bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in Hemoglobin and Hematocrit May Predict Mortality in Patients with Acute Coronary Syndromes.
Authors: Alirhayim Zaid , Qureshi Waqas , Donthireddy Vijaya , Hassan Syed , Khalid Fatima 
Published: October 2019

Abstract
Abstract 3171

Introduction:
Changes in plasma volume, the intravascular portion of the extracellular fluid volume, can be estimated by measuring changes in the levels of hemoglobin and hematocrit in the blood. In addition to hemoglobin & hematocrit levels, we are also able to use standard dilution techniques with radiolabeled albumin to accurately measure plasma volume changes. It is not known if plasma volume changes influence outcomes in patients with acute coronary syndromes. The aim of this study was to evaluate the effects of plasma volume changes in patients presenting with the acute coronary syndromes.

Methods:
Consecutive patients presenting to a single tertiary care center from January 2001 to December 2010 with non ST elevation myocardial infarction (NSTEMI) or ST elevation myocardial infarction (STEMI) were enrolled. Admission hemoglobin (Hbpre) & hematocrit (Hctpre) and discharge hemoglobin (Hbpost) and hematocrit levels (Hctpost) were obtained. Plasma volume changes were measured and a validated formula (ΔPV = ((Hbpre/Hbpost) × (100-Hctpost/100-Hctpre)-1) × 100%) was used to calculate the changes in plasma volumes. A detailed chart review was performed to collect information about baseline variables such as age, gender, hypertension, diabetes, hyperlipidemia, smoking status, and congestive heart failure. The Framingham Risk score was also calculated for each individual. Survival analysis was carried out for plasma volume changes of -20% - 0%, 0 – 20%, and ≥20%. Mortality data was collected from the social security death index for the first 60 days post-discharge.

Results:
A total of 9770 patients with confirmed NSTEMI or STEMI (mean age 61.8 ± 4.8 years, 48.8% women) were included in the final analysis. Mean pre admission hemoglobin (Hbpre) was 10.2 ± 1.4 g/dl and post admission hemoglobin (Hbpost) was 10.4 ± 1.3 g/dl. Change in plasma volume, ΔPV, was categorized into one of four categories, with 131 (1.3%) ≤20%, 6126 (62.7%) -20% - 0%, 3393 (34.7%) 0 – 20%, and 120 (1.2%) ≥20%. There were 509 deaths within 60 days of discharge. Change in plasma volume was found to be an independent predictor of mortality (HR 5.71; 95% CI 4.75 – 6.86, p = 0.0001) in a Cox proportional hazard model. Most of the deaths occurred during the first thirty days as demonstrated by the Kaplan – Meier's survival curve (Figure 1). Receiver operating curve showed an area under the curve of 0.876 for changes in plasma volume.

Conclusion:
This study shows that hemoglobin and hematocrit, although simple tests, can provide important prognostic information strongly predictive of short term mortality in patients with acute coronary syndromes. Further studies are required to see if monitoring of plasma volume and correction with pharmacological agents such as diuretics may lead to better outcomes.

Disclosures:
No relevant conflicts of interest to declare.
Temporal Differences in Terminal Erythroid Differentiation of Human CD34+ Cells Derived From Cord Blood and Adult Peripheral Blood
Authors: Jaffray Julie , Hu Jingping , Li Jie , An Xiuli , Narla Mohandas 
Published: October 2019

Abstract
Abstract 3200
Purified CD34+ cells derived from either cord blood (CB) or peripheral blood (PB) are currently being used to further our molecular and mechanistic understanding of human terminal erythroid differentiation. What is unclear is whether there are differences in the kinetics of terminal erythroid differentiation of CD34+ cells from these two sources. In the present study, we document that terminal differentiation in cultured CD34+ cells purified from peripheral blood is faster than that of CD34+ cells from cord blood. For these studies, we optimized an 18 day, three phase, in vitro culture system using CD34+ cells to obtain enucleated reticulocytes. In this system, proerythroblasts are generated starting at day 6 which further differentiate during the duration of culture to eventually generate reticulocytes. Based on the expression of various membrane surface markers, we used flow cytometry to quantitatively monitor terminal erythroid differentiation from proerythroblasts to enucleated reticulocytes during culture. The three surface markers, alpha-4 integrin, band 3 and CD36 enabled us to clearly distinguish between all distinct stages of terminal erythroid differentiation – proerythroblasts, early- and late- basophilic erythroblasts, polychromatic and orthochromatic erythroblasts. These analyses enabled us to show that CD34+ cells purified and cultured from peripheral blood underwent terminal erythroid differentiation at a faster rate than CD34+ cells from cord blood. Terminal erythroid differentiation in cord blood cultures was delayed on an average of 2 to 3 days compared to peripheral blood. For example, the surface protein expression pattern seen on days 11–12 of cell culture of peripheral CD34+ cells was not achieved in cord blood cultures until day 14. This delay in terminal differentiation was also reflected by increased extents of enucleation in peripheral blood cultures compared to cord blood (culture day 12: 33% enucleation in PB and 7% in CB and on day 14: 45% enucleation in PB and 19% in CB). These findings have enabled us to document significant differences between the kinetics of terminal erythroid differentiation of CD34+ cells derived from fetal cord and adult peripheral blood. While at the present time we do not have a mechanistic understanding for this difference, we are currently exploring if the observed differences may be related to differences in cell cycle dynamics between fetal and adult erythropopiesis.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Method Using Extracted Human Neutrophil Antigens From HNA Gene-Transfected Cell Lines for Detection of Antibodies Against Human Neutrophil Antigens
Authors: Onodera Rie , Nakamura Kazuhiro , Taniguchi Kikuyo , Kurita Emi , Hiraoka Asako , Yasui Kaduta , Matsuyama Nobuki , Hirayama Fumiya , Kobayashi Masao 
Published: October 2019

