bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Study of GPIb-VWF Mediated Early-Stage Platelet Activation Triggering On a Single Cell
Authors: Ju Lining , Zhu Cheng , Cruz Miguel A. , Chen Yunfeng 
Published: October 2019

Abstract
Abstract 1069
Binding of GPIbα to VWF tethers platelets to disrupted vascular surface during the haemostatic process. The GPIbα –VWF interaction can also trigger outside-in signaling cascade, resulting in platelet activation, characterized by morphological transformation from discoid to a more spiky shape as well as activation of integrin α IIbβ3. Using the adhesion frequency assay with a biomembrane force probe (BFP), we studied signal initiation by repeated brief contacts of a single platelet with a glass bead coated with VWF-A1 domain and/or fibronectin III 7–10 domain (FNIII7–10) in a precisely controlled fashion (Fig. A). Contacting platelets with beads coated VWF-A1 only resulted in adhesion kinetics mediated by GPIbα –VWF interaction kinetics independent of the activation stage of the platelet. Contacting platelets with beads coated FNIII7–10 only resulted in adhesion kinetics that correlated with the activation stage of the platelet. Discoid-shaped platelets yielded low level adhesions mediated by FN interaction with inactive α IIbβ3 (Fig. B, blue). By comparison, spiky-shaped platelets produced high level adhesions mediated by FN interaction with activated α IIbβ3 (Fig. B, red)that was four times stronger than the interaction with inactive α IIbβ3. Contacting platelets with beads coated both VWF-A1 and FNIII7–10 resulted in two-stage adhesion kinetics. The first stage was mediated by GPIbα –VWF binding, which triggered a second stage consisting of an increase in adhesion after a sub-second delay. The second-stage binding coincided with morphological changes characteristic of platelet activation and matched that mediated by FN interaction with activated α IIbβ3. On the other hand, the concurrent calcium imaging showed as the platelet target was brought to the A1 bead in a repeating manner, the recorded calcium fluorescence intensity climbed up as the repeated touches continue (Fig. C). The peak temporally correlates with the morphological change. Our data indicates that binding of VWF-A1 to platelet GPIbα initiates outside-in signaling, leading to rapid irreversible platelet shape changes and calcium mobilization within a few seconds.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Kinetic Study (PKS) May Early Identify a Subset of Patients with Immune Thrombocytopenia (ITP) Probably Cured by Romiplostim. Two Years Follow-up
Authors: Iuliano Francesco , Perricelli Alessia , Iuliano Eleonora , Infusino Stefania , Pomillo Angelo , Luci Maria , Barbano Filippo 
Published: October 2019

Abstract
Abstract 1099

Background
(TPO)-receptor agonists (romiplostim and eltrombopag) are new therapeutic modalities in the treatment of ITP. Romiplostim treatment was associated with a number of benefits compared with the standard of care in non splenectomized ITP patients: higher platelet response rate, lower rates of treatment failure and splenectomy, fewer bleeding events, and fewer blood transfusions. Nevertheless there are not enough data on the long-term side effects and economic consequences of prolonged treatment. Furthermore, there are no criteria to identify patients potentially cured and that could stop therapy. For all these reasons we have carried out a study of platelet kinetics in all patients treated with TPO- receptor agonists and we have identified a subset who achieved a normal platelet kinetics and that is no longer relapsed two years after discontinuation of the drug

Patients and Methods
A total of 18 adult patients, female (63%),median (range) age 55 (31 – 78) years, median (range) baseline platelet count 19 (3 – 32) × 109/L.,median of 4 (1 – 7) prior ITP therapies, received romiplostim administered once weekly sc, with dose adjustments to maintain platelet counts in the target range of 50–150×109/L. The median time since ITP diagnosis was 6,8 years (range, 0.6–12.8 years) and 10% had undergone a splenectomy. Patients received romiplostim for a median of 98 weeks (range, 18–104); taking the average weekly dose of all patients, the median was 4 mcg/kg. Home administration was started by 16% of patients (3/18) but 2/3 patients discontinued home administration and resumed weekly outpatient injection. All patients achieved a platelet count ≥50×109/L.

Results
3 out of 18 experienced thrombocytosis and rebound thrombocytopenia. A PKS with (111)In oxine-labeled autologous platelets was performed in all patients failing steroid treatment, before romiplostim was started. A gamma function was used for the calculation of platelet mean life span (MLS) that was greatly reduced in 100% of patients.3 patients, who had achieved early a stable platelet count ≥150×109/L. despite the discontinuation of romiplostim maintain normal platelet count after 24 months of follow-up. Stricking a second PKS six months after starting the treatment showed in these subset, a normal platelet half-life with normal uptake on the spleen and liver.

