bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Factor XI Receptor on Collagen Activated Platelets Is gC1qR/P33.
Authors: Baglia Frank A. , Bradford Harlan R. , Colman Robert W. , Peerschke Ellinor I.B. , Ghebrehiwet Berhane , Walsh Peter N. 
Published: April 2020

Abstract
Factor XI (FXI) binds to high affinity sites on the surface of thrombin-stimulated platelets where it is efficiently activated by thrombin. We have previously shown that FXI binding to thrombin-activated platelets is mediated by residues in the FXI Apple 3 (A3) domain interacting with leucine-rich repeat sequences within the NH2-terminal globular domain of glycoprotein (GP) Ibα (J. Biol. Chem.277:1662–1668, 2002; J. Biol. Chem.279:45470–45476, 2004; J. Biol. Chem.279:49323–49329, 2004). The cell surface receptor protein gC1qR/P33, expressed on collagen-stimulated platelets, binds FXII and high molecular weight kininogen (HK). We now provide evidence that the FXI binding site on collagen-activated platelets is gC1qR/P33: 1) two anti-gC1qR/P33 mAbs, 60.11 and 74.5.2, inhibited FXI binding to collagen-stimulated platelets whereas SZ-2 (a monoclonal antibody directed against GPIbα) does not; 2) by surface plasmon resonance, FXI was bound specifically to gC1qR/P33 in a Zn2+-dependent fashion (Kdapp ~5 nM); 3) soluble gC1qR/P33 inhibited the binding of FXI to collagen-stimulated platelets with an IC50 ~10 nM. Optimal binding of FXI to collagen-activated platelets (~1,500 binding sites per platelet; Kd ~10 nM) was achieved in the absence of HK. A synthetic peptide comprising residues Asn 235-Arg 266 within the FXI A3 domain inhibited FXI binding to collagen-activated platelets with a Ki ~10 nM which is identical to the Kd for FXI binding to platelets, whereas A1, A2 and A4 peptides had no effect. We then investigated whether gC1qR/P33 could promote activation of FXI by thrombin or FXIIa. In the presence of Zn2+ and Ca2+ ions, and the absence of HK, FXI was activated by thrombin in the presence of gC1qR/P33 at rates comparable to those seen in the presence of thrombin-activated platelets, whereas rates of FXI activation by FXIIa in the presence of gC1qR/P33 were 8-fold lower. Thus, thrombin is the preferred activator of FXI bound to gC1qR/P33. We conclude that whereas FXI binds to GPIbα on thrombin-activated platelets, it binds to gC1qR/P33 on collagen-activated platelets. Finally, we show here that both collagen and vonWillebrand factor can inhibit FXI binding to GPIbα, suggesting that on collagen-activated platelets gC1qR serves as an alternative FXI receptor that promotes FXI activation by thrombin.
Primary Acute Myeloid Leukemic Blasts Display Constitutive Serine 585 Phosphorylation within the 14-3-3 Binding Motif of the GM-CSF/IL-3 Receptor Required for Survival Signalling.
Authors: Thomas Daniel , Powell Jason , Lee Szu-Hee , Lopez Angel F. , Mark Guthridge 
Published: April 2020

Abstract
Acute myeloid leukemia (AML) is characterised by defects in the regulation of hematopoietic cell survival, differentiation and proliferation. IL-3 and GM-CSF exert potent survival and proliferative effects on a range of myeloid progenitor cells. Previously we have identified a novel cytoplasmic motif of the human GM-CSF/IL-3 receptor beta chain responsible for coordinating survival and proliferative responses in primary hematopoietic cells. The motif contains a serine residue (585) that is phosphorylated in response to cytokine and couples to the PI3K/AKT pathway via the 14-3-3 adaptor protein and is required for cell survival (Guthridge et al, Molecular Cell6:99, 2000;Blood103:820, 2004). An adjacent tyrosine residue (577) within the motif is phosphorylated via Jak2 and associates with proliferative function. We investigated the phosphorylation status of the beta chain motif in response to GM-CSF stimulation in blasts cells from 12 patients with AML, and in blood monocytes from 6 healthy volunteers. Leukemic samples consisted of &gt;99% blasts after buffy coat separation according to cytospin morphology. All leukemic blasts and blood monocyte samples expressed alpha and beta chain by flow cytometry. The phosphorylation status of residues 577 and 585 were examined utilising beta chain phospho-specific antibodies, after stimulation of AML blasts or blood monocytes with GM-CSF and beta chain immunoprecipitation. Human monocyte preparations all showed inducible serine 585 phosphorylation and tyrosine 577 phosphorylation. In contrast AML blasts displayed strong constitutive serine 585 phosphorylation in 10 out of 12 patients. No discernible tyrosine 577 phosphorylation in the absence of cytokine was observed for the AML samples. Beta chain phosphorylation pattern was independent of FAB classification. Inhibition of Jak2 did not prevent serine 585 phosphorylation of the receptor. Constitutive Akt activation was observed in AML blasts. The data provides evidence for dysregulation of a novel GM-CSF/IL-3 receptor mediated survival pathway involving serine/threonine kinases, 14-3-3 and Akt that is active in primary AML blasts in the majority of patients. This data would suggest that dysregulated activation of serine/threonine kinases and/or phosphatases at the receptor level may contribute to leukemogenesis.
Validation of the Prognostic Significance of the Disease Specific Gene CLLU1 in Chronic Lymphocytic Leukemia.
Authors: Josefsson Par , Jurlander Jesper , Geisler Christian H. , Pedersen Lone B. , Bach Lone , Andersen Mette K. , Leffers Henrik , Buhl Anne Mette 
Published: April 2020

