bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Akt3 in Mediating Outside-in Signaling of the Platelet Integrin αIIbβ3
Authors: O'Brien Kelly A , Hay Nissim , Du Xiaoping 
Published: October 2019

Abstract
Abstract 1134
Ligand binding to integrins mediates cell adhesion and transmits “outside in” signals that lead to cell spreading, migration, and proliferation. In platelets, the prototype integrin aIIbb3-mediated outside-in signaling is required for platelet spreading and retraction, and greatly amplifies platelet activation. Previous studies suggest that phosphoinositide 3-Kinases (PI3K) are activated upon binding of integrin αIIbβ3 to its ligand fibrinogen, and is important in outside-in signaling leading to platelet spreading. However, the mechanism by which PI3K transmits outside-in signals has been unclear. A major known downstream effector of PI3K is the Akt (protein kinase B) family of serine/threonine kinases, including Akt1, Akt2, and Akt3. We have recently shown that platelets not only express Akt1 and Akt2 as previously reported, but also express a substantial amount of Akt3. To investigate whether Akt3 is a downstream effector mediating PI3K-dependent integrin outside-in signaling, platelets from Akt3 knockout mice were compared with wild type platelets for their spreading on fibrinogen. Platelets from Akt3−/− mice showed partially, but significantly reduced spreading on fibrinogen, indicating that Akt3 is important in integrin-mediated outside-in signaling leading to platelet spreading. Consistent with the results of Akt3 knockout, treatment of platelets with a pan Akt inhibitor also significantly inhibited spreading of human and mouse platelets on fibrinogen. Akt becomes phosphorylated upon platelet spreading on fibrinogen, which is significantly reduced in Akt3 knockout platelets, and is abolished by PI3 Kinase inhibitor, wortmannin, or Src Family Kinase (SFK) Inhibitor, PP2, suggesting that Akt activation is downstream from PI3K, and SFK during integrin outside-in signaling. Thus, our data reveals that Akt3 is an important downstream effector of PI3K-dependent integrin outside-in signaling promoting platelet spreading.

Disclosures:
No relevant conflicts of interest to declare.
Characterization of the Binding Between the Polymeric Platelet Adhesive Proteins, Multimerin 1 and Von Willebrand Factor
Authors: Parker D'Andra , Tasneem Subia , Fuller Nola , Sadler J. Evan , de Groot Philip G , Hayward Catherine PM 
Published: October 2019

Abstract
Abstract 1144

Introduction:
Multimerin 1 (MMRN1) is a massive variably-sized homopolymeric protein that is stored in platelet and endothelial cell secretion granules, for release with vascular injury. Recently, MMRN1 was identified to support platelet adhesion in vitro and in vivo. At high shear, MMRN1 supports platelet adhesion by a von Willebrand factor (VWF)-dependent, but integrin-independent mechanism, involving platelet glycoprotein (GP) Ibα. Direct binding of MMRN1 to GP Ibα has not been demonstrated. These data led us to postulate that VWF binds MMRN1 at site(s) distinct from the GP Ibα binding site, and test the roles of VWF A domains in MMRN1 binding.

Methods:
Modified enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were used to assess binding interactions between wildtype (WT) MMRN1 and WT or domain deleted VWF constructs, and VWF polypeptides. Protein constructs tested included: multimeric VWF deletion constructs ΔA1A2A3-VWF, ΔA1A3-VWF, and ΔA1-VWF, and monomeric VWF polypeptides A1A2A3, A1A2, A1 and A3. Bovine serum albumin (BSA) coated surfaces were used as the negative control.

Results:
Unlike WT-VWF, VWF lacking the A domains (ΔA1A2A3-VWF) or the combination of the A1 and A3 domains (ΔA1A3-VWF) did not detectably bind to MMRN1 (p < 0.001). VWF lacking the A1 domain (ΔA1-VWF) showed MMRN1 binding comparable to WT-VWF (p = 0.39), excluding the possibility that MMRN1 binding site is located in VWF A1 domain (the region that binds GP Ibα). VWF polypeptides A1A2A3, A1A2 and A3 bound to MMRN1 (p < 0.001), unlike the VWF polypeptide A1 (p = 0.137), although the A1A2 polypeptide showed reduced binding compared to A1A2A3 (p < 0.001). SPR analyses confirmed that MMRN1 binding was supported by VWF peptides containing the A3 and/or A2 domains.