Conclusions
In our opinion PKS, in romiplostim responding patients who discontinued treatment for the stability of response, may easily detect patients probably cured

Disclosures:
No relevant conflicts of interest to declare.
Prospective, Open-label clinical Trial of Bivalirudin in Children with Venous Thromboembolism
Authors: O'Brien Sarah H. , Yee Donald , Lira Jessica , Goldenberg Neil A , Young Guy 
Published: October 2019

Abstract
Abstract 1165

Background:
Bivalirudin (BIV) is a direct thrombin inhibitor with a favorable pharmacologic profile in comparison to heparin. In a previous study in children <6 months of age with venous thromboembolism (VTE), bivalirudin was demonstrated to be a safe and potentially more effective anticoagulant. The present study's aims were to assess the safety, dosing, pharmacokinetics, pharmacodynamics, and efficacy of BIV in children >6 months of age.

Methods:
This prospective, open-label study evaluated the use of BIV for initial treatment of a new VTE in children between 6 months and 18 years of age. Eligible subjects received a BIV bolus of 0.125mg/kg followed by an initial continuous infusion of 0.125mg/kg/hour which was adjusted as needed to achieve a PTT of 1.5–2.5x their baseline PTT. BIV levels by high performance liquid chromatography and PTTs were tested simultaneously 5 times in the first 24 hours, daily thereafter and following all dose adjustments. Subjects remained on BIV until they were transitioned to long-term anticoagulant treatments. Imaging was required to confirm the presence of a VTE and follow-up imaging was obtained at 2–3 days and 30 days following initiation of anticoagulation. Subjects were closely monitored for bleeding and adverse events.

Results:
18 subjects (11M:7F) ranging in age from 9 months-17 years completed the study. There were no major bleeding events and one minor bleeding event. Outcome of VTE: at 2–3 days, 2 subjects had complete resolution and 7 subjects had partial resolution while at 30 days, 7 subjects had complete resolution and 9 subjects had partial resolution. There were no symptomatic recurrent events or any asymptomatic local progressions by surveillance imaging. Seven subjects required no dose adjustments during their course of treatment ranging between 2–5 days. See figure for correlation of PTT with BIV levels [each circle represents one subject with their mean PTT and BIV level with the standard deviations (r2=0.64)].

Conclusions:
BIV offers a potentially safer, more effective and pharmacologically more predictable alternative to heparin for initial anticoagulation of children with VTE. Pharmacokinetic-pharmacodynamic correlation was excellent suggesting PTT as an acceptable monitoring strategy for BIV in this population. Further studies comparing BIV to heparin should be conducted.

Disclosures:
Off Label Use: Bivalirudin is not approved for use in children. Goldenberg:Eisai Inc: Research Funding; Pfizer Inc: Membership on an entity's Board of Directors or advisory committees; CPC Clinical Research: Consultancy.
Rescue and Primary ITI in Children and Adults with a Single FVIII/VWF Product
Authors: Aledort Louis M. , Oldenburg Johannes , Santagostino Elena , Yuste Victor Jimenez 
Published: October 2019

Abstract
Abstract 1174

Introduction
Inhibitors are the most serious complication of hemophilia A (HA) treatment. Current optimal intervention for inhibitor eradication is immune tolerance induction (ITI), the goal of which is for the patient to resume therapy with FVIII. Basic and clinical research have strongly supported that VWF containing FVIII concentrates may be less immunogenic and are effective in primary ITI as well as for rescue ITI for those who fail to develop tolerance with recombinant proteins.

Objective
To assess the outcome of rescue and primary ITI in children (<18 years old) and adults with a single FVIII/VWF product (Fanhdi®, Grifols, Barcelona, Spain).

Methods
Retrospective data from HA patients (FVIII< 2%) with inhibitors from 22 centers in Spain, Italy and Germany, who underwent primary or rescue ITI with FVIII/VWF concentrate were evaluated. Success, partial success and failure were defined based on the criteria of the consensus recommendations of the 2006 International ITI Workshop.

Results
The largest international multicenter study on ITI using a single product has evaluated 41 cases of primary ITI (32 children and 9 adults) and 19 cases of rescue ITI (17 children and 2 adults).