Abstract
We have recently identified and cloned a novel disease specific gene CLLU1, with a strong prognostic significance in a small test cohort of CLL patients (ASH 2004, #770). To validate this finding in a larger series, 176 newly diagnosed, previously untreated CLL patients referred to Rigshospitalet, Copenhagen in 1991–1999, were investigated. Clinical characteristics of the population were: 116 stage A patients (66 %), median age 69.9 years, 98 males (56%), median follow up 59 months. The end points for the statistical analysis were overall survival and time to initiation of treatment. At time of analysis 91 (52%) patients had died and 104 (59%) had commenced treatment. Frozen patient samples, taken at time of diagnosis, were investigated for CLLU1 expression, as well as for mutational status, CD38 expression, ZAP-70 expression and cytogenetic aberrations. CLLU1 expression was assayed by QRT-PCR using the cDNA1 splice variant, and expressed as fold upregulation above the CLLU1 level in normal B-cells. CD38 and ZAP-70 analysis was performed by flow cytometry, using a 20 % cut-off level. Cytogenetic analysis was performed by FISH. The previously reported (ASH 2004, #770) median upregulation of CLLU1 in CLL cells was accurately reproduced and confirmed (25.5-fold (N = 176) vs. 27.7-fold (N = 59) in test population). Also in accordance with the pilot study, segregation of the patients based on the median CLLU1 expression level divided the population in two groups with significantly different times to initiation of treatment and borderline significant difference in overall survival. However, further analysis found an optimal cut-off level at 40-fold upregulation of CLLU1 expression; use of this cut-off demonstrated a highly significant difference in overall survival for patients with upregulated expression of CLLU1 (p = 0.0023). The median overall survival was 60.2 months for patients with CLLU1 expression above 40-fold compared to 100.8 months for the group with lower expression level. Likewise the time to initiation of first treatment was significantly shorter in the high-level expression group (p = 0.0018, median time to first treatment 9.3 months vs. 54.9 months). Furthermore, CLLU1 expression was upregulated in all poor prognostic subgroups investigated: Binet stage B or C, IgVH unmutated, unfavorable cytogenetics, high CD38 expression and high ZAP-70 expression. Our new study confirms that CLLU1 is a strong prognostic factor in CLL, comparable to other established prognostic markers. The exclusive upregulation of CLLU1 expression in CLL suggests a putative role for CLLU1 in the pathogenesis of CLL, and makes this gene a strong candidate for future gene-targeted treatment strategies.
Figure Figure
17p Deletion Predicts for Inferior Overall Survival after Fludarabine - Based First Line Therapy in Chronic Lymphocytic Leukemia: First Analysis of Genetics in the CLL4 Trial of the GCLLSG.
Authors: Stilgenbauer Stephan , Kröber Alexander , Busch Raymonde , Eichhorst Barabara , Kienle Dirk , Winkler Dirk , Hopfinger Georg , Lichter Peter , Emmerich Bertold , Hallek Michael , Döhner Hartmut ,   The GCLLSG
Published: April 2020

