bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detailed Mapping of the Binding Site within Platelet Glycoprotein Ibα for Leukocyte Integrin Mac-1 (αMβ2).
Authors: Han Qiang , Peng Yuandong , Chen Zhiping , Ustinov Valentin A. , Ugarova Tatiana P. , Plow Edward F. , Simon Daniel I. , López José A. 
Published: October 2019

Abstract
We have shown that the platelet counter-receptor for integrin Mac-1 (αMβ2, CD11b/CD18) is glycoprotein (GP) Ibα, the largest component of the GP Ib-IX-V complex, better known for its role in mediating platelet attachment to sites of vessel wall injury. The GP Ibα-Mac-1 interaction mediates the firm adhesion of leukocytes on platelet thrombi, enabling their subsequent transmigration into the vessel wall. Our previous studies showed that the I-domain of Mac-1 binds the C-terminal flanking sequences (Phe201–Gly268) of GP Ibα, as demonstrated by the fact that binding is almost completely inhibited by the I domain ligands fibrinogen and heparin, the I-domain monoclonal antibody LPM19c, and the GP Ibα-specific monoclonal antibody AP1. The latter antibody is of interest because it also inhibits the binding of von Willebrand factor and thrombin. We have mapped the AP1 epitope to a 10-amino acid sequence spanning Arg218 to Tyr228. In the current investigation, we constructed a series of cell lines expressing mutants of human GP Ibα, either by replacement of the human sequence with the corresponding dog sequence (dog GP Ibα does not bind human Mac-1) or by targeted mutagenesis, and tested their ability to bind the recombinant Mac-1 I domain. The GP Ibα region Phe201-Asn223 was crucial for Mac-1 binding, with residues Arg218, Asp222 and Asn223 playing vital roles. In addition, a peptide containing the AP1 epitope (Leu214-Val229) bound Mac-1 I-domain specifically and saturably. Peptide binding was blocked by LPM19c and soluble GP Ibα, and by the M2 peptide, which corresponds to the GP Ibα-binding site in the Mac-1 I domain (Phe201-Lys217). Peptide binding was also blocked by an antibody against the M2 sequence. The AP1 peptide inhibited the attachment of GP Ib-IX-V complex-expressing CHO cells to immobilized Mac-1 I domain, and the adhesion of THP-1 cells—a monocytoid line expressing Mac-1—to immobilized GP Ibα. This peptide did not inhibit attachment of the cells VWF surfaces, or thrombin-induced platelet aggregation. In summary, we have defined the GP Ibα sequence Arg214 to Val229 as a critical binding site for Mac-1. Because a peptide corresponding to this region inhibits GP Ibα binding to Mac-1 but blocks neither platelet adhesion to immobilized VWF nor thrombin-induced platelet aggregation, it has potential to guide the development of agents that will specifically inhibit leukocyte-platelet complexes that promote vascular inflammation.
Interaction of a SNARE-Associated Protein, Snapin with the Cytoplasmic Domain of the Platelet Membrane Glycoprotein V.
Authors: Nozoe Akiko , Fujimoto Tetsuro-Takahiro , Fujimura Kingo 
Published: October 2019

Abstract
Glycoprotein V (GPV) is a subunit of the platelet GPIb-IX-V receptor for von Willebrand factor (vWF) and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in platelet activation is still unknown. It is reported that the GPV knockout mouse did not suffer from a bleeding syndrome, and the GPV-deficient platelets showed normal GPIb expression and vWF-mediated adhesion. One report showed an increased sensitivity of these platelets to activation by low concentrations of thrombin, suggesting that GPV may act as a negative modulator of the thrombin response. Another showed that GPV-deficient platelets exhibited defective adhesion to a collagen-coated surface, suggesting GPV binds to collagen and participates in platelet adhesion and aggregation responses to this agonist. To define the signaling pathway through GPV, we searched for proteins that bind to the GPV. We preformed a yeast two-hybrid screening in a human placenta library, using the sequence for GPV cytoplasmic domain as a bait. As a result, we isolated 2 independent positive clones, and the both clones encoded a partial and a full length sequence of the same protein, named snapin (SNAPAP), which was previously reported as one of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)-associated proteins. By RT-PCR analysis, snapin mRNA was detected in megakaryocyte cell lines and peripheral blood platelets. When the snapin was expressed as a GFP-fusin protein in K562 cells, which expressed intrinsic GPIb-IX-V complex, the protein localized at perinuclear region and cytosol. By immunoprecipitation-western blotting study, GPV was co-immunoprecipitated with GFP-snapin from the lysates of these cells by anti-GFP antibody, whereas GPV was not detected in the precipitates by a control antibody. Previous reports showed that snapin is a binding protein of SNAP-25 (synaptosome-associated protein-25) and SNAP-23, and influences the release response of neurons and chromaffin cells by enhancing the synaptotagmin binding to the SNARE complex. Therefore, the novel interaction between GPV and snapin, identified in this study, might be involved in the intracellular membrane-fusion events and the granule release reaction during the platelet activation.
The Interaction of Platelet Integrin αIIbβ3 and ICln Is Activation Dependent.
Authors: Larkin Deirdre , Moran Niamh 
Published: October 2019