Conclusions:
The regions of VWF that support MMRN1 includes the A3, and possibly A2 domains, which respectively contain binding sites for collagen and ADAMTS-13. Our data suggest that the mechanism by which GP Ibα and VWF support platelet adhesion to MMRN1 at high shear include: VWF binding to GP Ibα via the A1 domain, and to MMRN1 via the A3 and possibly A2 domains. These findings have implications for the molecular mechanisms that support platelet adhesion at sites of vessel injury.

Disclosures:
No relevant conflicts of interest to declare.
Sustained Responses to Thrombopoetin Analogs in HIV Patients with Refractory ITP
Authors: Ochoa Roberto , Akunyili Ikechukwu , Milone Mary Anne , Harrington Thomas J. , Byrnes John J. 
Published: October 2019

Abstract
Abstract 1165
Thrombocytopenia(TP) is common in patients with Human Immunodeficiency Virus (HIV) infection, reported in as many as 10–30% of patients and seen more often in IV drug users and advanced HIV infection even in post-HAART era. Both decreased production of platelets and increased destruction have been proposed as mechanisms of thrombocytopenia and at least 2 different clinical scenarios have been described: patients with very advanced AIDS, being thrombocytopenia another manifestation of bone marrow suppression; and patients without significant T-cell depletion in whom thrombocytopenia develops likely as an immune phenomenom, “ITP-like” HIV-related thrombocytopenia (HIV-TP).
In HIV-TP, common treatments for classical ITP have been reported including corticosteroids, danazol, splenectomy, Intravenous Gammaglobulin, interferon, Anti-D, dapsone and splenic radiation with varying degrees of success with the highest responses seen with the use of HAART.
Romiplostim (RMP) and Eltrombopag (ETP) are thrombopoeitin agonists (TA) approved in 2009 for refractory ITP and recently proposed to be used as first-line agents. No reports of the use of TA in patients with HIV-ITP are available to date.
5 patients with HIV-related ITP were treated with TP in our centers between 2009 to 2011. Mean age was 48 years (25–61), 4 males and 1 female. All of the patients had failed or relapsed after prednisone, 3 of the patients had also failed multiple treatments including splenectomy (1 patient), IVIG, Rho(D) immune globulin, Rituximab, danazole and vincristine. All but 1 of the patients were on HAART by the time of treatment and CD4 counts were <200 cells/μL in 2 patients (10–157 cells/ μL).
All 5 patients were treated initially with RMP at the starting dose of 1 mcg/Kg and titrated as per guidelines. One patient requested to be switched to ETP and developed an acute lower extremity DVT and symptomatic PE 1 week after starting the medication.
Responses to treatment are shown in graph 1. Mean baseline Platelet count was 27 × 103/μL (13–40 × 103/μL) and it increased to 66 × 103/μL (45–86 × 103/μL) within 1 week and 76 × 103/μL (45–107 × 103/μL) after 1 month of therapy, 2.8 times the baseline (2.1 – 3.4 × baseline value). 4 patients achieved a platelet count ≥50 × 103/μL within 1 month. 4 of the patients had durable responses for ≥ 12 months not requiring dose adjustments or modifications; the other patient had several compliance issues and missed several doses of RMP, his platelet count improved every time the medication was restarted.
We present the first report of the successful and sustained use of TA in patient with HIV- ITP.

Disclosures:
Byrnes: Amgen: Honoraria, Speakers Bureau.
Critical Role for Stat5 in the Initiation and Maintenance of Polycythemia Vera in a Jak2V617F Knock-In Mouse Model
Authors: Yan Dongqing , Mohi Golam 
Published: October 2019

