bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nematode Antocoagulant Protein c2 (NAPc2) Interferes with Factor Xa Dimerization: Structural Alteration of Factor Xa Upon Dimerization.
Authors: Chattopadhyay Rima , Koklic Tilen , Majumder Rinku , Lentz Barry R. 
Published: October 2019

Abstract
Abstract 1128
Nematode Anticoagulant protein c2 (NAPc2) from the hematophagous hookworm, Ancyclostoma canimum, is a potent inhibitor of factor × activation to Xa by the Xase complex composed of VIIa and tissue factor. Factor Xa (FXa) plays a crucial role in blood coagulation; including formation of a prothrombin activating complex with its co-factor Va. NAPc2 also inhibits activation of prothrombin by this complex. FXa forms a functionally inactive dimer upon binding to either membrane bound or solution phosphatidylserine (PS) in the presence of calcium, and that the structure of FXa in the dimer is altered (Chattopadhyay et al, 2009; Koklic et al., 2009). The dimer interface predicted from mass spectroscopy involves a residue in the hirudin-binding site. NAPc2 interacts with two regions in FXa, one overlapping with the hirudin site and another in the substrate binding exosite (Murakami et al., 2007; Yegneswaran et al., 2003; Wilkens et al., 2002). We show that NAPc2 at low concentration does not interfere with dimerization, but at high concentration it destabilizes the dimer as documented with homoFRET studies with FEGRXa (Fluorescein-GLU-GLY-ARG-chloromethylketone-Xa). Proteolytic activity (prothrombin as substrate) suggested that the species Xa · NAPc2 (NAPc2 binding to FXa monomer) and FXa2 · NAPc2 (NAPc2 binding to FXa dimer) have activity comparable to FXa dimer. A model is suggested in which initial NAPc2 binding to the substrate-binding region in FXa alters interactions in the hirudin-binding region and thus alters dimer formation. Supported by USPHS grant HL072827 to BRL.

Disclosures:
No relevant conflicts of interest to declare.
TFPI Antagonist Aptamer ARC19499 Is a Procoagulant In Vitro and In Vivo.
Authors: McGinness Kathleen E. , Waters Emily K. , Genga Ryan M. , Olson Karen A. , Nelson Jennifer A. , Kurz Jeffrey C. , Schaub Robert G. 
Published: October 2019

Abstract
Abstract 1134
Hemophilia is a bleeding disorder that results from deficiencies in coagulation factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B), primarily impacting the intrinsic coagulation pathway. The extrinsic coagulation pathway remains intact in hemophiliacs and is negatively regulated by tissue factor pathway inhibitor (TFPI). The primary role of TFPI is regulation of the FVIIa:tissue factor complex through inhibition of FVIIa and FXa. Inhibition of TFPI may provide an effective treatment for hemophilia by allowing sufficient thrombin generation via the extrinsic coagulation pathway to bypass the defect in clot propagation caused by deficiency of FVIII or FIX.
ARC19499 is an aptamer that is a potent, specific inhibitor of TFPI. We have employed a variety of in vitro and in vivo methods to demonstrate that ARC19499 mediates a procoagulant effect in hemophilic plasma and in a model of hemophilia A in non-human primates (NHP). Plasma-based experiments that measure thrombin generation over time demonstrate that ARC19499 mediates a procoagulant response in both hemophilia A and hemophilia B plasma, restoring thrombin generation to near normal levels at 10 – 100 nM aptamer. Experiments in TFPI-depleted plasma and in vitro binding experiments demonstrate that this procoagulant effect is dependent on the specific interaction between ARC19499 and TFPI. ARC19499 restores thrombin generation to a level that is equivalent to or better than 14% FVIII replacement at a concentration of 30 nM in hemophilia A plasma and has an additive effect on thrombin generation when used in combination with FVIII. In a NHP model of hemophilia, cynomolgus monkeys acquire a hemophilia A-like state following administration of an anti-FVIII antibody as measured by ex vivo thromboelastography (TEG). TEG coagulation parameters are corrected following administration of ARC19499. In addition, antibody-mediated FVIII depletion moderately prolongs saphenous-vein bleeding time. ARC19499 corrects bleeding times to normal.
These experiments demonstrate that the anti-TFPI aptamer ARC19499 mediates a procoagulant hemostatic effect both in vitro and in vivo in hemophilia model systems that is dependent on specific interaction between the aptamer and TFPI. ARC19499 may provide an effective alternative to replacement factors and bypassing agents for the treatment of hemophilia. Additionally, aptamer therapeutics have the advantage of subcutaneous bioavailability which provides an opportunity for improved treatment regimens in hemophilia.