Abstract
Abstract 3272
Antibodies against Human Neutrophil Antigen (anti-HNA antibodies) have been implicated in immune neutropenia and transfusion-related acute lung injury. Those have been usually analyzed by Granulocyte indirect immunofluorescence test by flow-cytometry (GIFT-FCM), which requires fresh blood for panel neutrophils that cannot be preserved more than a few hours. Furthermore, specificity for anti-HNA antibodies cannot be easily identified by GIFT-FCM because of several antigens on membrane of panel neutrophil. In this study, we have developed a novel method designated as KY-Luminex Method, which was modified from Monoclonal antibody-specific immobilization of granulocyte antigens.
For KY-Luminex Method, extracted human neutrophil antigens (eHNAs) were established from gene-transfected cell lines (KY-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b) that express HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b, respectively. These eHNAs were immobilized by monoclonal antibodies on microspheres (eHNA-microspheres). Sera were tested to the eHNA-microspheres in wells of a 96-well microplate, respectively. The reactivities of the sera were analyzed by Luminex100.
From May 2010 to March 2012, sera were obtained from 101 patients with autoimmune neutropenia in children. The age distribution was from 2 months to 72 months. Sixty-three patients were females and 38 were males.
As shown in Table, the sera from 74 patients showed positive reaction both in standard GIFT-FCM and KY-Luminex method. The sera from 11 patients showed positive in GIFT-FCM, and negative in KY-Luminex. These sera finally proved to have antibodies against antigens other than neutrophil such as anti-HLA antibody. The sera from 13 patients showed negative in GIFT-FCM, and positive in KY-Luminex. The sera from 3 patients showed negative both in GIFT-FCM and KY-Luminex. Sera with positive reaction against each panel neutrophil in GIFT-FCM have tendency to react against multiple antigens in KY-luminex method. Especially, majoryty of analyzed sera had antibodies against HNA-4a, 4b, 5a and 5b. No antibodies against each antigens were detected in sera from normal controls by KY-luminex method.
These results indicate that KY-luminex method has superior to GIFT-FCM in both sensitivity and specificity of anti-HNA antibodies. Furthermore, eHNA-microspheres are stable at 4 centigrade for more than a half year, and many samples such as 96 samples could be analyzes at once in simple and easy method.
Table. Relationship of reactivities of patient's sera between GIFT-FCM and KY-luminex method. Data represent the number in 101 patients GIFT-FCM KY-Luminex method positive negative positive 74 11 negative 13 3

Disclosures:
No relevant conflicts of interest to declare.
Induction of Endothelial Cell Apoptosis by Cleaved High Molecular Weight Kininogen Is Mediated Through a Lck and p53-Dependent Pathway
Authors: Betapudi Venkaiah , McCrae Keith R. 
Published: October 2019

Abstract
Abstract 3305

Background and objective:
High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. HK normally circulates as in the single chain form, but may be cleaved by plasma kallikrein to release the nonapeptide bradykinin, resulting in the formation cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring after kallikrein cleavage result in increased exposure of histidine and glycine-rich regions with kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, the receptors that mediate the antiangiogenic activity of HKa remain controversial, and the signaling pathways that lead to apoptosis have not been defined. Previous studies suggested possible involvement of SRC family kinases (SFK) in this process, and the purpose of this work was to further define the activation of SFKs and their downstream targets during HKa-induced endothelial cell apoptosis.

