in subjects receiving rhTPO than those who did not (9.7±3.1 vs. 2.6±4.2, p=0.002) after three months. The percentage of nucleated erythroid cells in bone marrow was also higher in subjects receiving rhTPO after three months (22.2±13.2% vs. 13.6±13.9% in those who did not receive rhTPO; p=0.007). The percentage of reticulocytes in peripheral blood was higher in subjects receiving rhTPO (1.9±1.4% vs. 0.7±0.4% in those who did not receive rhTPO; p=0.001) after three months. Myeloid percentage in bone marrow did not differ at any time points (3, 6, or 9 months). The need for platelet transfusion was lower in subjects receiving rhTPO (transfusion interval: 13.8±14.3 vs. 6.9±5.2 and 26.3±28.9 vs. 15.7±13.1 days in those who did not receive rhTPO during the first three and six months, respectively; P=0.004, P=0.03). The need for red cell transfusion was also lower in subjects receiving rhTPO (interval: 32.5±22.0 vs. 11.9±7.2 days and 50.4±27.9 vs. 23.9±20.1 days during the first three and six months, respectively; p=0.001 P=0.009). Time to independence from platelet transfusion was significantly shorter in subjects receiving rhTPO (99.9±49.9 vs. 156.3±14.5 days in those who did not receive rhTPO; p=0.01).

Conclusion
rhTPO improves hematologic response and promotes bone marrow recovery in SAA patients receiving IST.

Disclosures:
No relevant conflicts of interest to declare.
A NUP98-HOXD13 Leukemic Fusion Gene Leads To Aberrant Actin Localization In Dysplastic Megakaryocytes
Authors: Caudell David L. , Okyere Benjamin , Cawley Jacob , Puthiyaveetil Abdul Gafoor A. , Heid Bettina 
Published: October 2019

Abstract
Myelodysplastic syndrome (MDS) is a hematopoietic malignancy characterized by peripheral cytopenias due to bone marrow (BM) failure. Megakarypoiesis, megakaryocyte (MK) production, and platelet release are impaired in in some cases of MDS. Patients often have fewer, but larger circulating platelets, which have abnormal demarcation membrane systems (DMS); the DMS, which determines the number and size of platelets released, is dependent on actin formation. However, the precise role of actin during megakaryopoiesis is poorly understood. Transgenic mice that express the fusion gene NUP98-HOXD13 (NHD13) is a model for MDS and have dysplastic MKs in BM, and macro platelets in circulation. We hypothesized that expression of NHD13 disrupts actin localization during megakaryopoiesis resulting in reduced platelet release and macro platelet formation. To test the hypothesis, BM from wild type (WT) and NHD13 mice were flushed and cultured in media supplemented with Thrombopoietin for 5 days. Following in vitro propagation, MKs were harvested over a discontinuous gradient for downstream experiments. Sternums were also fixed in paraformaldehyde, stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 BM contained many dysplastic MKs. Harvested MKs and BM cores from one femur were processed and analyzed by transmission electron microscopy (TEM) and the ultrastructural properties of the DMS detailed. TEM of MKs showed NHD13 leads to formation of an irregular DMS along with abnormal distribution of unusually large granules in MK cytoplasm. Cultured MKs were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin and Myosin IIa and the cytoskeleton visualized by confocal microscopy. WT MKs in vitro had two phenotypes: (1) MKs with myosin and actin evenly dispersed in the cytoplasm and (2) MK with actin predominantly in the periphery of the cytoplasm. In contrast, transgenic MKs displayed only the former phenotype suggesting that actin localization is impaired in NHD13 MKs. Finally, MKs were stimulated with estrogen and adhered to fibrinogen matrices to determine their proplatelet formation functionality. Our results showed impaired proplatelets formation in NHD13 MKs. These data suggest that expression of NHD13 leads to aberrant actin localization leading to dysplastic MK differentiation and macro platelet release. Understanding molecular mechanisms of abnormal megakaryopoiesis in MDS is important as many MDS patients die of hemorrhagic complications. Further studies using this model system will provide a platform for translational research and should reveal potential therapeutic targets in MDS, leading to improved patient care/survival.

Disclosures:
No relevant conflicts of interest to declare.
Expression Pattern Of Mirnas Is Not Predictive For Poor Prognosis In BCR-ABL1-Like Childhood Acute Lymphoblastic Leukemia
Authors: Akbari Moqadam Farhad , Lange-Turenhout Ellen , Veer Arian van der , Marchante João R.M. , Pieters Rob , Den Boer Monique L. 
Published: October 2019

