bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome-Wide Association Study of Platelet Function in African Americans
Authors: Qayyum Rehan , Becker Lewis , Becker Diane , Yanek Lisa , Leal Suzanne M , Mathias Rasika , Bray Paul F. 
Published: October 2019

Abstract
Abstract 1068

Background:
We have previously shown that platelet aggregation, an essential component of pathogenesis of acute coronary syndromes, has higher heritability in African Americans than Caucasians. However, a genome-wide association study of native platelet function in African Americans has not been reported.

Methods:
Platelet-rich plasma (PRP) was isolated from blood samples obtained from the discovery (GeneSTAR, N=835) and replication (Platelet Genes and Physiology Study, N=119) cohorts. Optical aggregation was used to measure maximal aggregation in PRP after samples were stimulated with arachidonic acid, collagen, ADP, or epinephrine. Genotyping was conducted with Illumina 1M arrays. For each cohort, age- and sex-adjusted linear mixed models were used to test for association between each SNP and each phenotype under an additive model.

Results:
Of the 117 SNPs that were significant (P< 5 × 10−8) in the discovery sample, 50 SNPs in 4 regions were also significant in the replication sample (Table). Among the replicated SNPs, a previously reported SNP in European Americans, rs12041331, in the PEAR1 gene was associated with ADP- and epinephrine-mediated aggregation. 47 SNPs in the 17q21.31 region, in one LD block, were associated with collagen-mediated aggregation.

Conclusions:
In this first GWAS of native agonist-mediated platelet aggregation in African Americans, we have discovered, and replicated in an independent sample, 4 regions that are associated with platelet aggregation. Several genes in the identified regions are expressed in platelets and further study of these genes may provide novel insights in platelet biology.

Disclosures:
No relevant conflicts of interest to declare.
Direct Observation of Von Willebrand Factor Elongation and Fiber Formation On Collagen During Acute Whole Blood Exposure to Pathological Flow
Authors: Colace Thomas Vincent , Diamond Scott L. 
Published: October 2019

Abstract
Abstract 1070
Under conditions of pathological shear rate, von Willebrand Factor (vWF) undergoes conformational changes and self aggregation. We sought to visualize this phenomenon using a novel microfluidic model of stenosis and understand its role in thrombus formation in elevated shear rate environments. In severe stenosis, vWF experiences millisecond exposures to pathological wall shear rates (gw). Distinct from shear experiments that last many seconds, we deployed microfluidic devices for single-pass perfusion of whole blood or platelet free plasma (PFP) over fibrillar type 1 collagen (< 50 msec transit time) at pathological gw or spatial wall shear rate gradient (grad gw). Using fluorescent anti-vWF, long thick vWF fibers (>20 mm) bound to collagen were visualized at constant gw > 30,000 s−1 during perfusion of PFP, a process enhanced by EDTA. Rapid acceleration or deceleration of EDTA-PFP at grad gw = ± 5.5 × 105 to 4.3 × 107 s−1/cm did not promote vWF deposition when gw < 30,000. At 19,400 s−1, EDTA-blood perfusion resulted in rolling vWF-platelet nets, while blood perfusion (normal Ca2+) generated large vWF/platelet deposits that repeatedly embolized and were blocked by anti-GP1b or the aIIbβ3 inhibitor GR144053 and did not require shear gradients. Blood perfusion at venous shear rate (200 s−1) produced a stable platelet deposit that was a substrate for massive but unstable vWF-platelet aggregates when flow was increased to 7800 s−1. Supported by collagen and enhanced by platelet GP1b and aIIbβ3, vWF fiber formation occurred during acute exposures to pathological gw but did not require wall shear rate gradients.
Figure 1 A, Platelet free citrated-plasma was treated with 1 μg/mL fluorescently labeled anti-vWF and 5 mM EDTA. The plasma samples were perfused over a collagen type 1 surface at local wall shear rates of 30,000, 62,400 and 125,000 s−1 from left to right. Long fibers of vWF (>20 μm) appeared at shear rates above ∼30,000 s−1, with more fibers appearing at higher shear rates. The bar indicates 15 μm. Figure 1. A, Platelet free citrated-plasma was treated with 1 μg/mL fluorescently labeled anti-vWF and 5 mM EDTA. The plasma samples were perfused over a collagen type 1 surface at local wall shear rates of 30,000, 62,400 and 125,000 s−1 from left to right. Long fibers of vWF (>20 μm) appeared at shear rates above ∼30,000 s−1, with more fibers appearing at higher shear rates. The bar indicates 15 μm.