Results:
We first assessed the activation of SFKs in proliferating endothelial cells stimulated with bFGF before and after incubation with HKa (6–20 nM). SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region mediated by the negative regulator C-terminal Src kinase (Csk). Exposure of endothelial cells to HKa caused downregulation of Csk in a dose-dependent manner within 60 minutes. In parallel, we observed a significant increase in expression of the proapoptotic SFK Lck in endothelial cells exposed to HKa, though expression of other SFKs including Lyn, Fyn, Src, Hck and Blk were not significantly altered. Increased expression of Lck was associated with activation of p53 and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bak. Endothelial cell lysates prepared within 60 minutes of exposure to HKa demonstrated significant increases in the activity of caspases 3 and 7, as well as depletion of DNA fragmentation factors (DFF) 45 and 35, which cleave and inactivate DFF40, a major endonuclease involved in apoptosis. In parallel studies, endothelial cells depleted of Lck by treatment with Lck siRNA displayed loss of p53 phosphorylation, caspase 3 and 7 activity, and expression of Bax and Bad with no effects on the expression of Bad and Bid.

Conclusion:
These findings demonstrate a critical role for Csk in regulation of SFK activation and endothelial homeostasis, and demonstrate that downregulation of Csk by HKa leads to activation of a Lck-dependent, p53-mediated apoptotic pathway. Increasing the expression of Lck may represent a novel mechanism for regulation of aberrant angiogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Complement Activation in Patients with Acquired Thrombotic Thrombocytopenic Pupura (TTP)
Authors: Yang Shangbin , Cataland Spero R , Holers V. Michael , Lundberg Ante S , Wu Haifeng 
Published: October 2019

Abstract
Abstract 3323

Background:
Although severe deficiency(<10%) of the ADAMTS13 protease is central to the pathophysiology of acquired TTP, recently published data have suggested a role for complement activation in the pathogenesis of acquired TTP (Reti et al, JTH 2012; 10:791–798).

Patients:
We performed a retrospective study in longitudinally banked plasma samples from 9 patients with acquired TTP and severely deficient ADAMTS13 activity at presentation. Patients were randomly selected from our cohort of acquired TTP patients who achieved remission and were followed longitudinally with plasma samples banked at regular intervals. The presenting platelet count, LDH, serum creatinine, and ADAMTS13 activity of these 9 subjects were comparable to the entire cohort of acquired TTP patients in our research program at the time of their initial presentation.

Methods:
Samples were obtained at the time of their initial presentation prior to the initiation of plasma exchange therapy (PEX), at remission (median 2 weeks after the last PEX), and at sustained remission (median 2 months after remission sample) were utilized in this study. The complement proteins Bb (alternative pathway), C3a and C3 (all pathways of complement activation), and C5b-9 (terminal pathway) were measured.

Results:
At presentation, 4 subjects had evidence of complement activation as defined by increased levels of C3a and decreased C3, and all 9 subjects exhibited activation of the alternative pathway (Bb). In serially measured samples, the complement protein Bb was significantly higher, and C3 significantly lower, at presentation, returning to normal at the time of sustained remission (Table 1). C5b-9 and C3a demonstrated similar trends as Bb. ADAMTS13 activity, severely deficient in all subjects at presentation, was also significantly higher at remission and sustained remission.

Conclusions:
These data from a cohort of ADAMTS13 deficient, acquired TTP patients provide additional evidence for the role of complement activation in the pathogenesis of acquired TTP. Further study of our complete cohort of patients with acquired TTP is presently underway to better define the role complement activation in patients with acquired TTP.

Disclosures:
Cataland: Alexion: Consultancy, Research Funding. Holers:Alexion: Consultancy. Lundberg:Alexion: Employment. Wu:Alexion: Consultancy, Research Funding.
The Molecular Mechanisms of Serotonin in the Regulation of Megakaryocytopoiesis and TPO Production
Authors: Yang Mo , Meng Fanyi , Ye Jie Yu , Xu Yue , Xiao Bin , Chong Beng 
Published: October 2019

Abstract
Abstract 3347
We have reported that serotonin (5-HT) show a promoting effect on cord blood CD34+ stem/progenitor cells (Yang et al, Stem Cells 2007). We also demonstrated that serotonin enhances murine megakaryopoiesis via 5-HT2 receptors (Yang et al. Blood Coagul Fibrinol 1996). In this present study, we explored how serotonin regulated human megakaryocytopoiesis, proplatelet formation, and thrombopoietin (TPO) production. Our results indicated that serotonin significantly promoted human CFU-MK formation and reduced apoptosis in megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryocytopoiesis through stimulating release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO by q-PCR, ELISA and cytokine-array assays. Our findings demonstrated an important role of serotonin played on megakaryocytopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs, which indirectly promoted megakaryopoiesis. In present studies, we have demonstrated a positive “feed-back” control loop between MK-derived granule- serotonin and megakaryocytopoiesis. These findings improved our knowledge on megakaryocytopoiesis regulation and provided new clues on identifying novel thrombopoietic agents. It also deepened our understandings on how TPO production is regulated.