Abstract
Abstract 121
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The JAK2V617F mutation has been identified in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of multiple signaling molecules/pathways. However, the key signaling downstream of JAK2V617F required for transformation, induction and maintenance of MPNs remains elusive. Using a mouse genetic strategy, we found that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. Whereas inducible expression of Jak2V617F in mice resulted in all the features of human PV, including increase in red blood cells, hemoglobin, hematocrit, white blood cells, platelets, and splenomegaly, deletion of Stat5 in the Jak2V617F knock-in mice normalized all the blood parameters and the spleen size. Histopathologic analyses revealed that Stat5 deficiency blocked the development of PV in mice expressing Jak2V617F. In addition, deletion of Stat5 completely abrogated erythropoietin (Epo)-independent erythroid colony formation evoked by Jak2V617F, a hallmark feature of PV. Flow cytometric analysis revealed that concomitant deletion of Stat5 reduced the Jak2V617F-induced expansion of LSK (lin−Sca-1+c-kit+) and MEP (megakaryocyte-erythroid progenitors) as well as CD71+Ter119+ and Gr-1+Mac-1+ populations to normal levels. Unlike Jak2V617F knock-in mice, which developed myelofibrosis at old age, Stat5-deficient Jak2V617F-expressing mice failed to develop myelofibrosis. Re-expression of Stat5 in Stat5-deficient Jak2V617F knock-in mice bone marrow by retroviral transduction completely rescued the defects in transformation of hematopoietic progenitors and the PV phenotype. Furthermore, deletion of Stat5 after establishment of PV disease in the transplanted animals expressing Jak2V617F by injection with polyinosine:polycytosine (pI:pC) normalized the blood parameters and inhibited the progression of the disease. Together, these results indicate a critical function for Stat5 in the induction and maintenance of PV. Biochemical analyses revealed that Stat5 deficiency significantly inhibited constitutive phosphorylation of p70S6 kinase and markedly reduced expression of Bcl-xL, Cyclin-D2 and Pim-1 mediated by Jak2V617F. These suggest that p70S6 kinase, Bcl-xL, Cyclin-D2 and Pim-1 are downstream targets of Jak2V617F-Stat5 signaling, and they may play a role in hematopoietic transformation induced by Jak2V617F. These findings provide strong support for the development of Stat5 inhibitors as targeted therapies for the treatment of PV and other JAK2V617F-positive MPNs.

Disclosures:
No relevant conflicts of interest to declare.
Effects of Strong P2Y12 Receptor Inhibition by Prasugrel on Platelet Inhibition During Endotoxemia: A Randomized Controlled Trial
Authors: Spiel Alexander O , Derhaschnig Ulla , Jilma-Stohlawetz Petra , Jilma Bernd 
Published: October 2019

Abstract
Abstract 1245

Background:
P2Y12 receptor antagonists have become a mainstay for the treatment of cardiovascular diseases. Yet, they have rarely been evaluated under pathophysiological conditions apart from arterial diseases.

Objectives:
We hypothesized interactions between prasugrel and enhanced von Willebrand Factor (VWF) release in a model of systemic inflammation, and compared the pharmacodynamic effects of prasugrel versus placebo on agonist-induced platelet aggregation and shear-induced platelet plug formation.
Subjects/Methods: Twenty healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Each volunteer received either placebo or a 60 mg-loading dose of prasugrel two hours before endotoxin infusion. Platelet inhibition was measured with Multiple Electrode Aggregometry (MEA), the Platelet Function Analyzer-100 (PFA-100) and the Vasodilator Stimulated Phosphoprotein (VASP) phosphorylation assay, respectively. Results: Prasugrel reduced the platelet reactivity index in the VASP assay from 79% to 5–7%, and unequivocally prolonged the closure times of the Innovance cartridge to >300s, but also the CADP-CT to >300s in the majority of subjects. Prasugrel not only blunted platelet aggregation induced by ADP (−81%), but also other pathways including arachidonic acid (−60%), ristocetin (−75%; p<0.001 for all), and to a lesser degree collagen or thrombin receptor activating peptide (TRAP). Prasugrel decreased shear-induced platelet plug formation but VWF release during endotoxemia partly antagonized the inhibitory effect of prasugrel as measured with the PFA-100. Endotoxemia acutely decreased ristocetin and TRAP induced platelet aggregation, and enhanced ristocetin induced aggregation after 24h.

Conclusions:
These data for the first time demonstrate that strong in vivo blockade of P2Y12 by prasugrel inhibits a broad spectrum of platelet aggregation pathways. However, VWF release may reduce prasugrel's effects under high shear conditions.

Disclosures:
No relevant conflicts of interest to declare.
Carbon Monoxide Therapy Modulates Hematopoietic Stem Cell Development in Heme-Oxygenase-1 Knockout Mice
Authors: Beckman Joan Denise , Marker Paul H , Nguyen Julia , Belcher John D , Croatt Anthony J. , Nath Karl A. , Hebbel Robert P , Vercellotti Gregory M 
Published: October 2019