Conclusion
The response rate of 87% (CS+PS) in primary ITI exceeds that previously reported with any product class. The response rate (CS+PS) of 74% in the higher risk profile of rescue ITI is what one might expect for primary ITI. These excellent results, using a single VWF containing FVIII product, should encourage the use of this class of product for immune tolerance.

Acknowledgments:
The authors acknowledge Grifols financial support of the study. The authors also acknowledge Drs Jeffrey Spears and Roser Peiro, Grifols, for their editorial assistance in preparation of the abstract.

Disclosures:
Aledort: Grifols: Advisory Board Other, Honoraria; Kedrion: Advisory Board, Advisory Board Other; Baxter: DSMB, DSMB Other; Octapharma: DSMB, DSMB Other. Off Label Use: This FVIII is not licensed for ITI - nor is any other product. Oldenburg:d and e: Baxter, Bayer, Biotest, CSL-Behring, Grifols, Inspiration, NovoNordisk, Octapharma, Pfizer e: Biogen IDec, Swedish Orphan Biovitrum: Honoraria, Research Funding. Santagostino:Bayer, Baxter, Pfizer, CSL Behring, Novo Nordisk, Biotest, Kedrion and Grifols: Speakers Bureau; Pfizer, Baxter, Bayer, CSL Behring, Kedrion, Grifols and Novo Nordisk: Consultancy; Novo Nordisk and Pfizer: Research Funding. Jimenez Yuste:Grifols, Baxter, Pfizer, NovoNordisk: Advisory Board Other, Consultancy.
Platelet Factor 4 (PF4) Causes Cell Cycle Arrest in Megakaryocytes (Megs) by Inactivating CDC2 (CDK1) and CDK2
Authors: Xiao Liqing , Poncz Mortimer , Lambert Michele 
Published: October 2019

Abstract
Abstract 1238
PF4 (CXCL4), a platelet specific chemokine released in large amounts from activated platelet α -granules, is a negative regulator of megakaryopoiesis. In mouse studies, we have shown that PF4 levels regulate steady-state platelet count and impact chemotherapy and radiation-induced thrombocytopenia. In a clinical study in leukemia patients, we found that PF4 levels were inversely related to steady-state platelet count and to recovery after chemotherapy. The molecular basis for the effect of PF4 in megakaryopoiesis is largely unknown. Our studies in cell models suggested that PF4 might act through the cell surface receptor low-density lipoprotein related protein-1 (LRP1). Using an early megakaryoblastic cell line, which expresses LRP1, Meg-like cell line (Meg01), we show that PF4 exerts an anti-proliferative effect on the cells through inactivation of cell cycle regulators CDC2 (CDK1) and CDK2. PF4 treatment (200 μg/ml for 48 hrs) of Meg01 cells induced a decrease in cells in G1 (from 68% of cells to 51%, p=0.001) with a concurrent increase in the percentage of cells in S (12% of cells to 21%, p = 0.02 for no PF4 vs. PF4 treatment) and G2 (from 20% to 28% of cells) phase, without significant bromodeoxyuridine (BrdU) incorporation by the cells in the S phase, suggesting that PF4 causes a cell cycle arrest resulting in decreased cell proliferation. The cell cycle arrest and lack of BrDU incorporation was confirmed in primary murine Megs. No apoptosis was detected in PF4 treated Meg01 or primary cells. To determine the molecular mechanisms by which PF4 causes cell cycle arrest, we used Western blots interrogating cell cycle proteins. We detected a transient increase in the inhibitory phosphorylation (at Tyr15) of CDC2 after PF4 treatment, as well as a decrease in phosphorylation of the activating site (Thr160) on CDK2. In addition, we found PF4 treatment resulted in the degradation of Cdc25c, the upstream phosphatase of Tyr15 of CDC2. In primary murine Megs, we detected a significant decrease of total CDC2, biologically equivalent to the CDC2 inactivation seen in Meg01 cells. The CDK inhibitor Roscovitine inhibited Meg01 cell proliferation and had minimum additive effect with PF4. Overexpression of the constitutively active CDC2 mutant CDC2AF with the inhibitory phosphorylation sites Thr14 and Tyr15 replaced by Ala and Phe, respectively, desensitized the cells to PF4 treatment. These results suggested that PF4 inhibits megakaryopoiesis by decreasing the proliferation of megakaryocytes in their early developmental stage by inactivating cell cycle regulators CDC2 and CDK2. Unraveling the mechanisms by which PF4 inhibits megakaryopoiesis may lead to the development of novel therapeutics to regulate platelet counts.

