bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endothelial Cell-Derived Microparticles in the Pathogenesis of Chemotherapy-Associated Hypercoagulability.
Authors: Lechner Daniel , Kollars Marietta , Eichinger Sabine , Kyrle Paul Alexander , Weltermann Ansgar 
Published: October 2019

Abstract
Background: Cisplatin-based chemotherapy is a risk factor of venous thromboembolism in cancer patients. The underlying pathogenesis remains unclear. We hypothesized an apoptotic effect of cisplatin on endothelial cells (EC) inducing a release of small membrane vesicles, so-called microparticles (MP) which are known to cause hemostasis activation.
Objectives:
To quantify the release of MP from EC following administration of cisplatin and to investigate MP-associated procoagulant mechanisms.
Methods: Two EC lines (HUVEC, HMVEC-L) were exposed to cisplatin (1, 2.5, 5, 10, and 20 μM) for up to 120 h. Cell viability was assessed by quantification of mitochondrial dehydrogenase activity, counts and procoagulant activity of MP were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen levels were determined by ELISA.
Results: EC viability decreased in a dose- and time-dependent manner and was accompanied by an increasing release of MP into culture media (maximum: HUVEC + 544%; HMVEC-L + 1738%). In parallel, procoagulant activity of media increased by up to 150% (HUVEC) and 493% (HMVEC-L), respectively. The procoagulant activity was almost abolished by annexin V but was not suppressed by a monoclonal TF-antibody. TF antigen levels on MP were persistently low even at high cisplatin concentrations.
Conclusion: At pharmacologically relevant concentrations, cisplatin induced a marked release of procoagulant MP from EC. Negatively charged phospholipids but not TF on MP were decisive for total thrombin generation. Further studies are warranted to investigate the cisplatin-induced release of EC-derived MP in vivo.
Regulation of Tissue Factor by NF-kB Transcription Factor p50 Is Essential for the Pathogeneses of Deep Vein Thrombosis and Arterial Restenosis.
Authors: Geng Jian-Guo , Wang Jian-Guo , Mackman Nigel , Slungaard Arne , Huo Yuqing , Key Nigel S. 
Published: October 2019

Abstract
NF-kB transcription factors regulate the expression of tissue factor (TF), a principal initiator for the coagulation cascade. Dominant among the five cellular members of NF-kB transcription factors is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a 350-dalton antagonist that targets reduced cysteine62 of p50 for inhibition of NF-kB activation) and genetic deletion of p50 potently attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. The direct binding of p50/p65 heterodimer to the TF-kB site in human TF promoter was demonstrated by p50 and p65 antibody ‘supershift’ using electrophoretic mobility shift assay and immunoprecipitation of the promoter of the human TF gene from chromatins of TNF-a-stimulated human umbilical vein endothelial cells. Andro-treated and p50 null mice both exhibited suppressed TF expression, blunted fibrin deposition, reduced venous thrombosis, and decreased neointimal hyperplasia. Blockade of TF activity by an anti-murine TF antibody also attenuated venous thrombosis and neointimal proliferation in vivo. Our findings thus indicate that NF-kB transcription factor p50 critically regulates TF activity in the pathogeneses of deep vein thrombosis and arterial restenosis, and suggest that specific inhibitors of p50, such as Andro, have the potential to be therapeutically valuable for preventing and perhaps treating arterial and venous thrombosis.
Heparin-PF4 Antibodies Are Frequent after Vascular Surgery, but Not a Frequent Cause of Graft Thrombosis or Thrombocytopenia.
Authors: Alexy Tamas , Tucker Sony , Boyle Soames , Rochanda Leanne , Hood Douglas , Rowe Vincent L. , Weaver Fred A. , Liebman Howard A. 
Published: October 2019