Disclosures:
No relevant conflicts of interest to declare.
Targeting Thrombomodulin to Endothelial ICAM-1 Rather Than PECAM-1 Allows Partnering with the Endothelial Protein C Receptor and Enhances Protection of Endothelial Barrier Function
Authors: Greineder Colin F , Zaytsev Sergei , Zern Blaine , Chacko Ann Marie , Muzykantov Vladimir R 
Published: October 2019

Abstract
Abstract 3351
The thrombomodulin-protein C pathway has important antithrombotic and anti-inflammatory roles in a variety of disease models and human illnesses, including severe sepsis, acute lung injury, and focal ischemia & reperfusion. We recently reported that anchoring recombinant thrombomodulin (TM) to PECAM-1 on the luminal surface of the vascular endothelium is protective in a mouse model of inflammatory lung injury. To this point, it has been unclear whether targeted thrombomodulin is able to partner with the endothelial protein C receptor (EPCR) in the same way as its endogenous counterpart. Here we demonstrate that anchoring TM to endothelial ICAM-1, rather than PECAM-1, results in approximately 10-fold greater activation of protein C. Furthermore, blocking protein C binding to EPCR results in marked reduction of protein C activation when TM is targeted to ICAM-1, whereas protein C activation is largely independent of EPCR when PECAM-1 is used as the target ligand. Consistent with this in vitro observation, anti-ICAM/TM fusion protein provides greater in vivo protection in a mouse model of inflammatory lung injury than its PECAM-targeted analogue, more potently blocking expression of pro-inflammatory cytokines and more effectively stabilizing endothelial barrier function. Since ICAM, endogenous TM, and EPCR are all thought to localize to microdomains on the apical surface of endothelial cells, we hypothesize that ICAM-targeting more effectively mimics the natural configuration and brings TM within sufficient proximity to allow access to its membrane co-factor. These observations could have profound implications for the therapeutic efficacy of a whole series of endothelial-targeted proto-drugs and their potential for translational success.

Disclosures:
No relevant conflicts of interest to declare.
Structure-Activity Relationship of Pro- and Anticoagulant Effects of Fucus Vesiculosus Fucoidan
Authors: Dockal Michael , Till Susanne , Zhang Zhenqing , Knappe Sabine , Reutterer Sabrina , Quinn Catherine , Redl Christoph , Ehrlich Hartmut J. , Scheiflinger Friedrich , Szabo Christina 
Published: October 2019

Abstract
Abstract 3353
Fucoidans are sulfated polysaccharides extracted from brown algae. Fucoidans have a wide variety of biological activities including pro- and anticoagulant activities, which occur at different concentration ranges. Therefore, fucoidans have also been described as non-anticoagulant sulfated polysaccharides (NASPs, Liu et al. Thromb Haemost 2006; 95:68). Fucoidans have complex structures due to their large molecular weight (Mw), wide Mw distribution, variable degree and pattern of sulfation, diverse monosaccharide composition, branching of the sugar chain and different monomer linkages. This structural complexity challenges identification of the components responsible for fucoidan activities.
The aim of the presented study was to fractionate Fucus vesiculosus (F.v.) fucoidan by size and to de- and oversulfate it to obtain compounds of varying Mw or degree of sulfation (DS), respectively. The fucoidans were then to be analyzed for their pro-and anticoagulant activities and structure, and the effect of modified fucoidan on the target protein Tissue Factor Pathway Inhibitor (TFPI) investigated.
Two approaches were applied to generate F.v. fucoidan with different structural properties to the original. Firstly, fucoidan was fractionated by size using ultrafiltration. The Mw of the resulting fractions ranged from 8–180 kD. NMR, elemental analysis and HPAEC showed that other structural features, such as sulfate content and monosaccharide composition, were similar to those of the original fucoidan. Thrombin generation (CAT) assays showed an EC50 for procoagulant activity of 0.3–3 μg/mL; aPTT increased by 50% at 4–25 μg/mL. Generally, higher Mw increased procoagulant activity. Below 15 kD, this activity was markedly reduced. The minimum active length of F.v. fucoidan was 70 carbohydrate units.
De- and oversulfated F.v. fucoidans were used to investigate the impact of charge on pro- and anticoagulant activities. In the CAT assay, oversulfated fucoidans showed improved procoagulant activity with an EC50 of 0.09–0.12 μg/mL, while desulfated fucoidans demonstrated reduced procoagulant activity compared to the original. A DS of 0.5 was estimated to be the limit for procoagulant activity. Inhibition of TFPI by fucoidan was assessed with a dilute prothrombin time assay (dPT) with added full-length TFPI. TFPI-blocking activity was mainly dependent on the DS and less on the fucoidans' Mw. Interestingly, oversulfation also stimulated an undesired activation of the contact pathway.
The presented study determined the minimal structural requirements for procoagulant activity of fucoidan molecules and identified features causing undesired biological effects.

Disclosures:
No relevant conflicts of interest to declare.