Abstract
The major platelet integrin αIIbβ3 plays a critical role in thrombus formation and has been a major target for anti-thrombotic therapy. The precise molecular mechanism of activation of the platelet integrin αIIbβ3 is not understood although a critical role for the conserved αIIb-integrin cytoplasmic motif, KxGFFKR, has been identified. We have recently shown, using a synthetic KVGFFKR peptide probe, evidence for a direct and specific interaction between the platelet integrin αIIbβ3 and a protein involved in regulatory volume decrease, ICln. ICln is a volume regulating chloride channel protein that is capable of forming a pore structure within the membrane. Previously we demonstrated by surface plasmon resonance that purified αIIbβ3 binds to His-tagged recombinant ICln (His-ICln). We now show that purified αIIbβ3 binds to His-ICln in protein pulldown assays using a western blot detection system. In addition, His-ICln selectively pulls down endogenous αIIbβ3 from platelet lysates, but in an activation dependent manner. These comparisons were carried out in lysates from resting platelets and platelets activated with U46619 (10μM), TRAP (5μM) and thrombin (0.2U/ml) for 3 minutes at 37°C, unstirred. Reduced integrin precipitation was observed in lysates from platelets treated with acyclovir (100μM), a known pharmacological inhibitor of ICln, consistent with an activation dependent interaction.
As the channel function of ICln is nucleotide sensitive we set out to further identify if ICln regulates the activation of αIIbβ3 or if the integrin contributes to the activation of ICln. Low dose nucleotides were found to effect platelet function. Gel-filtered platelets treated with ATP (2nM) and apyrase (1U/ml) showed an increase in PAC-1 binding that could be inhibited with acyclovir (1mM). U46619 stimulation caused an increase in PAC-1 binding and enhanced platelet aggregation in the presence of ATP/apyrase that could also be inhibited by acyclovir in a dose-dependent manner (10μM–1mM). Thus, activation of ICln by low dose nucleotides can induce αIIbβ3 activation without further stimulus. However maximum αIIbβ3 activation is observed upon stimulation of both αIIbβ3 and ICln. The presence of an ICln inhibitor, acyclovir, interferes with platelet aggregation and integrin activation with and without specific ICln stimulation, suggesting a co-dependence between αIIbβ3 and ICln to obtain maximum platelet activation. Taken together with results of the protein- protein interaction studies this data suggests an activation-dependent, functional interaction between the volume regulating ICln protein and the platelet integrin αIIbβ3.
Dose-Adjusted EPOCH-Rituximab Is Highly Effective in the GCB and ABC Subtypes of Untreated Diffuse Large B-Cell Lymphoma.
Authors: Wilson Wyndham H. , Dunleavy Kieron , Pittaluga Stefania , Grant Nicole , Steinberg Seth , Raffeld Mark , Chabner Bruce , Grossbard Michael , Jaffe Elaine , Staudt Louis , Janik John 
Published: October 2019