Abstract
The CLL4 trial evaluated first line treatment with fludarabine (F) or F + cyclophosphamide (FC) among 375 CLL patients up to 65 years enrolled between 1999 and 2003. FC resulted in a higher overall response rate (94% vs. 83%; P=0.001), longer median progression free survival (PFS) (48 vs. 20 months (m); P=0.001), but no difference in overall survival (OS) (Eichhorst et al., ASH 2003 and submitted). At enrollment, genomic aberrations and the VH mutation status have been analyzed for 307 and 280 cases thus far. The most frequent genomic aberrations were 13q-: 50.3%, 13q- single: 34.1%, 11q-: 20.3%, +12q: 13.7%, 6q-: 8.7%, 14q-: 6.1%, and 17p-: 4.9%. VH was mutated in 33.9% and unmutated in 66.1% of cases. The aberrations 13q- and 13q- single were more frequently observed in the VH mutated subgroup (45.4% vs. 32.7% and 35.5% vs. 18.8%, respectively), while 11q- (26.9% vs. 12.2%) and 6q- (14.2 vs. 1.7%) were more common in the VH unmutated subgroup. Clinical outcome was evaluated in subgroups defined by genomic aberrations and VH status for both treatment arms combined. In univariate analyses, significant associations were found for the following parameters: the overall response rate was significantly lower in the subgroup with 17p- (53.8% vs. 89.6%, P=0.001), the median PFS was significantly shorter in the subgroups with 11q- (17.4 vs. 26.8 m, P=0.044), 14q- (14.5 vs. 24.5 m, P=0.018), and 17p- (11.0 vs. 24.1 m, P=0.002), and the median OS was significantly shorter in the subgroup with 17p- (15.9 m vs. not reached, 75% survival at 43.8 m, P&lt;0.001, Figure 1). Multivariate analysis was performed including the treatment arms, specific genomic aberrations, and the VH mutation status as possible prognostic factors: the parameters with significant adverse impact were F monotherapy (HR 1.70, 95%CI 1.12-2.58, P=0.013) and 17p- (HR 3.67, 95%CI 1.66-8.14, P=0.001) regarding PFS, but only 17p- (HR 7.32, 95%CI 2.44-21.90, P&lt;0.001) regarding OS. In conclusion, this first analysis of the prospective CLL4 trial shows that 17p- CLL is characterized by a short OS after F-based first line therapy. In future trials, alternative primary treatment strategies of proven efficacy in 17p- CLL such as alemtuzumab should be considered for these patients.
Figure Figure
Early Results from LRF CLL4: A UK Multicenter Randomized Trial.
Authors: Catovsky Daniel , Richards Susan , Hillmen Peter 
Published: April 2020

Abstract
On behalf of the UK NCRI CLL Trials Group and the Adult Leukaemia Working Party. The CLL4 trial opened in 2/99 and closed in 10/04. 783 previously untreated patients were randomized and 6 were excluded. 12% of patients were entered from outside the UK (Argentina, Ireland, Italy, New Zealand and Russia). Median follow-up is 21 months. The male:female ratio was 2.8:1 and the distribution by Binet stages was A progressive 25%, B 45% and C 30%. One third of cases were aged &lt;60 years, one third 70 years or over. Patients were randomized between chlorambucil (10mg/m2 x 7 days)(n=387) and fludarabine alone (FDR)(n=194) or in combination with cyclophosphamide (FC) (n=196). FDR and FC were given intravenously (IV) in the first 2 years (FDR 25mg/m2 x 5 days and FDR 25mg/m2 plus cyclophosphamide 250mg/m2 for 3 days). Early in 2001 oral FDR became available and the protocol was modified to allow its use: FDR 40mg/m2 x 5 days and FDR 24mg/m2 plus cyclo 150mg/m2, both for 5 days. Differences between the IV and oral formulations will be presented elsewhere in this meeting (Hillmen et al). Responses were assessed by bone marrow biopsies. Responses to treatment are summarized in the table below in 661 patients with available data. Within each age group treatment affected response (CR+nodPR v PR/NR/PD: p=0.0003 for &lt;60 years, p&lt;0.0002 for 60–69 years and p=0.002 for ≥70 years) with highest responses with FC and lowest with chlorambucil. The toxicities profile showed more neutropenia with FC (55%) than FDR (40%) and chlorambucil (29%) and more hospitalisations with FC and FDR. However, and as reported in ASH 2004, there were more hemolytic anemias after chlorambucil (13%) than with FDR (10%) and FC (4%). There was more nausea/vomiting and alopecia with FC than with the other regimens. Progression free survival (PFS) up to 10/04 showed fewer events with FC (O/E 0.5) than FDR (O/E 1.1) and chlorambucil (O/E 1.3); FDR+FC v Chl p&lt;0.00005; FC v FDR p&lt;0.0005. PFS at 3 years is chlorambucil 23%, FDR 31% and FC 62%. Analysis of survival thus far showed no differences between the three regimens. We conclude that the combination of FC is associated with a greater quality of responses, more myelotoxicity and less hemolytic anemia than FDR and chlorambucil and it could become the new standard regimen for CLL upon which future regimes will build. CLL4 has generated a wealth of prognostic information: FISH, VH mutations, expression of CD38 and ZAP-70 which will allow a better characterization of risk groups and their response to first-line therapy. Some of these analyses will be presented in this meeting (Oscier et al).
Chlorambucil FDR FC * within the first year and not assessable for response No. patients 309 176 176 Complete Response (CR) 8% 15% 37.5% Nodular partial response (NPR) 18.5% 27% 22% Partial response (PR) 42.5% 35% 31% No response/progressive disease (NR/PD) 28% 18.5% 6.5% Died early* 3% 4.5% 3%
Shigatoxin Triggers Thrombotic Thrombocytopenic Purpura in Genetically Susceptible ADAMTS13-Deficient Mice.
Authors: Motto David G. , Chauhan Anil K. , Zhu Guojing , Homeister Jonathon , Lamb Colin B. , Desch Karl C. , Tsai Han-Mou , Wagner Denisa D. , Ginsburg David 
Published: April 2020