Disclosures:
No relevant conflicts of interest to declare.
Drug Metabolites Formed in Vivo Promote the Clearance of Human Platelets by Metabolite-Dependent Antibodies in NOD/SCID Mice
Authors: Bougie Daniel W. , Nayak Dhirendra , Aster Richard H. 
Published: October 2019

Abstract
Abstract 1083
Drug induced immune thrombocytopenia (DITP) is a relatively common and often serious complication of treatment with many drugs. It can be extremely helpful clinically to identify the responsible agent, especially in patients taking multiple drugs. It is sometimes possible to demonstrate a drug-dependent antibody (DDAb) that binds to platelets only when drug is present but results are often negative even in patients with a history that strongly favors DITP. An important reason for this is that a drug metabolite rather than the parent drug can be the sensitizing agent. Because drugs often have many metabolites, most of which are not readily available, identification of the metabolite-specific, DDAbs can be very difficult.
Animal models for studying immune clearance of human cells have not been available because of naturally occurring xenoantibodies. However, recent studies have shown that destruction of human platelets by human antibodies can be studied in the NOD/SCID mouse, which lacks xenoantibodies (Boylan et al 2009). Metabolism of many drugs is similar in mice and humans. We have shown that mice injected with acetaminophen and naproxen produce metabolites that promote in vivo destruction of human platelets by DDAbs specific for metabolites of these drugs (Bougie et al 2010). These findings suggested that the NOD/SCID mouse might be a useful screening tool to identify putative DDAbs specific for metabolites of other drugs in patients suspected of DITP.
To detect metabolite-specific antibodies, mice are transfused with human platelets suspended in test serum. After collecting a baseline blood sample to define the relative number of human platelets in each mouse, the implicated drug is injected (intraperitoneal). Drug metabolites made by the mouse diffuse into the circulation and shorten the survival of human platelets if a metabolite-specific, platelet-reactive antibody is present in the test serum. For a result to be considered “positive” for DDAbs, human platelet survival should be unaltered in mice given test serum alone and normal serum plus drug and should be significantly shortened in mice given test serum plus drug (Figure).

Findings made to date with 94 archived serum samples from DITP suspects exposed to 5 widely used medications are summarized in Table 1. No platelet-reactive antibodies were detected in vitro using unmodified drug. However, 13 patient samples caused accelerated clearance of human platelets when mice were injected with the implicated drug (2 furosemide, 6 clopidogrel, 2 pantoprazole, 3 ibuprofen).

These findings suggest that DITP resulting from sensitization to drug metabolites may be much more common than has been thought and indicate that the NOD/SCID mouse model provides a highly efficient tool to screen candidate sera for metabolite-specific, platelet-reactive antibodies.

Disclosures:
No relevant conflicts of interest to declare.
Thrombin Generation Mediators and Markers in Sepsis Associated Coagulopathy and Their Modulation by Recombinant Thrombomodulin
Authors: Hoppensteadt Debra , Cunanan Josephine , Khan Hussein , Kaul Inder , Osawa Yutaka , Fareed Jawed 
Published: October 2019