Abstract
Surgical intervention for atherosclerotic disease of the aortoiliac and femoral-popliteal arteries has significantly reduced the incidence and complications of peripheral ischemia. However, surgical correction of obstructive arterial lesions can be complicated by either early graft failure due to acute thrombosis or late stenosis of the vascular bypass graft. Despite improvements in surgical technique, early graft failure is still observed in up to 10% of patients and up to 30 % can subsequently develop a graft stenosis. The etiology of early graft closure is less clearly understood. Anatomic or structural defects in the new graft may result in early thrombosis. Underlying prothrombotic conditions have also been suggested as contributing factors to early graft thrombosis. Recently, we have observed recurrent thrombotic events occurring in vascular grafts in two patients who were subsequently diagnosed with the syndrome of Heparin-Induced Thrombocytopenia. Because of these events we undertook an IRB approved, prospective evaluation of HIT antibodies in patients undergoing vascular surgery. Patients undergoing infra-inguinal bypass surgery for atherosclerotic vascular disease were enrolled after informed consent. Blood samples were obtained the day before surgery, day of hospital discharge, at days 14 and 28 after surgery. Platelet counts were obtained on the day of discharge and on post-surgery day 14. Plasma samples were assayed for PFA-heparin antibodies by ELISA (Stago Diagnostica) and platelet activating antibodies by platelet aggregometry. All assays were performed in duplicate.
Seventy-nine patients were enrolled. Six (7.7%) patients provided only the pre-surgical blood specimen and only 68 (86%) patients provided all four blood specimens. All but 5 patients reported previous heparin exposure. Of the 73 patients in which post-surgical blood samples were taken, 28 (38%) became ELISA positive for PF4-Heparin antibodies. When tested for platelet activating antibodies, 7 (10%) were positive. All patients who developed a positive ELISA had antibody detected by day 14. Four patients tested positive by ELISA prior to surgery, with 3/4 having positive aggregation assays. One ELISA negative patient developed early graft closure. No patient developed thrombocytopenia or a 50% or greater decrease in platelet count. Patients undergoing vascular surgery frequently develop PF4-Heparin antibodies of which up to 10% patients have platelet activating antibodies. However, both thrombocytopenia and vascular graft thrombosis due to HIT is a rare event.
Hepatocytes Ingest Longterm Refrigerated Platelets: A Novel Platelet Clearance Mechanism.
Authors: Rumjantseva Viktoria , Josefsson Emma C. , Wandall Hans , Hartwig John H. , Stossel Thomas P. , Hoffmeister Karin M. 
Published: October 2019

Abstract
We previously reported that the lectin domain of αMβ2 receptors on hepatic macrophages mediates rapid clearance of washed murine platelets transfused after refrigeration for 2 hours, recognizing exposed βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered platelet GPIbα molecules and that the same receptors elicit phagocytosis of refrigerated human platelets human macrophages in vitro. A platelet-associated galactosyltransferase catalyzes the covering of βGlcNAc residues with galactose in the presence of UDP-galactose, thereby blocking clearance of cold mouse platelets in vivo and phagocytosis of human platelets in vitro. These intriguing findings contradicted earlier evidence that refrigeration of human platelets procured for transfusion only promotes their rapid clearance after prolonged (>8h) incubation and also are inconsistent with the well-known recognition system for exposed galactose residues through asialoglycoprotein (ASGP) receptors. Reconciling these contradictions, we report that the absence of plasma during storage accounts for the differences in time of exposure to cold to promote clearance and that mouse platelets cold-stored in plasma also only clear rapidly after long-term (48h) storage. We also found that hepatic clearance of long-term cold-stored (LTCS) mouse platelets occurs in hepatocytes. Streptavidin-POD staining revealed abundant LTCS biotinylated platelets in hepatocyte phagosomes. Furthermore, cells of the hepatocyte HepG2 line avidly ingest fluorescently-labeled LTCS human platelets (7-fold above the baseline of room-temperature-stored platelets), as evidenced by flow cytometry, fluorescent microscopy and by time-laps video microscopy. Long-term cold storage increases by ~1.7-fold platelet binding of the galactose-specific lectin RCA I, implying that with long-term cold storage, exposed galactose residues cluster sufficiently to induce recognition by hepatocyte ASGPR receptors. The results define a new clearance mechanism, representing the first example of blood cell removal by a non-myeloid cell. Since we find that human platelets also express a cell surface sialotransferase that adds sialic acid to galactose residues, we suggest that a combination of sialylation and glactosylation, achievable by addition of sugar substrates alone, might accommodate long-term cold storage of platelets for transfusion.
Inhibition of Thrombin-Induced Platelet Aggregation Using Human Single Chain Fv Antibodies Specific for TREM-Like Transcript-1.
Authors: Washington A. Valance , Giomarelli Barbara , Chisholm Maia M. , Quigley Laura , McMahon James B. , Mori Toshiyuki , McVicar Daniel W. 
Published: October 2019