Disclosures:
McGinness: Archemix Corporation: Employment. Waters:Archemix Corporation: Employment. Genga:Archemix Corporation: Employment. Olson:Archemix Corporation: Employment. Nelson:Archemix Corporation: Employment. Kurz:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.
Structural Insights Into How Clotting Proteins with GLA Domains Bind to Membrane Surfaces.
Authors: Davis-Harrison Rebecca L , Tavoosi Narjes , Clay Mary , Boettcher John M , Rienstra Chad M , Morrissey James H. 
Published: October 2019

Abstract
Abstract 1141
Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. In spite of their importance, a clear picture of how GLA domains bind to the membrane interface has yet to emerge. A further intriguing aspect of the membrane's role in blood coagulation is that certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. The mechanisms of this synergy, and of PE's contribution to GLA domain binding, are poorly understood – although a number of hypotheses have been put forward. We now propose a new hypothesis to explain GLA domain binding to membranes, which we term the ABC (Anything But Choline) hypothesis; it invokes two main types of protein-phospholipid interactions: a single L-serine-specific binding site in each GLA domain; and multiple “phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. We have utilized liposomes and nanoscale phospholipid bilayers (Nanodiscs) in studies employing a series of techniques including solid-state NMR (SSNMR) and surface plasmon resonance (SPR) to address the mechanism of GLA domain-membrane interactions. We provide direct evidence in favor of the ABC hypothesis for GLA domain binding to membrane surfaces. Using SSNMR, we demonstrate that two distinct PS headgroup conformations are induced by binding of calcium ions, and that a third, novel PS headgroup conformation is induced when the prothrombin GLA domain engages the membrane. SPR studies have allowed for the determination of thermodynamic profiles of GLA domains interacting with phospholipid bilayers containing PS and/or PE, providing further insights to the mechanisms of GLA domain-membrane interactions.

Disclosures:
No relevant conflicts of interest to declare.
Loss of Foxo3a In FA Mice Leads to Hematopoietic Stem Cell Exhaustion That Can Be Attenuated by Natural Anti-Oxidant Quercetin.
Authors: Li Jie , Sipple Jared , Pang Qishen 
Published: October 2019

Abstract
Abstract 1166
Fanconi anemia (FA) is a genetic disorder characterized by genomic instability, bone marrow (BM) failure and predisposition to cancer. However, FA mouse models do not show spontaneous genetic instability. Previous study shows that FOXO3a is associated with the FA pathway through oxidative stress-specific interaction with FANCD2. To address the consequence of loss of FOXO3a function in FA hematopoiesis, we generated Foxo3a-/-Fancd2-/- and Foxo3a-/-Fancc-/- double-knockout (DKO) mice by crossing Foxo3a+/− with Fancd2+/− or Fancc+/−; mice. Reactive oxygen species are increased in low-density BM (LDBM) cells isolated from DKO mice compared to those from single KO (SKO) or wt mice. Analysis of hematologic parameters shows significantly increased number of nucleate cells and high ratio of eosinophils in peripheral blood of DKO mice. CFU assay shows more progenitor cells in peripheral blood isolated from DKO mice. Moreover, BM progenitor cells from DKO mice exhibit lower adhesion but higher migration activity, compared to those from wt or SKO mice. Consistent with this, Cdc42 pull-down assay shows lower Cdc42 activity in DKO LDBM cells than in wt or SKO cells, indicating that decreased Cdc42 may contribute to the observed aberrant adhesion and migration activities. DKO mice show significant decrease in primitive progenitor (Lin-Sca-1+c-kit+; LSK) cells, increase in BrdU+ and G1-phase LSK cells, and impaired repopulating capacity after competitive BM transplantation, which can be attenuated by the anti-oxidant Quercetin. Taken together, loss of Foxo3a in FA mice results in FA-like syndrome, which may be resulted from increased reactive oxygen species accumulation.