Abstract
Abstract 1318
Previous studies have demonstrated that heme-oxygenase-1 (HO-1), the rate limiting enzyme in the catabolism of heme, is a regulator of the balance between hematopoiesis and stem cell preservation under stress. HO-1−/− mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO), a by-product of HO-1 activity with antioxidant and anti-apoptotic properties; will reduce stress hematopoiesis in HO-1−/− mice, reducing inflammation and redistributing hematopoietic potential. In order to test this hypothesis we treated 25 week old HO-1−/− mice and HO-1+/+ mice (n=7/strain) with 250 ppm inhaled CO for 1 h/day, 3 days/week for eight weeks and compared them to an equal number of untreated HO-1−/− and HO-1+/+ mice. After 8-weeks of treatment the mice were sacrificed and flow cytometry was performed on bone marrow to assess hematopoietic potential. Cell cycle analysis of the bone marrow demonstrates that untreated HO-1−/− mice have a significantly decreased percent of cells in S-phase compared to untreated HO-1+/+ mice. Treatment with CO significantly (p<0.05) increases the percent of cells in S-phase in HO-1−/− mice but not HO-1+/+ mice. Reactive oxygen species (ROS) production in lineage−, c-kit+, Sca-1+ (KLS) cell population was assessed using 5-(and 6-)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate dye. HO-1−/− mice have a significantly (p<0.001) increased proportion of ROS positive KLS cells compared with HO-1+/+ mice. Analysis of long-term (LT), short-term, and multipotent (MPP) progenitor cell populations was conducted. Treatment with CO significantly (p<0.05) increases the percent of LT-HSC and MPP progenitor cells in HO-1−/− mice but not HO-1+/+ mice. Concordantly, the total white blood cell count of the CO-treated mice increased significantly. The differential of the mature blood cells demonstrates significant shift in cell maturation, with significant increase in red blood cells, platelets, and lymphocytes and a significant decrease in monocytes. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1−/− mice leading to a change in mature cell populations. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation, ultimately leading to a change in the inflammatory milieu of the mice.

Disclosures:
Belcher: Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.
Regulatory Networks Governed by MicroRNAs in T-ALL Oncogenesis and Normal T-Cell Development
Authors: Van der Meulen Joni , Mets Evelien , Mestdagh Pieter , Pipelers Peter , Taghon Tom , Trujillo David Camacho , Avran David , Van Peer Gert , Van Roy Nadine , Thas Olivier , Benoit Yves , Vandesompele Jo , Poppe Bruce , Van Vlierberghe Pieter , Rondou Pieter , Soulier Jean , Speleman Frank 
Published: October 2019

Abstract
Abstract 1366
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes affecting children, adolescents and adults. T-ALL is characterized by a differentiation arrest at specific stages of T-cell development, primarily originating from the ectopic expression of T-cell oncogenes. Based on the mutation status of these genes and mRNA profiles, at least 5 different molecular-cytogenetic subgroups have been delineated. In addition, genetic abnormalities affecting cell cycle, proliferation, survival and self-renewal are present in all T-ALL subgroups. In a recent study, we discovered five oncogenic microRNAs (miRNAs) co-regulating the expression of key tumor suppressor genes implicated in the pathogenesis of T-ALL (Mavrakis et al., Nature Genetics, 2011). In this study, we established the extended miRNAome (756 miRNAs) in a genetically well characterized T-ALL patient cohort (n=65), 20 T-ALL cell lines as well as 9 different subsets of sorted T-cell populations from 3 human donors in parallel to mRNA and gene copy number profiles thus allowing integrative data mining. Cross-comparison between the different datasets resulted in the identification of miRNAs with presumed function in normal T-cell development as well as novel candidate miRNAs in T-ALL pathogenesis. The candidates were validated in an independent patient series (n=50). To better understand our previously established miRNA-mRNA regulatory T-cell/T-ALL network, we performed miRNA-mRNA correlation analysis followed by gene set enrichment analysis in order to assign putative functions to the selected miRNAs (http://www.mirnabodymap.org/; Mestdagh et al., Nucleic Acids Research, in press). In addition, the regulatory network was consolidated and extended through identification of miRNAs targeting selected key T-ALL oncogenes and tumor suppressor genes using a robust high throughput 3'UTR screening assay. In conclusion, this study further details the regulatory networks controlling normal T-cell development and T-ALL oncogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Significance of Quantification of the PML-RARa Transcript in Children with Acute Promyelocytic Leukemia: A Single Center Experience
Authors: Zhang Li , Cao Zeng , Zou Yao , Ruan Min , Li Qinghua , Wang Jianxiang , Zhu Xiaofan 
Published: October 2019

Abstract
Abstract 1474

Objective:
Minimal residual disease (MRD) monitoring based on the detection of PML/RARa transcripts employing PCR technology has clearly demonstrated its benefit in the diagnosis and follow-up of acute promyelocytic leukemia (APL) patients. So far, real-time quantitative PCR (RQ-PCR) has been investigated to provide prognostic indexes for APL management in many adults studies. However, there are still no data on the use of such assays for child APL therapy. The aim of this study was conducted to clarify the relationship between the level of MRD and outcome in children with newly diagnosed PML/RARa-positive APL and identify the subgroups at low-risk of relapse.