Abstract
Diffuse large B-cell lymphoma (DLBCL) is subdivided by microarray into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. These subtypes have distinct pathways of lymphomagenesis and outcome with 5-year survival of 59% and 31%, respectively, using CHOP. A molecular predictor showed high tumor proliferation and the ABC subtype are associated with a poor outcome. We showed DA-EPOCH overcomes the adverse effects of high tumor proliferation (BLOOD 99:2685, 2002) and rituximab overcomes the adverse effects of BCL-2, which is associated with the ABC subtype (Proc Am Soc Heme 102:abstr 356, 2003). To assess DA-EPOCH-R with G-CSF in GCB and ABC subtypes, we employed a validated immunohistochemistry (IHC) algorithm (BLOOD: 103:275, 2004). GCB was defined as CD10+ or BCL-6+ and MUM1-. ABC was defined as CD10- and BCL-6- or CD10-, BCL-6+ and MUM1+. Eligibility includes de novo DLBCL, no prior chemotherapy, any PS, HIV- and stage I thymic DLBCL > 5 cm and all stage II-IV. Characteristics of 77 enrolled pts include median (range) age 48 (12-85) and PS 1 (0-3); stage III/IV 48 (62%); and LDH > nl 42 (55%); extranodal sites >1 27 (35%); and HI/H IPI 26 (34%). Including all 77 pts, response is CR/CRu 72 (94%). At the median (range) follow-up of 28 (4-60) mos, PFS is 82% and OS is 83% (Fig 1). DFS is 87% at 28 mos with no relapses beyond 18 mos. PFS at 28 mos according to IPI risk is 95% for 0-2 factors and 54% for 3-5 factors. Using IHC, 44 pts were subdivided in GCB (35) and ABC (9) subtypes. The proportion of the ABC subtype vs. GCS is low and likely due to incomplete IHC for the ABC subtype. Specifically, 16 cases that were CD10- did not have adequate BCL-6 and/or MUM1 IHC for categorization. PFS at 28 mos for the GCB and ABC subgroups were 84% and 62%, respectively (Fig 2). DA-EPOCH-R employs a pharmacodynamic design where doses of doxorubicin, etoposide and cyclophosphamide are adjusted (i.e. normalized) to achieve neutrophil nadirs < 500/mm3. PK analysis shows that clearance of doxorubicin and etoposide varies by up to 1 log and that dose-adjustment compensates for these differences. This approach increases dose intensity in pts with high drug clearance while limiting toxicity. Based on 451 cycles, the target nadir ANC < 500/mm3 was achieved on 277 (61%) cycles but with 67 (15%) episodes of fever/neutropenia. Non-hematological toxicity was low and there were 2 treatment related deaths. These results suggest DA-EPOCH-R is highly effective in all subtypes of de novo DLBCL. A phase III study comparing R-CHOP and DA-EPOCH-R with microarray analysis of tumor biopsies is under development.
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Normal Prion Protein Colocalises with the Tetraspanin CD63 in Cultured Human Erythroblasts.
Authors: Griffiths Rebecca E. , Anstee David J. 
Published: October 2019

Abstract
Detailed information regarding the intracellular distribution and trafficking of prion protein (PrP) in human haemopoietic cells is lacking. In order to address this deficiency we have investigated the expression of PrP in cultured erythroid cells. CD34+ cells derived from peripheral blood were cultured in the presence of IL3, SCF and EPO and aliquots removed from the cultures at different time points for examination by confocal microscopy with monoclonal antibodies. PrP was demonstrated in the plasma membrane and, by colocalisation with organelle-specific markers, in Golgi, ER and lysosomes at all time points. PrP mRNA expression was profiled throughout the culture by QRT-PCR. PrP mRNA expression is high in the immature cells and decreases throughout the culture. PrP mRNA expression is different from erythroid specific protein expression profiles. Monoclonal antibodies to PrP and another GPI-linked protein DAF (CD55) showed distinct, non-overlapping, staining in the plasma membrane suggesting they are located in different microdomains. CD63 is a widely expressed tetraspanin thought to function in trafficking membrane proteins between the plasma membrane and intracellular organelles. All CD63 staining observed in cultured erythroid cells was colocalised with PrP. A similar concordance was not observed with CD63 and DAF. These results disclose a previously unrecognised property of PrP and suggest a general mechanism whereby PrP is recycled within cells. This mechanism may be relevant to the pathogenesis of transmissible spongiform encephalopathies.
E47 Binds to PU.1 Inhibiting Its Ability To Bind DNA and Activate Gene Expression.
Authors: Dahl Richard , Iyer Sangeeta R. , Simon M. Celeste 
Published: October 2019

Abstract
The transciption factor PU.1 is required for the development of macrophages, granulocytes, and B lymphocytes. Additionally its protein concentration in multipotential hematopoietic progenitor cells regulates cell fate decisions with high levels of PU.1 directing myeloid cell fate acquisition and low levels directing B cell fate acquisition. Potentially high levels of PU.1 are required for myeloid development in order to overcome repressive effects of B cell lineage specific factors. The essential B cell factor BSAP (Pax-5) was shown to associate with PU.1 and repress its transactivation activity. Here we investigate whether two other essential B cell factors, E2A and EBF could affect PU.1 activity. We observed that the E2A component, E47, but not EBF could associate with PU.1. Additionally E47 could repress PU.1 dependent transactivation of the M-CSFR promoter and that this repression is mediated by inhibiting PU.1 DNA binding. Exogenous E47 expression in an IL-3 dependent multipotential cell line could block PU.1 induced differentiation and expression of endogenous M-CSF receptor protein. Our data suggests that high PU.1 concentration is required for myeloid development in order to overcome repressive actions of other lineage specific transcriptional regulators such as E47.
Severely Impaired Erythropoiesis in Mice Lacking Mediator Subunit Med1/TRAP220.
Authors: Borggrefe Tilman , Waskow Claudia , Roeder Robert G. , Stumpf Melanie 
Published: October 2019