Disclosures:
No relevant conflicts of interest to declare.
Disturbed NK Cell Compartment In Human CML and Bcr-Abl Positive Mice.
Authors: Chen Christiane I-U , Koschmieder Steffen , Kamp Linda , Altvater Bianca , Pscherer Sibylle , Maecker Holden , Berdel Wolfgang E. , Juergens Heribert , Lee Peter P , Rossig Claudia 
Published: October 2019

Abstract
Abstract 1207
Targeted pharmacologic therapy with tyrosine kinase inhibitors (TKIs) has become the first-line treatment for patients with CML. However, BCR-ABL+ stem cells resist elimination by continuous TKI treatment in most patients. By contrast, graft-versus-leukemia effects during allogeneic hematopoietic stem cell transplantation can eradicate the disease, suggesting an important role of the immune system in this disease. Besides cytotoxic T cells, natural killer (NK) cells may be involved in immune control of CML. Here, we explored numbers and functionality of NK cells in CML patients and in a transgenic inducible BCR-ABL mouse model.
In 18 patients with newly diagnosed chronic-phase CML, the relative proportions of peripheral blood CD56+CD3- NK cells within the lymphocyte gate were significantly reduced compared to age-matched healthy controls (7.0 ± 5.8% versus 13.1 ± 5.1, p=0.005) and did not recover to normal levels during imatinib-induced remission (9.2 ± 5.9%, p=0.024, follow-up 10–59 months). Functional experiments showed reduced in vitro expansion of CML NK cells at diagnosis in response to stimulation with 4-1BBL/mbIL-15 transduced K562 cells (23.5 ± 14.46 fold vs 41.2 ± 7.2 fold, p=0.013) and under imatinib treatment (31.5 ± 10.5 fold, p=0.03), and a reduced degranulation response to K562 target cells by CD107 upregulation (2.8 ± 2.7% at diagnosis and 9.0 ± 13.2% under treatment, vs. 19.1 ± 8.0% in controls, p=0.003 and p=0.045, respectively).
To investigate, whether the defective NK-cell compartment in CML is a consequence of the characteristic BCR-ABL-induced myeloproliferation, we addressed the quantity and functionality of NK cells in a double transgenic mouse model of human CML. Consistent with the results in human CML, the relative proportions of NK1.1+ NK cells among total splenic lymphocytes were significantly reduced in BCR-ABL induced mice (6.4 ± 3.5% vs. 14.7 ± 1.8%, p=0.005). Moreover, compared to NK cells isolated and expanded from BCR-ABL-non-induced control mice, the degranulation response of splenic NK cells from BCR-ABL+ mice to YAC-1/NIH-3T3 cells was significantly decreased (25.7 ± 1.6% vs 42.4 ± 5.6%, p=0.002), and analogous results were obtained with NK cells expanded from bone marrow of these mice (7.7 ± 4.9% vs. 25.0 ± 7.1%, p=0.033).
These results suggest both quantitative and qualitative defects within the NK cell compartment in CML. Further work will aim at identifying the underlying mechanisms of the NK cell deficiency in CML, and the development of strategies to utilize NK cells for immunotherapy of CML.