Methods:
Since January 2004, we have analyzed 40 child patients treated with all-trans-retinoic acid (ATRA)±arsenic trioxide (ATO) in induction, with a median follow-up of 47 months. They were monitored by RQ-PCR. Hematologic and molecular relapses were recorded. We then looked for associations between relapse risk and RQ-PCR results in children.

Results:
The pretreatment characteristics of the 40 patients are listed in Table 1. Thirty-nine patients (97.5%) entered complete remission (CR). The 5-year probabilities of disease-free survival (DFS) and overall survival (OS) were 73.1% and 91.4%, respectively. By employing the standardized real-time quantitative polymerase chain reaction (RQ-PCR) for minimal residual disease (MRD) monitoring, no significant difference were observed in the PML/RARa normalized copy number (NCN) between patients in continuous complete remission and those who relapsed (neither at diagnosis nor after induction). After induction therapy, eight out of 25 cases with positive RQ-PCR (more than 1 NCN) relapsed in contrast to none out of 13 patients with negative RQ-PCR(100% and 55.2% DFS at 5 years in the negative and positive RQ-PCR groups, respectively; P=0.018, Fig.1). Also of note, in the positive RQ-PCR group, the patients treated with ATRA+ATO in induction had a lower relapse rate when compared with those treated with ATRA alone (P=0.03).

Conclusions:
PML/RARa - based MRD monitoring by RQ-PCR might allow us to identify subgroups of patients at low risk of relapse after induction in childen. Patients with a low risk of relapse could be monitored less frequently. Combination of ATO and ATRA might decrease the relapse rate compared with ATRA alone in induction therapy for childhood APL.

Disclosures:
No relevant conflicts of interest to declare.
Early Assessment of Minimal Residual Disease (MRD) by Qualitative PCR for NPM-ALK Identifies Patients At Very High Risk of Relapse in Anaplastic Large Cell Lymphoma
Authors: Damm-Welk Christine , Mussolin Lara , Pillon Marta , Zimmermann Martin , Reiter Alfred , Woessmann Wilhelm , Rosolen Angelo 
Published: October 2019

Abstract
Abstract 1565
25–35% of children and adolescents with ALK-positive anaplastic large cell lymphoma (ALCL) suffer a relapse. Detection of minimal disseminated disease (MDD) by qualitative RT-PCR for NPM-ALK in bone marrow (BM) or blood (PB) confers a relapse risk of 50%. We set out to address the issue of whether detection of minimal residual disease (MRD) by qualitative RT-PCR for NPM-ALK in BM or PB early during treatment allows identification of patients at highest risk of relapse.
ALCL-patients were treated according to protocols NHL-BFM95, LNH97 or ALCL99, all BFM-type protocols consisting of comparable cytoreductive prephase and chemotherapy courses between 8/1998 and 12/2008. The qualitative RT-PCR for NPM-ALK with a sensitivity of 10−5 was applied as previously described. PB and/or BM of 184 patients with NPM-ALK-positive ALCL were screened for the presence of NPM-ALK transcripts (MDD) at diagnosis. BM or PB was requested from those patients with detectable MDD in BM or PB for the measurement of MRD before the second course of chemotherapy (course 2).
MDD was detected in BM or PB or both media in 103 of 184 patients with NPM-ALK positive ALCL. The cumulative incidence of relapse (CIR) of 81 MDD-negative patients was 16±5% compared to 51±5% of 103 patients who were MDD-positive in either BM or PB or both (p<.001). MRD before course 2 could be evaluated in either BM or PB of 52 MDD-positive patients. CIR of the 26 MRD-positive patients (81±8%) was significantly higher than CIR of the 26 MDD-positive, but MRD-negative patients (31±9%) (p<.001). CIR of MDD-positive patients without material for MRD available was 35±7%. 5-year survival of MDD-negative patients and MDD-positive but MRD-negative patients were similar (92±2% and 92±5%, respectively) whereas survival of MRD-positive patients was 65±9% (p<.001).
Our data suggest that early evaluation of MRD by a sensitive RT-PCR in NPM-ALK positive ALCL with detectable MDD at diagnosis identifies patients with a very high risk of relapse.

Disclosures:
No relevant conflicts of interest to declare.