Abstract
Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease in which the 5-years event-free survival rates are currently above 80%. We recently identified a poor prognostic set of patients within unclassified BCP-ALL patients. These patients have a high risk of relapse and a gene expression profile similar to BCR-ABL1-positive ALL cases. This so-called BCR-ABL1-like (or Ph-like) group of patients showed similar high frequencies of deletion in B-cell development genes (den Boer et al, Lancet Oncology, 2009 and Mullighan et al. New Eng J Med, 2009). Here, we investigate the miRNA expression pattern in BCR-ABL1-like ALL. Further, we addressed whether altered expression levels of discriminative miRNAs of BCR-ABL1-like ALL has prognostic importance. MiRNA expression levels of leukemic cells of 91 newly identified children with BCP-ALL: i.e. 15 BCR-ABL1-like, 14 BCR-ABL1-positive, 9 TCF3-rearranged, 14 hyperdiploid (>50 chromosomes), and 15 B-other patients (negative for BCR-ABL1-like ALL and other known genetic lesions) were measured by Taqman® Array Human miRNA cards (TLDA cards, Applied Biosystems). Using the R statistics program (R-2.15, release June 2012), the Limma package was applied to compare the expression levels of miRNAs between the groups. Each genetic subtype was compared to the remaining cases to identify subtype-specific miRNAs.
Our data revealed that children with ETV6-RUNX1-positive, hyperdiploid, TCF3-rearranged and MLL-rearranged ALL demonstrate a subtype-specific miRNA signature. In contrast, BCR-ABL1-positive and BCR-ABL1-like cases showed a more variable miRNA expression pattern, resulting in 2 clusters of patients. The majority of the children with BCR-ABL1-like ALL (11 out of 15) showed a miRNA expression pattern different from that of BCR-ABL1-negative genetic subtypes of pediatric BCP-ALL and cluster with 43% of BCR-ABL1-positive patients (6 out of 14, cluster-I). The remaining 8 BCR-ABL1-positive and 4 BCR-ABL1-like cases showed more heterogeneous expression patterns (cluster-II). The prognosis of patients in both clusters, however, did not differ. Similarly, there was no significant difference in IKZF1, PAX5, JAK2 or CDKN2A/B status between these two clusters. Top-10 most differentially expressed miRNAs of the children with BCR-ABL1-like ALL were miR-324-5p, miR-345, miR-190, miR-130a, miR-545, miR-152, miR-103, miR-191, miR-197 and miR-101. None of these discriminative miRNAs was predictive for clinical outcome of BCR-ABL1-like patients. In conclusion, our data suggest that a miRNA signature is not suitable to dissect good and poor prognostic BCR-ABL1-like cases.

Disclosures:
No relevant conflicts of interest to declare.
Circulating t(14;18)+ Cells As Predictive Markers Of Follicular Lymphoma Development
Authors: Roulland Sandrine , Morgado Ester , Kelly Rachel , Sungalee Jocelyne Stephanie , Colombat Philippe , Solal-Celigny Philippe , Salles Gilles Andre , Vineis Paolo , Nadel Bertrand 
Published: October 2019

Abstract

Background
The (14;18) translocation constitutes both a genetic hallmark and critical early event in the natural history of Follicular Lymphoma (FL). However, t(14;18) is also detectable in the blood of otherwise healthy persons, and the relationship with progression to disease remains unclear. A key gap in our understanding is whether such t(14;18)+ cells in healthy individuals constitute committed FL precursors, and if high t(14;18) frequencies in blood represent a suitable predictive biomarker of FL development.

Methods
Among 520,000 healthy participants enrolled in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort, we identified 100 subjects who developed FL 2-161 months after enrolment. Blinded Prediagnostic blood from these and 218 controls were screened for t(14;18) by sensitive PCR-based assays. Results were subsequently validated on an independent cohort (65 cases, 128 controls). Clonal relationships between t(14;18) cells and FL biopsy were also assessed by molecular backtracking of paired prediagnostic/tumor samples.

Results
Clonal analysis of t(14;18) junctions in paired prediagnostic blood vs. tumor demonstrated that progression to FL occurred from t(14;18)+ committed precursors. Furthermore, we show that the prevalence of t(14;18) was significantly higher in individuals that subsequently develop FL compared to the controls (56% vs. 28.9%, p<10-3). Using a ROC curve analysis, we estimated that an optimal frequency value for predicting FL development was 10-4. Findings from the discovery cohort were fully validated in an independent cohort and when combined, we confirmed the optimal frequency value of 10-4. Overall, the combined analysis reports a 23-fold higher risk of subsequent FL associated with the 10-4 threshold (OR: 23.17; 95% CI, 9.98-67.31; P=7.7x10-9). Remarkably, stratified analyses on time to diagnosis revealed that risk estimates remained high and significant up to 15 years before diagnosis.

Conclusion
We demonstrated that high t(14;18) frequencies in blood from healthy individuals discriminates commitment to FL development up to 15 years before diagnosis and define the first predictive biomarker for FL. Providing they represent bona fide precursors, this molecular blood-based biomarker could therefore be of great value in identifying groups of at-risk individuals and their detailed geno-phenotypic characterization should provide important insights into the factors and kinetics of events driving early FL progression.

Disclosures:
No relevant conflicts of interest to declare.