Disclosures:
No relevant conflicts of interest to declare.
IFNγ Licensed Human Bone Marrow Derived Mesenchymal Stromal Cells Inhibit T Cell Cytokine Production by a Mechanism Independent of Indoleamine 2 3-Dioxygenase (IDO) Activity
Authors: Chinnadurai Raghavan , Copland Ian , Patel Seema , Galipeau Jacques 
Published: October 2019

Abstract
Abstract 1255
Human Bone marrow derived Mesenchymal Stromal Cells (MSCs) inhibit activation-mediated T cell proliferation. This functional interaction serves nearly universally as a surrogate assay for immune suppressive activity and potency of MSCs in vitro. However the role of MSCs in inhibiting T cell function, cytokine production in particular, has not been investigated thoroughly. In the present study we have compared resting MSCs with IFNγ licensed MSCs for their ability to inhibit activated T cell function such as cytokine secretion and degranulation through intracellular cytokine staining assays. PBMCs cocultured with resting or inflammatory cytokine licensed MSCs were stimulated with the super antigen Stephylococcal Enterotoxin B (SEB) for 12–14hours in the presence of Brefeldin A. Intracellular cytokine staining was performed on the T cells to detect the Interferon gamma (IFNγ), Tumor Necrosis Alpha (TNFα) and Interleukin-2 (IL-2). Our results demonstrate that resting MSCs do not inhibit T cell cytokine secreting function. However, IFNγ and IFNγ plus TNFα licensed MSCs dose dependently inhibit IFNγ secretion by T cells. Our results also show that IFNγ and IFNγ plusTNFα licensed MSCs not only inhibit cytokine secretion but also inhibit dregranulation of activated T cells. Boolean gating analysis demonstrates that IFNγ and IFNγ plusTNFα licensed MSCs inhibit triple cytokine producing highly inflammatory IFNγ +TNFα +IL2+T cells. Phenotypic analysis of MSCs demonstrates that IFNγ licensing upregulate indoleamine 2 3-dioxygenase (IDO) and B7-H1. Blocking of IDO activity with 1-Methyl D,L Tryptophan (1MT) will abolish MSC-mediated blockade of T cell proliferation. However, 1MT does not reverse the MSC blockade of T cell cytokine secretion. Phenotypical analysis of T cells demonstrate that PD1+ cells are inhibited while PD1- cells are not inhibited suggesting the role of B7-H1 and PD-1 inhibitory pathway is involved in the inhibitory potential of IFNγ licensed MSCs. Interestingly, the inhibitory potential of IFNγ licensed MSCs has been observed only with the SEB stimulation but not with anti-CD3, CD28 stimulation. This suggests that MHC-TCR interaction is necessary for the inhibitory effect of IFNγ licensed MSCs. Our results demonstrate the superiority of IFNγ licensed MSCs in inhibiting multiple cytokine producing T cell function independent of IDO activity. Our results also provide the rationale for use of IFNγ licensed MSCs in autologous immunosuppressive cell therapy.

Disclosures:
No relevant conflicts of interest to declare.
Rb Protects B-Lineage Hematopoietic Progenitor Cells From Oxidative Stress and Exhaustion
Authors: He Zhiwen , Wang Yinan , Luo Jun , Lu Lan , Su Mack Y. , Vij Ravi , Bhattacharya Deepta , Tomasson Michael H. 
Published: October 2019

Abstract
Abstract 1315

Background:
Multiple myeloma (MM) is the second most common hematologic malignancy in the United States. Chromosome 13 deletions are found in approximately one half (50%) of all patient samples and is associated with worse outcomes. The retinoblastoma gene (Rb1) has been implicated as a candidate tumor suppressor at 13q14, however, unmutated Rb1 remains expressed from retained alleles in the majority of cases. We sought to explore the role of Rb1 in plasma cells by generating mice with deficiency of Rb1 targeted to late B-cell development.

Methods:
The Rb family member p107 can compensate for Rb1 function, so we crossed Rb1Flox/Flox and P107−/−mouse strains with Cγ1-Cre knock-in mice that express the Cre recombinase specifically in germinal center (GC) B-cells to generate mice with tissue-specific deletion of Rb in germinal center cells (Cg1-Rb-KO), and we aged these mice to assess disease development.