Disclosures:
Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.
Combination of Mycophenolate Mofetil with Cyclosporine A and Methotrexate as Gvhd Prophylaxis In Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Zhu Xiaoli , Lai Xiaoyu , Luo Yi , Shi Jimin , Tan Yamin , Huang He 
Published: October 2019

Abstract
Abstract 1280
Graft-versus-host disease (GVHD) is the dominant complication limiting the efficacy and safety of hematopoietic stem cell transplantation (HSCT), especially from unrelated donors. The most widely used regimen of prophylaxis is the combination of cyclosporine A (CSA)and a short-course of methotrexate (MTX). But the patients receiving grafts from matched unrelated donors still have a 50–80% risk of clinical significant GVHD. Thus, more effective prophylaxis is needed. In this study, we try to evaluate the efficacy and safety of the new GVHD prophylaxis regimen combining mycophenolate mofetil (MMF) with CSA and MTX in unrelated donor allogeneic stem cell transplantation. Between November 1998 and June 2008, 153 patients with a variety of hematologic malignancies were enrolled. There were 102 males and 51 females, median age 26 (range, 8–52) years. Diagnoses were acute myeloid leukemia (n=54), acute lymphoblastic leukemia (n=47),myelodysplastic syndrome (n=8), multiple myeloma (n=1) and chronic myeloid leukemia (n=53). One hundred and eight patients had HLA compatible unrelated donors, 37 patients had single locus-mismatched unrelated donors and 8 patients had 2 loci-mismatched unrelated donors. GVHD prophylaxis consisted of CSA,MTX and MMF. CSA was started on day -7 with the initial dosage of 2.5mg/kg daily, and the dose was adjusted to maintain a whole blood steady-state level of 200–400ng/ml. The dose was tapered during the second to third month post-transplant depending on GVHD status. MTX was given at a dosage of 10mg on days+1, +3 and +6. MMF was administered at an oral dose of 500mg daily from day 0 to day +100. The cumulative incidence of grades II-IV aGVHD and grades III-IV aGVHD for all patients were 43.9% and 17.9% respectively for entire group. The cumulative incidence of grades II-IV aGVHD for patients who had HLA-matched donors and mismatched donors were 40.2% and 51.9% respectively (P=0.079). The cumulative incidence of grades III-IV aGVHD for patients who had HLA-matched donors and mismatched donors were 14.0% and 26.9% respectively (P=0.026). The cumulative incidence of cGVHD was 41.4%, with 19.3% extensive cGVHD. At 3 years, the incidence of relapse for entire group was 24.0%. The cumulative incidence of transplant-related mortality at 100 days and 3 years were 12.5% and 34.9%, respectively. The probabilities of overall survival at 2 years and 5 years were 59.2% and 50.2%, respectively. The probabilities of disease-free survival at 2 years and 5 years were 56.3% and 46.7%, respectively. From these results, we conclude that the combination of MMF with CSA and MTX is an effective prophylaxis regimen for acute and chronic GVHD in unrelated donor allogeneic stem cell transplantation without increasing the risk of relapse.

Disclosures:
No relevant conflicts of interest to declare.
Long Term Study of the Impact of Quantitative Molecular Monitoring of Bcr-Abl Transcripts on the Risk of Relapse of CML After Allogeneic HSCT.
Authors: Arpinati Mario , Amabile Marilina , Bochicchio maria Teresa , Poerio Angela , Bandini Giuseppe , Bonifazi Francesca , Stanzani Marta , Castagnetti Fausto , Rosti Gianantonio , Martinelli Giovanni , Baccarani Michele 
Published: October 2019

Abstract
Abstract 1287

Background:
Monitoring of minimal residual disease through RT-PCR analysis of bcr-abl transcripts allows early detection of CML relapse after allogeneic HSCT. However, the introduction of more sensitive techniques, such as quantitative PCR, may result in decreased specificity, leading to false positive results.