Abstract
Platelets play a duel role in our cardiovascular system. They are important in maintaining our vascular integrity. They also facilitate the progression of cardiovascular diseases (CVD) such as arteriosclerosis. In more severe cases, displaced platelet aggregates can lead to myocardial infarction or stroke. Because of their prominent role, anti-platelet therapies are used to reduce mortality associated with CVD. However, the use of these therapies is sometimes associated with bleeding complications, thrombocytopenia or non-responsiveness and the search for new therapeutic targets continues. Some of the recent treatment strategies include molecules that target platelet membrane receptors. The success shown by this class of therapeutics has prompted us to evaluate the recently characterized murine platelet receptor, TREM like transcript 1 (TLT-1), as a therapeutic target. Murine studies suggest that TLT-1 is a platelet specific protein found in α-granules of platelets and megakaryocytes. Here we asked if human TLT-1 was also linage specific and if platelet aggregation could be modulated through interaction with TLT-1 specific antibodies. We evaluated human TLT-1 (hTLT-1) for pattern of expression in the peripheral blood and we found that hTLT-1, like its mouse counterpart, is platelet specific. To address TLT-1 function we panned a naïve human library of single chain fragments (scFvs) with a TLT-1 extracellular domain/human fc fusion protein to produce monoclonal antibodies specific for hTLT-1. We isolated six scFv clones using ELISA to detect reactivity to the fusion protein and confirmed their specificity by flow cytometry on cells transfected with TLT-1 and purified human platelets. These scFvs were then tested to see if they would disrupt or enhance platelet aggregation. Here we demonstrate, for the first time, inhibition of thrombin induced platelet activation using scFv that are specific for TLT-1. Taken together these results suggest TLT-1 is a potential target for therapeutic intervention of cardiovascular disease.
Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.
Authors: Zorca Suzana M. , Josefsson Emma C. , Rumjantseva Viktoria , Hartwig John H. , Hoffmeister Karin M. 
Published: October 2019

Abstract
We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation.
The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.
Intravenous (IV) Iron Supplementation in Patients with Chemotherapy-Induced Anemia (CIA) Receiving Darbepoetin alfa Every 3 Weeks (Q3W): Iron Parameters in a Randomized Controlled Trial.
Authors: Lerchenmueller Christian , Husseini Faress , Gaede Bernd , Mossman Tony , Suto Tamas , Vanderbroek An 
Published: October 2019