Abstract
Abstract 1131
Severe sepsis remains the most common cause of death in critically ill patients and thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). The purpose of this study was to profile prothrombin fragment (F1.2), thrombin antithrombin complex (TAT) and d-Dimer (DD) throughout the course of hospital stay in subjects identified with sepsis. Plasma samples from seven hundred and fifty eight patients enrolled in the ART-123 study, a phase 2b, international, multicenter, randomized controlled trial, separated into treatment and placebo groups were analyzed for various parameters using ELISA methods. Plasma levels of F1.2, DD, and TAT were recorded at several time points following administration of recombinant thrombomodulin or placebo and the results are tabulated as the median values, in the following tables. All parameters in both treatment groups showed a decrease from the baseline values at day 14. In the group treated with thrombomodulin the median F1.2 levels demonstrated a marked decrease on day 14 exhibiting a 50% decrease, whereas the F1.2 levels fluctuated throughout the study period and showed slight change on day 14, in the placebo group. Although both groups showed a gradual decrease in the DD, the group treated with thrombomodulin demonstrated a stronger decrease over the 14 day period compared to the placebo treated group. While the data was widely scattered, these results show that DIC represents a hypercoagulable state along with other hemostatic abnormalities and the activation of the inflammatory process. Modulation of these activation processes through such targets as DD, F1.2 and TAT may play an important regulatory role in the pathogenesis of sepsis associated coagulopathy. Moreover, this study validates the hypothesis that thrombomodulin down regulates the thrombin generation mediators/markers in sepsis associated DIC.
Median Value For Patients Treated with Placebo in the DIC Study Parameter Baseline Post Day1 Pre Day 3 Post Day 3 Day 7 Day 14 F1.2 (pmol/L) 304.96 304.73 392.90 372.75 328.34 290.19 DD (ng/ml) 4669.5 4825.5 4610 4378.5 3740 3468 TAT (ng/ml) 7.66 6.95 7.28 6.73 6.31 6.04
Median Value For Patients Treated with Recombinant Thrombomodulin in the DIC Study Parameter Baseline Post Day1 Pre Day 3 Post Day 3 Day 7 Day 14 F1.2 (pmol/L0 278.33 240.96 293.42 281.98 228.82 142.59 DD (ng/ml) 5152.50 4465.00 3726.00 3289.00 2785.00 3275.50 TAT (ng/ml) 9.03 6.49 5.99 5.77 5.96 7.53

Disclosures:
Kaul: Asahi Kasei Pharma America Corporation: Employment. Osawa:Asahi Kasei Pharma America Corporation: Employment.
Structural Comparability Between Recombinant FVIII-Fc and Its Isolated FVIII and Fc Constituents
Authors: Kulman John , Tsutakawa Susan , Bou-Assaf George , Berkowitz Steven , Light David R. , Mezo Adam , Wang Deping , Whitlow Marc , Culyba Liz , Tainer John , Peters Robert 
Published: October 2019

Abstract
Abstract 1135
rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protection from bleeds for patients with hemophilia A receiving prophylaxis or on-demand treatment, or undergoing surgery. In this study, we evaluated the structural comparability of BDD rFVIII and rFVIIIFc using small angle X-ray scattering (SAXS) and hydrogen/deuterium exchange (HDX) mass spectrometry. SAXS analysis of BDD rFVIII and rFVIIIFc, performed at the Advanced Light Source Synchrotron, provided the electron pair radius distribution of each molecule and accurate but inherently low resolution structural information. Both BDD rFVIII and rFVIIIFc were monomeric in solution. BDD rFVIII had a radius of gyration (Rg) of 39.7 Å, compared with a Rg of 38 Å calculated from its published atomic coordinates (2R7E.pdb). rFVIIIFc had a larger Rg of 51 Å, which was consistent with a simple computational model of BDD rFVIII tethered to Fc that predicted an Rg of 50 Å. HDX mass spectrometric analysis yielded 94% coverage of the BDD rFVIII component and 87% coverage of the Fc component. Rates of deuterium exchange within peptides derived from each of these components in the context of the rFVIIIFc fusion protein were very similar to those of the isolated BDD rFVIII and Fc constituents. Taken together, the results of SAXS and HDX analyses support the conclusion that fusion of the Fc moiety to BDD rFVIII does not alter the higher order structure of either component.