Patients and methods:
In this study we reviewed the results of molecular analysis of bcr-abl transcripts in all patients with p210+ CML who underwent allogeneic HSCT from 1983 through 2007. Q-PCR analysis was started in 2002. Out of 189 patients, 87 patients had available Q-PCR data; of these, 63 patients with at least three separate Q-PCR data were included in the study. Median time to the 1st Q-PCR analysis was 2196 days (35-7823). Median age was 36 years (13-56), 62%/38% received transplant from a related/unrelated donor, 62% with BM. 32% were in accelerated phase (AP). Patients with at least one positive Q-PCR value (measured as a ratio of bcr-abl to abl of > 0.001) were classified as Major Molecular Remission (MMR) patients. Each event was defined as one or more consecutive positive results.

Results:
60/63 patients are alive after a median follow up of 3693 days (898-9405). 6 have relapsed 2142 (1419-3746) days after transplant. 52 (83%) patients had at least one positive result (28 with a value of >0.01, 24 with a value of <0.01), whereas 11 (17%) had persistent undetectable transcripts. In MMR patients, 94 events occurred. 29 patients had only one event, while 6 had >3 events. In 10 patients, the event occurred within 1 year after transplant, whereas in 28 it occurred after >5 years. 6/52 MMR patients relapsed, as compared to 0/11 with persistent undetectable transcripts (p=0.19). Relapse did not correlate with the Q-PCR value, the number of events or the time to the event. Finally, of 46 MMR patients who did not relapse, 35 had undetectable transcripts at last follow up. Positive Q-PCR had low specificity (19%) and positive predictive value (12%) in predicting relapse after allogeneic HSC transplantation.

Conclusion:
Our data confirm that the detection of low levels of bcr-abl transcripts (as based on Q-PCR) has a poor accuracy in predicting relapse, and it should not be considered as the sole indication to start treatment. It appears that fluctuation of bcr-abl transcript levels is common as late as > 10 yrs after transplant, possibly suggesting the long term persistence of CML stem cells.

Disclosures:
Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.
The Cutoff Leukemic Cell Burden to Influence Outcomes of Allogeneic Stem Cell Transplantation for Acute Myelogenous Leukemia In Hematological Complete Remission.
Authors: Lee Sung-Eun , Kim Hee-Je , Min Woo-Sung , Yahng Seung-Ah , Cho Byung-Sik , Eom Ki-Seong , Kim Yoo-Jin , Min Chang-Ki , Lee Seok , Cho Seok-Goo , Kim Dong-Wook , Lee Jong-Wook , Park Chong-Won 
Published: October 2019

Abstract
Abstract 1298

Background:
Pre-transplant disease status is considered a critical prognostic factor of patients undergoing allogeneic stem cell transplantation (SCT) for AML. The previous studies about the impact of leukemic cell burden on transplant outcomes were based on hematological complete remission (CR), which requires <5% blasts in a bone marrow aspirate. This study was performed to evaluate 2 questions: (1) Can we discriminate the impact of leukemic cell burden from CR status according to current hematological CR definition? (2) Is it possible that the poor outcomes by leukemic cell burden are overcome by the graft-versus-host disease (GVHD)-related graft-versus-leukemic (GVL) effect?

Methods:
We retrospectively analyzed 363 patients with de novo AML who underwent allogeneic SCT between January 2002 and December 2008. There were 183 men and 180 women with a median age of 37 years (range 15–68).