Results:
In Cg1-Rb-KO mice, we demonstrated the Rb1 locus was successfully recombined in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). Recombination was also detectable in bone marrow B-cell but not myeloid cell progenitors. Cg1-Rb-KO mice developed normally but became ill starting at 40 weeks of age. Sick mice demonstrated weight loss, hypoplastic spleens, osteopenia and significant lymphopenia. Thorough analysis of bone marrow and spleen hematopoietic progenitor cell subsets by multi-parameter flow cytometry at the time of death revealed dramatic reductions in marrow B-cell (Hardy fractions C-D, from 3.18×103to 40 cells and from 1.4×106 to 7.5×104cells respectively), suggesting an effective collapse of the B-cell lineage in mice lacking Rb. Examination of Cg1-Rb-KO mice prior to becoming sick (age 30–35 weeks) demonstrated increased numbers of B-cell progenitors in the bone marrow, consistent with Rb's known role in cell cycle regulation. Together, our results suggest Cg1-Rb-KO mice develop benign B-cell hyperplasia followed by a lethal collapse of the B-cell lineage. To help explain this B-cell exhaustion phenotype, we measured reactive oxygen species (ROS) in the B-cell of young Cg1-Rb-KO mice. We observed significantly increased ROS in Cg1-Rb-KO mice at baseline and following LPS stimulation ex vivo and in vivo compared to p107-KO control mice, suggesting that B-cells from Cg1-Rb-KO mice have elevated levels of oxidative stress.

Conclusion:
Rb1 is important for maintenance of B-cell homeostasis and protects B-lineage progenitor cells from oxidative stress and exhaustion. Rb1 deletion was not sufficient for B-cell transformation and additional mutations are likely required for MM development in the context of complete RB1 loss.

Disclosures:
Vij: Millennium: Speakers Bureau.
Tumor Suppressor Capacity of Krüppel Like Factor 4 (KLF4) in NOTCH1-Induced T-Cell Acute Lymphoblastic Leukemia
Authors: Shen Ye , Suppipat Koramit , Park Chun Shik , Yamada Takeshi , Lacorazza H. Daniel 
Published: October 2019

Abstract
Abstract 1325
Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children, among which T-cell ALL (T-ALL) exhibits worst prognosis. Studies on genetic factors supporting T-ALL leukemogenesis will contribute to the development of alternative therapies. Previously our laboratory described that the Krüppel like factor 4 (KLF4) restricts proliferation in normal CD8+ T cells. In addition, we have found that loss of KLF4 increases self-renewal and enhances survival of normal hematopoietic stem cells, which is a feature that cancer cells acquire during transformation. There are also emerging evidences showing reduced levels of KLF4 in bone marrow of T-ALL patients compared to healthy individuals and a correlation of KLF4 expression with response of ALL patients to standard treatment. Collectively, these findings suggest a potential role of KLF4 in tumor suppression and drug resistance in hematological malignancies. To study whether KLF4 functions as a tumor suppressor in T-ALL, we used Klf4 conditional knockout mice and NOTCH1-induced T-ALL model. Bone marrow (BM) cells from control (Klf4fl/fl) and Klf4 deleted (Klf4fl/fl; Vav-iCre) mice injected with 5-FU were transduced with retrovirus carrying the NOTCH1-L1601P-DP mutant found in human T-ALL patients and then transplanted into irradiated recipient mice. Mice transplanted with transduced control BM cells showed lower incidence of leukemia (21.7%, n=23) with longer median latency (130 days). In contrast, mice transplanted with transduced Klf4-deleted BM cells showed significantly higher penetrance (76.5%, n=34) with shorter median latency (93 days, p<0.01). Moribund mice from both groups showed similar infiltration of leukemic cells in liver, kidney, testis, brain and spleen. Immunoblots of leukemic cells purified from BM of diseased mice showed increased levels of Cyclin D1 and phospho-Rb in Klf4-deleted cells, suggesting enhanced proliferation. In addition to increased proliferative activity, a global gene expression analysis revealed significant differences in metabolic pathways. Concomitantly, Klf4-deleted leukemic cells displayed increased levels of phosphorylated mTOR, an important signaling involved in the metabolic reprogramming of cancer cells. Collectively, our study shows that KLF4 has tumor suppressor function in a mouse model of NOTCH1-induced T-ALL, providing a novel molecular target to prevent expansion of pre-leukemic cells.

Disclosures:
No relevant conflicts of interest to declare.