Disclosures:
Kulman: Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Berkowitz:Biogen Idec: Employment. Light:biogenidec: Employment. Mezo:Biogen Idec: Employment. Wang:Biogen Idec: Employment. Whitlow:Colabrativ Inc.: Employment; Biogen Idec: Consultancy. Culyba:Biogen Idec: Employment. Peters:Biogen Idec: Employment.
Real Life Efficacy and Safety of Rivaroxaban for Acute VTE Treatment – First Results of the Prospective Noac Registry (NCT01588119)
Authors: Gelbricht Vera , Koehler Christina , Werth Sebastian , Haensel Ulrike , Schreier Thomas , Eulitz Maria , Tittl Luise , Beyer-Westendorf Ines , Beyer-Westendorf Jan 
Published: October 2019

Abstract
Abstract 1159

Background:
In the EINSTEIN study rivaroxaban (RX) has been found to be at least as effective and safe as warfarin in treatment of acute deep vein thrombosis (DVT), which lead to approval of RX in many countries. However, patients in RCT‘s present a selected population treated under a strict protocol and followed for a short period of time. Consequently, efficacy and safety of new oral anticoagulants (NOAC) need to be confirmed in unselected patients in daily care.

Objectives:
To evaluate the efficacy, safety and management issues of rivaroxaban anticoagulation in acute VTE in daily care.

Patients and methods:
A network of 200 physicians from private practice and hospitals enrol patients in the prospective NOAC registry. Inclusion criteria are: 1) indication for NOAC anticoagulation >3 month; 2) age > 18 years; 3) written informed consent; 4) availability for follow-up. No Exclusion criteria apply. In the registry, up to 2000 patients will receive prospective follow up (FU) by phone visits at day 30 day and quarterly thereafter to collect efficacy and safety data.

Results:
Until July 31th 2012, 938 patients were registered. Of these, 105 patients received RX for acute VTE treatment (demographic data in table 1). In our registry, the population receiving acute VTE treatment is older than the EINSTEIN population (62.2 vs. 55.8 years). Most patients are treated for major VTE (proximal deep vein thrombosis (DVT) pulmonary embolism (PE)), but about 20% are treated for isolated distal DVT.
The results of 1-, 3- and 6-months FU are shown in table 2. Until now, no recurrent VTE or VTE-related death occurred. Two patients (1.9%) experienced a major vascular event (acute limb ischemia) at the beginning of NOAC therapy and one patient experienced a minor vascular event (tachyarrhythmia). Bleeding events were frequent (22.3%) but only five patients (4.8%) experienced major bleeding events, one of which was a fatal intracranial bleeding. Three patients (2.9%) died during FU (1 intracranial bleed, 2 of underlying diseases). At 6 month, only eight patients (7.8%) were switched to other anticoagulants and one patient (1.0%) had an unscheduled discontinuation of anticoagulant therapy.

Conclusion:
In unselected patients in daily care, acute VTE treatment with RX is effective and safe with low rates of cardiovascular or bleeding events during the first 180 days of treatment.

Disclosures:
Werth: Bayer Healthcare: Honoraria. Beyer-Westendorf:Bayer Healthcare: Bayer provided a grant to support the NOAC registry in part Other, Honoraria; Boehringer Ingelheim: Boehringer provided a grant to support the NOAC registry in part, Boehringer provided a grant to support the NOAC registry in part Other, Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria.
The Balance of Positive and Negative Effects of TGF-Beta Signaling Regulates the Hemangioblast Development in Human Pluripotent Stem Cells
Authors: Bai Hao , Xie Yin-Liang , Gao Yong-Xing , Cheng Tao , Wang Zack Z 
Published: October 2019