Abstract
Background and Aim: The role of IV iron supplementation during treatment with erythropoiesis-stimulating agents (ESAs) in patients with CIA is of increasing interest as a possible means of improving response. This randomized, open-label, multicenter study was designed to evaluate the safety and efficacy of IV iron vs standard practice in CIA patients receiving darbepoetin alfa. Interim efficacy analyses showed a higher response rate for darbepoetin alfa with IV iron compared to darbepoetin alfa with standard iron practice with no difference in the safety profile between the treatment arms (Vanderbroek et al, EHA 2006). Iron parameters are reported here.
Methods: Eligible patients were diagnosed with a non-myeloid malignancy and had CIA with a baseline hemoglobin (Hb) value < 11g/dL. All patients received darbepoetin alfa 500 mcg administered Q3W with the SureClick™ prefilled autoinjector. Patients were randomized 1:1 to IV iron 200 mg (single dose Q3W at the same time as darbepoetin alfa or in 2 doses of 100 mg within 3 weeks) or standard practice (oral iron or no iron). Randomization was stratified by tumor type and baseline Hb category (< 10 or ≥10 g/dL).
Results: A total of 400 patients were randomized. Mean (SD) age of the study population was 61.4 (11.5) years; range, 20–86. Sixty percent (n=241) of participants were women; 28% (n=114) had lung or gynecological tumors; and 52% (n=208) had a baseline Hb value ≥10 g/dL. In the interim analysis population (n=196), the mean (SD) weekly dose of IV iron was 64.8 (6.6) mg in the IV iron group (n=100). In the standard practice group, 28 of 96 patients (29%) received oral iron and 2 (2%) received IV iron (these patients were analyzed as randomized). Mean (standard error) serum ferritin concentrations and percent transferrin saturation (TSAT) in the 2 groups from baseline (BL) to end of study (EOS) are shown in the figure.
Conclusions: The combination of darbepoetin alfa Q3W and IV iron appeared to be associated with a trend toward increased mean serum ferritin levels compared to the standard practice control arm. In contrast, mean TSAT surprisingly appeared to be similar in the 2 groups for most of the study period, perhaps suggesting that TSAT is influenced by other factors. Iron management appears to be an important factor in the response to ESAs and the findings presented here suggest the need for additional exploration into iron uptake and demand in cancer patients treated with darbepoetin alfa.
Serum Ferritin Concentration Serum Ferritin Concentration Transferrin Saturation (%) Transferrin Saturation (%)
Characterisation of the Laminin 10/11 Binding Site on the Lutheran Glycoprotein Suggests a Novel Type of Protein-Protein Interaction.
Authors: Mankelow Tosti J. , Burton Nicholas , Stefansdottir Fanney O. , Spring Frances A. , Parsons Stephen F. , Gilstring C. Fredrik , Brady R. Leo , Narla Mohandas , Chasis Joel Anne , Anstee David J. 
Published: October 2019

Abstract
The Lutheran glycoprotein is a five domain member of the immunoglobulin superfamily (IgSF) with a wide tissue distribution. It is a ligand for Laminin isoforms containing the alpha5 chain (Laminins 10 and 11). Lutheran glycoprotein on erythrocytes is thought to play a role in vasocclusive events that are a serious cause of morbidity in sickle cell anaemia. We have investigated the molecular basis of the Lutheran:Laminin 10/11 interaction. Lutheran binding to Laminin 10/11 is pH and salt dependent suggesting the interaction is influenced by surface charge. Since Laminins are known to contain areas of positive charge that are of importance in binding to other ligands (heparin, alpha-dystroglycan), a molecular model of Lutheran glycoprotein was constructed to identify surface exposed areas of negatively charged aspartic and glutamic acid residues. Selected residues were mutated to alanine and the mutant proteins examined for binding to Laminin 10/11 using ELISA and Surface Plasmon Resonance. Mutations E309A and D310A greatly reduced binding to Laminin 10/11 while D312A completely abolished binding. The Lutheran model predicts a rod-like structure with a flexible hinge region of 6–8 residues between the 2nd and 3rd IgSF domains. Residues E309, D310 and D312 are located on domain 3 proximal to the hinge region. Mutations (H235P, and delta 233–235) within the hinge region also abolished Laminin binding showing the hinge region to be essential for ligand interaction. Electron tomography on recombinant Lutheran-Fc chimeric protein bound to Laminin 10/11 suggested Lutheran glycoprotein bends at the hinge region to expose the critical negatively charged residues on domain 3 and thereby allow Laminin binding. These data suggest Lutheran-Laminin 10/11 interaction is a novel type of protein:protein interaction and provide a foundation for further investigation of its biological significance.
Comprehensive Analysis of Murine and Human RBC Ghost Proteomes.
Authors: Wooden Jason M. , Finney Greg , MacCoss Michael , Gilligan Diana M. 
Published: October 2019