Abstract
GATA transcription factors are essential for diverse developmental processes including erythropoiesis. Med1/TRAP220 was shown to interact with GATA factors in vitro (Crawford et al. (2002): J Biol Chem. 277(5):3585-92.). Med1/TRAP220 is part of the multiprotein Mediator complex that forms a bridge between gene-specific regulatory proteins and the RNA polymerase II (Borggrefe (2004): Research Adv. in Biol. Chem. (2: 9-20), Bourbon et al. (2004) Mol. Cell 14(5):553-7). Mediator subunit Med1/TRAP220 deficient mice die during mid-embryonic development (day 11.5dpc) due to defects in placental, cardiac and hepatic development (Ito et al. (2000): Mol Cell. 5 (4):683–93., Landles et al. (2004): Mol Endocrinol. 17(12):2418–35). In order to analyze hematopoiesis in TRAP220-deficient embryos, AGMs (aorta-gonad-mesonephros) and Yolk-sacs were isolated from day 10.5 dpc embryos and analyzed by FACS and methylcellulose assays. TRAP220-deficient embryos contain c-Kit positive cells and BFU-E (Burst forming units erythrocytes) activity. However, CFU-E (Colony-forming units erythrocytes) activity was 10-fold reduced. This correlated with severe decrease of the CD71/Ter119 double-positive cells, the CFU-E containing fraction, in FACS-analysis.
Our data shows that Mediator subunit Med1/TRAP220 is required for erythropoiesis just before the CFU-E stage. Because GATA factors affect early erythropoiesis and because MED1/TRAP220 binds to GATA factors in vitro, our data suggest that the Mediator of transcriptional regulation forms the bridge between GATA factors and the RNA polymerase II machinery.
Lyn Kinase Supports Primary Erythroid Progenitor Cell Development.
Authors: Karur Vinit , Vincent Bethany , Lowell Clifford , Wojchowski Don M. 
Published: October 2019

Abstract
Several lines of investigation have implicated Lyn as an important positive effector of red cell development: Deregulated Src kinases promote erythroleukemia, and Lyn is the predominant Src kinase of erythroid cells; as critical erythropoietic factors, Kit and the Epo receptor each stimulate Lyn kinase; and in an insightful set of investigations in J2E cells, Lyn has been shown to be required for Epo-dependent late erythroid development. Based on these considerations, adult bone marrow-derived primary erythroid progenitor cells from Lyn −/− mice presently were assessed for their ex vivo growth, survival and differentiation potentials. Lyn −/− erythroid progenitors expanded efficiently in serum-free media, and showed essentially wild-type Epo dose-dependent proliferative responsiveness. When transferred to BSA/insulin/transferrin differentiation medium, however, Lyn −/− erythroid progenitor cells clearly faltered in their development to Ter119-high, CD71-low erythroblasts. For these Lyn −/− cells, annexin-V binding studies revealed that this defect was associated, in part, with a stage-specific loss in survival potential. Interestingly, however, this defect was not Epo-dose dependent. In addition, MACS-isolated Kit-positive early erythroid progenitor cells prepared from Lyn −/− mice (unlike preparations from wild-type mice) failed to support synergistic effects of SCF-plus-Epo in 3HdT incorporation assays. In response to phenylhydrazine, Lyn −/− mice exhibited expanded erythroid progenitor cell pools (including BFUe and CFUe), and this hyper-expansion may occur in response to the compromised survival of late Lyn −/− erythroblasts. Analyses of pp60-Src expression revealed elevated levels of activated PY416-Src specifically in Lyn −/− EPC, a finding that is consistent with the activation of apparent compensatory mechanisms. In contrast, no significant changes in the levels of GATA1 or other assessed erythroid defining factors were detected. In response to phenylhydrazine, Lyn −/− mice showed ≥2-fold enhanced splenomegaly, as well as enhanced frequencies of BFUe, CFUe and Ter119(+) cells. Overall, these studies in primary erythroid progenitor cells from Lyn −/− mice reveal a previously undiscovered positive role for Lyn as a late-stage specific positive effector of erythroid cell survival, and regulator of Epo receptor and Kit co-signaling.
Extracellular Nucleoside Diphosphate Kinase NDPK/Nm23 Protein Isoforms A and B Support Erythropoiesis and Have a Burst-Promoting Activity.
Authors: Berneman Zwi N. , Willems Roel , Nijs Griet , Lenjou Marc , Van Bockstaele Dirk R. 
Published: October 2019