Abstract
Abstract 1220
Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling human hemangioblast development are largely unknown. Our previous study demonstrated that CD34 progenitors from human embryonic stem cells (hESCs) contain a population of cells that give rise to hematopoietic cells and endothelial cells. In this study, we established a serum-free and feeder-free system to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including hESCs and induced-pluripotent stem cells (hiPSCs). The hemangioblast development in hPSCs was established in three major steps: i) the formation of uniformed embryoid bodies (EBs) in hanging-drops, ii) the development of multipotential mesoderm precursors in cultivation of EBs, and iii) the generation of CD34+CD31+VE-cadherin+ progenitors, which have hemangioblast potential to give rise to both hematopoietic and endothelial cells (via hemogenic endothelium). The hESC-derived hematopoietic cells expressed CD41, CD61, CD117, CD71, and CD235a. The endothelial cells were CD31+ and VE-cadherin+ cells. We found that inhibition of Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) by Y27632 was critical for hanging-drop EB formation. Transforming growth factor beta (TGF-beta) is a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. The expression of TGF-beta was increased during hPSC differentiation. Inhibition of TGF-beta signaling by the TGF-beta inhibitor, SB431542, significantly decreased the efficiency of CD34+ progenitors, suggesting a crucial role of TGF-beta signaling in hPSC differentiation. To further investigate when TGF-beta signaling affects hPSC differentiation to CD34+ progenitors, we added TGF-beta inhibitor at different stages of hPSC differentiation. We found that TGF-beta signaling is required for the early mesoderm induction. Inhibition of TGFb signaling before hemangioblast commitment during PSC differentiation significantly decreases the frequency of hemangioblasts (CD34+/CD31+/VE-cadherin+ cells) and the expression of mesoderm markers, such as Brachyury, Tie1, EOMES, and MIXL1. On the other hand, inhibition of TGF-beta signaling after mesoderm induction increases the number of CD34+/CD31+/VE-cadherin+ cells. EdU cell proliferation assays indicated that the proliferation of CD34+ progenitors were not affected by SB431542, suggesting that TGF-beta signaling suppresses hemangioblast generation. Our study demonstrated that TGF-beta signaling has the double-edged effect on hPSC differentiation to hemangioblasts.

Disclosures:
No relevant conflicts of interest to declare.
Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice
Authors: Moriya Kunihiko 
Published: October 2019

Abstract
Abstract 1311
Mixed-lineage-leukemia (MLL) fusion oncogenes are involved in de novo and secondary therapy-related acute leukemia in humans. To understand the pathogenesis of MLL-rearranged leukemia, several murine models for this disease have been established. However, the leukemia derived from murine hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. In vivo studies, in which primary human leukemia cells were transplanted into immunodeficient mice, provided us significant advances to understand the pathogenesis of human leukemia. However, these models are not suitable for studying physiological leukemogenesis because the initiation and progression processes are hard to be evaluated. Therefore, a new in vivo experimental model for analyzing leukemogenesis derived from primary human HSCs and by genetic hits is required.
Here we developed a humanized murine model for human leukemia by transplanting human cord blood-derived CD34-positive HSCs transduced with an MLL-AF10 oncogene and second hits into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice.
Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, however, MLL-AF10 alone could not induce leukemic transformation. Accumulating evidence points to a multistep pathogenesis for leukemia development and progression. In the multi-genetic step models of leukemogenesis, particularly in the two-hit model of leukemia, the initial genetic hit often leads to abnormal cell differentiation (Type II mutation), while subsequent mutations may activate specific signaling pathways that are involved in cell growth, such as the Ras/MAP kinase pathway (Type I mutation). Because active mutations in Ras genes are often found in MLL-related leukemia, we next transduced constitutively active form of K-Ras mutant along with the MLL-AF10 oncogene. Enforced expression of the mutant K-Ras alone was insufficient to induce leukemia. Eight weeks after transplantation, all of the recipient mice transduced both MLL-AF10 and mutant K-Ras developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We could also observe organ infiltration in liver and spleen in vivo that mimicked clinical symptoms observed in human leukemia.
We thus successfully established a murine model for human MLL-rearranged leukemia derived from human HSCs. The present model is a useful experimental platform for the multi-step leukemogenesis of human leukemia and for new therapeutic approaches that should be evaluated in vivo.

Disclosures:
No relevant conflicts of interest to declare.