Abstract
The life threatening disease TTP is associated with ultra-large von Willebrand Factor multimers (UL-VWF) in the circulation due to inherited or acquired deficiency of the ADAMTS13 metalloprotease. Here we show that ADAMTS13-deficient mice generated by gene targeting are viable and exhibit normal survival through 2 years of age. Despite the absence of VWF-cleaving protease activity (&lt;1% of normal), wild-type and ADAMTS13-deficient plasma exhibit identical VWF multimer distributions, and Adamts13−/− mice develop spontaneous TTP at an extremely low rate (2 cases out of 358 mice). However, intravital microscopy demonstrated that VWF-mediated platelet-endothelial interactions are significantly prolonged in Adamts13−/− mice. These observations suggested that additional environmental triggers and/or genetic modifying factors may be required to bring about TTP in the setting of ADAMTS13 deficiency. To address the effect of VWF level on development of TTP, Adamts13−/− mice were crossed to mice of the CASA/Rk strain which exhibit markedly elevated plasma VWF levels. Resulting CASA/Adamts13−/− mice demonstrated plasma VWF ranging from 150% to 600% of C57BL/6 controls, and we found that 21% of these mice were thrombocytopenic at baseline (vs. 0% of controls). Introduction of the CASA/Rk genetic background also resulted in the appearance of UL-VWF in CASA/Adamts13−/− mice, further prolonged VWF-mediated platelet-endothelial cell interactions, increased the rate of spontaneous TTP, and markedly decreased survival. Challenge of CASA/Adamts13−/− mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP, with thrombocytopenia, profound microangiopathic hemolytic anemia, and platelet- and VWF-thrombi seen in multiple organs. Surprisingly, we observed no correlation between plasma VWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from VWF. Our laboratory is pursuing the identification of these genes which may provide insight into the pathogenesis and treatment of TTP in humans. Finally, our data also suggest that microbial-derived toxins, or possibly other sources of endothelial injury, may be among the key factors required to trigger acute TTP in the setting of ADAMTS13 deficiency.
The Prothrombotic Polymorphism Prothrombin G20210A Is an Ancestral Mutation That Arose in Caucasians Approximately 20,000 Years Ago.
Authors: Zivelin Ariella , Mor-Cohen Ronit , Kovalsky Victoria , Conard Jacqueline , Peyvandi Flora , Pabinger Ingrid , Bertina Rogier , Peyvandi Ferial , Emmerich Joseph , Seligsohn Uri 
Published: April 2020

Abstract
The prothrombotic prothrombin G20210A polymorphism is associated with increased plasma levels of prothrombin, it confers a 2–5 fold increase in the risk of venous thromboembolism and is prevalent in Caucasians. We previously showed that prothrombin G20210A originated in a single ancestral founder (Zivelin et al. Blood92:1119, 1998). In this study we estimated the age of the G20210A polymorphism by assessing linkage disequilibrium (LD) between A20210 and 9 microsatellites markers or 5 SNPs located upstream and downstream of the prothrombin gene (Figure). The study group included 93 unrelated A20210 homozygotes (21 Israelis, 19 French, 17 Italians, 17 Austrians, 16 Dutch and 3 Germans) and 63 unrelated healthy Caucasian homozygotes for G20210. Calculation of the mutation age was done by the DMLE+ program (Reeve and Rannala, Bioinformatics18:894, 2002), which allows Bayesian inference of mutation age based on observed LD at multiple genetic markers. Assuming 20 years as a generation, analysis of the microsatellite markers yielded an estimated age of 19,750 years (95% CI 16,200–29,500), and analysis of the SNPs yielded an estimated age of 20,500 years (95% CI of 14,700–27,950). We previously estimated by a different method (Risch et al. Nature Gen9:152, 1995) that the age of factor V Leiden, also prevalent only in Caucasians, is 21,000–34,000 years (Zivelin et al. Blood89:397, 1997). In the present study we recalculated the age of factor V Leiden by the DMLE+ program in 117 homozygous for factor V Leiden and 160 controls and obtained a value of 20,600 years (95% CI of 15,400–29,800). These data suggest that both relatively common prothrombotic polymorphisms occurred about 20,000 years before our time following the evolutionary divergence of Africans from Non-Africans and of Caucasians from Oriental subpopulations.
Figure Figure
A Platelet Shedding Defect in Mice Lacking the Basic Helix-Loop-Helix Transcription Factor Scl.
Authors: McCormack Matthew P. , Hall Mark A. , Schoenwaelder Simone M. , Jackson Shaun P. , Ellis Sarah , Jane Stephen M. , Curtis David J. 
Published: April 2020