Abstract
We previously demonstrated the in vitro hematopoietic effects of different nucleoside diphosphate kinase NDPK/Nm23 proteins, i.e. increase of burst-forming units erythroid (BFU-E) and decrease of colony-forming units macrophage (CFU-M) (Willems R et al. Experimental Hematology2002;30: 640–648). The effect was especially pronounced in the marrows that had a particular low BFU-E count. Since these experiments were performed under optimal erythroid growth factor stimulation including adequately high concentrations of erythropoietin (Epo) and stem cell factor (SCF 100 ng/mL), subtle effects of NDPK/Nm23 proteins on erythropoiesis could have been missed. In the present series of experiments on 7 normal bone marrow mononuclear cell suspensions, we deliberately created suboptimal in vitro conditions for early (BFU-E) erythropoiesis: i.e. serum free conditions with a limited number of cytokines (only Epo and SCF) and a low concentration of burst-promoting activity (SCF at 5 ng/ml). The results show that addition of NDPK A/Nm23-H1 and NDPK B/Nm23- H2 (but not of NDPK C) at high (40 μg/mL), but not low, extracellular concentration is sustaining and improving in vitro erythropoiesis in a statistically significant manner (P<0.05). Red cell colonies increased by a factor of 1.9 and 1.4 following addition of NDPK A/Nm23-H1 and NDPK B/Nm23- H2 respectively. Especially with NDPK A/Nm23-H1, this approaches levels attained under optimal conditions for erythropoiesis, i.e. at SCF concentration of 100 ng/mL, where a 2.1-fold increase was seen, as compared to cultures with low concentrations of SCF (5 ng/mL) without addition of extracellular NDPK/Nm23. In cultures without Epo no erythroid colonies were seen, even following addition of NDPK A/Nm23-H1 or NDPK B/Nm23-H2. This study strongly suggests that NDPK/Nm23 has a burst-promoting activity. The fairly high concentrations of NDPK/Nm23 constitutively present in plasma supports the physiological role of this protein in stimulating early erythropoiesis. Of the (known) cytokines or proteins with burst-promoting activity (eg. SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), NDPK/Nm23 (this study)), only SCF and NDPK/Nm23 are produced in a constitutive fashion, strongly supporting their role in the steady-state support of early erythropoiesis.
Serum Erythropoietin and Aging: A Longitudinal Analysis.
Authors: Ershler William B. , Shan Sheng , McKelvey Julie , Artz Andrew S. , Denduluri Neelima , Tecson Josephine , Taub Dennis D. , Brant Larry , Ferrucci Luigi , Longo Dan L. 
Published: October 2019

Abstract
There are conflicting data regarding expected changes in serum erythropoietin (EPO) with advancing age, primarily because prior reports have been based upon cross-sectional analyses of diverse populations. To examine changes in serum EPO with age, we reviewed the clinical history, hematological parameters and serum EPO levels of 143 participants in the Baltimore Longitudinal Study on Aging (BLSA) who were studied at 1 – 2 year intervals for eight to thirty years. None had anemia at the time of the first visit (by WHO criteria: <12g/dL for women; or <13 g/dL for men) but it developed in 10 (7%) over the period of evaluation. An additional 20 individuals (14%) had a fall in hemoglobin of 1.5 g/dL or more, sustained over at least two visits, and for the purpose of this analysis were also considered anemic. EPO levels rose significantly with age for the group as a whole, and the slope of the rise was found to be greater for those who did not have associated diabetes or hypertension. Subjects who developed anemia but did not have hypertension or diabetes had the greatest slope in EPO rise over time. In contrast, those who had, or developed hypertension or diabetes during the period of follow-up within the BLSA, had significantly higher initial EPO levels and a reduced EPO slope over time. The increase in serum EPO with aging may be compensating for increased RBC turnover or increased EPO resistance of red cell precursors. We suspect that with very advanced age, or in those with compromised renal function (e.g., diabetes or hypertension), the compensatory mechanism becomes inadequate.
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