Abstract
Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. Mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton. Genetic knock-out of various components of this apparatus has led to the creation of mouse models which have contributed significantly to our understanding of these disorders in humans. However, the mouse and human RBC protein complements have not been comprehensively compared.
Using newly developed proteomic methodology, we conducted a peptide level ‘bottom-up’ analysis of the normal mouse and human RBC ghost (i.e., RBC membrane skeleton and associated proteins). RBCs were purified using cellulose acetate chromatography from whole blood taken from three genetically identical mice and two hematologically normal yet genetically diverse humans. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin and analyzed by shotgun proteomics using microcapillary liquid chromatography coupled with tandem mass spectrometry. In total, 400 and 491 unique proteins were identified in human samples A and B respectively while 469 proteins were found in common across the three mouse samples. All previously identified membrane skeleton proteins were found in the human and mouse samples. Likewise, well-known RBC membrane proteins were represented. Of interest, a surprising number of proteins were found associated with the RBC ghost involved in processes such as protein repair (15–20), protein degradation (30–43), oxidative stress response (4–6), Ras oncogene biology (28–30), and glycolysis (3–6). Collectively, the two human samples represented 544 unique proteins. These results affirm the usefulness of inherited anemia mouse models given the observed conservation of membrane skeleton components and the inherent challenges with doing normal versus diseased RBC analysis in humans due to genetic variation.
Genomic Composition of Howell-Jolly Bodies.
Authors: Gantt Nicole M. , Walker Bob , Lee Y. Terry , Jiang Yuan , Staker Pamela , Oneal Patricia A. , Bhanu Natarajan V. , Meltzer Paul S. , Miller Jeffery L. 
Published: October 2019

Abstract
Erythrocyte inclusion bodies that are now commonly referred to as Howell-Jolly Bodies (HJB) were originally discovered over 100 years ago by microscopy. These nuclear fragments are observed in the erythrocytes of patients lacking splenic function, but their precise genomic origins are vague. Here we report studies of the genetic content of HJB using a molecular cytogenetic method known as Comparative Genomic Hybridization (CGH). Peripheral blood samples from six splenectomized human donors were studied. All donors had undergone splenectomies prior to this study for diagnostic and therapeutic indications related to underlying neoplastic and autoimmune disease. Ultrasound testing was used to rule out the presence of accessory splenic tissue, and peripheral blood smears confirmed the presence of HJB. Nucleated cells were removed by density gradients and leukocyte filtration for use as patient-matched controls. DNA was isolated from the leukocytes and HJB-containing erythrocytes. The presence of high molecular weight HJB DNA was confirmed in each sample using gel electrophoresis. As expected, it was not possible to obtain sufficient quantities of DNA from the control erythrocytes of healthy individuals with normal splenic function. CGH was performed using equivalent amounts of reference genomic DNA co-hybridized with HJB DNA or matched donor leukocyte DNA. Agilent oligonucleotide CGH arrays were hybridized and scanned according to the manufacturer’s instructions. The control hybridizations (genomic DNA isolated from donor leukocytes) demonstrated standard patterns consistent with the donor’s karyotype. In contrast, the HJB hybridization signals demonstrated an unusual pattern of centromeric-to-telomeric enrichment on each chromatid of each chromosome. The HJB hybridization signals also increased as a function of distance from the centromere according to the chromatid size. The enhanced signal relative to centromeric distance varied considerably between chromosomes, but was remarkably consistent between donors. These data suggest Howell-Jolly Bodies contain a non-random distribution of centromere-depleted chromatin generated after DNA double-strand breakage during erythroid differentiation.