Abstract
Transcriptional regulation of megakaryocyte maturation including platelet shedding is poorly characterised. One potential regulator is Scl, a basic helix-loop-helix transcription factor essential for hematopoietic specification in embryonic development. Scl is expressed in megakaryocytes and erythroid cells, forming DNA binding complexes with other transcription factors including GATA-1. To overcome embryonic lethality of Scl knockout mice, we have generated conditional Scl knockout mice to allow deletion of Scl in cell lineages including megakaryocytes. Scl-null adult mice have mild thrombocytopenia (730±90x109/L compared with 1400±220x109/L in control mice) despite normal numbers of megakaryocytes. Megakaryocyte ploidy was increased in the absence of Scl (modal ploidy 32n compared with 16n in control mice) indicating that Scl is not required for megakaryocyte development and endomitosis. Platelet survival studies demonstrated a normal platelet half-life of Scl-null platelets, suggesting that thrombocytopenia was due to impaired platelet production. This defect was more pronounced in the setting of pharmacologic administration of thrombopoietin (Tpo) or 5-fluorouracil, with the platelet count in Scl-null mice remaining static despite appropriate increases in megakaryocyte number and ploidy. Ultrastructural studies of Scl-null megakaryocytes were performed to examine the function of Scl in later stages of megakaryocyte maturation. Transmission electron microscopy demonstrated markedly disordered platelet demarcation membranes and a lack of platelet granules, defects reminiscent of GATA-1-null megakaryocytes. Gene expression analyses of FACS-sorted Scl-null megakaryocytes demonstrated a 5-fold reduction in β-1 tubulin, a proposed target gene of GATA-1 and NF-E2 in megakaryocytes. Consistent with the role of this gene in maintaining platelet discoid shape, Scl-null platelets were abnormally round. The similarity of defects of GATA-1- and Scl-null mice implies that these genes act together to regulate platelet shedding and structure.
CTCF and Nucleophosmin Mediate Long Distance Cyclin D1 Deregulation in t(11;14) B Cell Malignancies.
Authors: Epner Elliot M. , Wang Jin , Liu Hui , Clurman Bruce 
Published: April 2020

Abstract
Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). Cyclin D1 is known not to be expressed in normal lymphocytes, yet is deregulated by IgH translocations and insertions. The molecular mechanisms of long distance cyclin D1 activation by IgH regulatory elements in B cell malignancies have been obscure. Chromatin immunoprecipitation (ChIP) assays demonstrated the presence of the CCCTC-binding factor (CTCF) at both the cyclin D1 promoter and 3′ IgH regulatory regions only in cyclin D1 expressing malignant B cell lines containing IgH translocations or insertions. The nucleolar protein nucleophosmin (B23; NPM), a newly identified CTCF interaction partner, exhibited a similar distribution in ChIP assays. Combined FISH/immunofluorescence labelling studies have shown the presence of the cyclin D1 loci colocalized with CTCF and nucleophosmin at the nucleolus in MCL cell lines and patient samples, “Knockdown” of NPM by shRNA shows a specific growth arrest in MCL and MM cell lines containing the t(11;14) and cell context dependent apoptosis. This growth arrest is not mediated by changes in cyclin D1 RNA or protein levels. Tethering of the cyclin D1 promoter to the 3′Cα IgH regulatory regions at the nucleolar membrane by CTCF/nucleophosmin allows spatial juxtaposition of the cyclin D1 promoter and 3′IgH regulatory elements and provides a mechanism for long distance cyclin D1 deregulation. Nucleophosmin is also identified as a target for novel therapies in t(11;14) B cell malignancies.