Results:
To compare outcomes according to blast counts in patients who were in CR at bone marrow aspirate just before allogeneic SCT, we divided the patients into subgroups which had >2 - <3% blasts (n = 17), >3 - <5% blasts (n = 8) by plotting a receiver operating characteristic (ROC) curve. At a median follow-up of 37.1 months (range: 0.2–90.1) for survivors, there were significant differences in overall survival (OS) (68.3 vs 37.5, P= 0.075), event-free survival (EFS) (68.3 vs 18.8, P= 0.022), relapse (14.1 vs 64.3, P= 0.063) between two groups. In Multivariate analysis for the patients in CR status just before allogeneic SCT, <3% blasts remained a significant favorable independent variable influencing EFS after adjusting for significant pre-transplant variables in compare with the group which had >3 - <5% blasts [HR (95% CI) 4.21 (1.46-12.1), P=0.008] and the development of chronic GVHD was associated with lower relapse [HR (95% CI) 2.93 (1.7-5.06), P=<0.000], which was translated into improved OS [HR (95% CI) 2.0 (1.28-3.16), P=0.003], EFS [HR (95% CI) 2.24 (1.46-3.43), P=<0.001].

Conclusion:
These results indicate that pre-transplant bone marrow status is an important prognostic factor for patients with AML, and even among the patients in CR, strictly more than 3% blasts is suggested as a poor prognostic factor. Therefore, the impact of leukemic cell burden on outcomes of allogeneic SCT for AML should be considered to make more defined SCT plan. Further studies for treatment strategies to enhance the GVL effect according to leukemic cell burden before transplant are needed.

Disclosures:
No relevant conflicts of interest to declare.
Poor FEV1 or DLco on Pre-Transplant PFT Does Not Compromise Outcomes In Patients Undergoing a Reduced-Intensity Allogeneic Progenitor Cell Transplant.
Authors: Sharma Manish , Saliba Rima M. , Qazilbash Muzaffar H. , Okoroji Grace-Julia , Popat Uday , de Lima Marcos , Champlin Richard , Hosing Chitra 
Published: October 2019

Abstract
Abstract 1322

Background:
Pre-transplant work-up prior to an allogeneic stem cell transplant (allo-SCT) generally includes pulmonary functions tests (PFT) with assessment of FEV1 and DLco. It has been previously published that patients with a DLco or FEV1 less than 60% of predicted have higher non-relapse mortality (NRM) when undergoing a myeloablative allo-SCT. However, the PFT criteria for selecting candidates for a reduced intensity (RI) allogeneic transplant allo-SCT remains poorly investigated. We performed a retrospective analysis to assess the impact of low FEV1 or DLco on NRM and overall survival (OS) in patients undergoing a RI allo-SCT.

Methods:
All patients who underwent a RI allo-SCT for hematological malignancies at MD Anderson Cancer Center from Jan 2000 to April 2009 were included. Using Cox's proportional hazards regression, we compared the rate of NRM and OS between patients who had a pre-transplant FEV1 or DLco > 50% (control group) prior to a RI allo-SCT and those with FEV1 and DLco < 50% (study group). The cumulative incidence of NRM was estimated considering disease progression as a competing risk. Actuarial OS was estimated by the Kaplan-Meier method.

Results:
Patient characteristics are presented in Table 1. There were no statistically significant differences between the study and control groups except for age and sex. A significantly higher proportion of patients were > 50 years in the control group (65%) compared with the low PFT group (45%, P=0.02) and there were more males in the control group (P=.001). The median FEV1 and DLco in the study group was 55% (28-111) and 47% (33-98) of predicted, respectively. On univariate analysis abnormal PFTs did not impact OS at 30 months (HR 1.1, 95% CI 0.7–1.8, P=0.7) or NRM at 1 year (HR 1.3, (95% CI 0.6–2.7, P=0.4). Active disease at SCT, and a diagnosis of CLL, NHL or HL were associated with significantly worse OS and NRM, whereas age >50 years was only associated with worse OS. Low PFT had no significant impact on OS and NRM when evaluated separately in patients older or younger than 50 years old.

Conclusion:
Our experience suggests that a low (<50% of predicted) FEV1 or a DLco on pre-transplant evaluation does not adversely impact NRM and OS after RI allo-SCT. These findings need to be validated in a multivariate analysis.

Disclosures:
No relevant conflicts of interest to declare.