bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinical Practice Patterns of Anti-Xa Monitoring During Low Molecular Weight Heparin Therapy
Authors: Stutsky Martha E. , Reardon David P. , Lee Alfred I 
Published: October 2019

Abstract
Introduction: Therapeutic low molecular weight heparin (LMWH) regimens were developed empirically based on patient weight and renal function without the need for laboratory monitoring. The role of anti-Xa monitoring for patients receiving LMWH has yet to be fully established and is routinely performed only in high risk patients. At our institution, guidelines for monitoring LMWH recommend drawing anti-Xa levels for patients with chronic kidney disease or those with obesity requiring long-term therapy. The purpose of this analysis was to evaluate compliance with institutional guidelines for monitoring anti-Xa levels for patients receiving LMWH, including indication for anti-Xa monitoring, timing of anti-Xa level in relation to drug administration, and follow up actions of providers in response to abnormal anti-Xa levels.
Methods: We conducted a retrospective chart review of adult patients who received therapeutic LMWH (enoxaparin or dalteparin) and were ordered for an anti-Xa level between September 1, 2014 and July 1, 2015. Patients were excluded if they were ordered for an anti-Xa level while on a prophylactic dose of LMWH or while on a non-LMWH factor Xa inhibitor. Data collected included patient demographics, LMWH dose and indication, ordering service, presence of a Hematology consult, reason for anti-Xa monitoring, timing of the level after the previous LMWH dose, and provider response to the level.
Results: One hundred patients met inclusion criteria, 99 of whom received enoxaparin. Venous thromboembolism was the most common reason for LMWH therapy (85%) followed by atrial fibrillation (11%). The most frequent doses of enoxaparin administered were 1 mg/kg every 12 hours (52%), 1 mg/kg every 24 hours (22%) and 1.5 mg/kg every 24 hours (21%). The most frequently specified indications for anti-Xa monitoring were renal dysfunction and acute kidney injury (30%), followed by abnormal body mass index (13%); no indication was specified by the prescriber or pharmacist in 36% of cases. Of the 100 anti-Xa levels in the study, 65 were drawn at the correct time (3-5 hours after the previous dose of enoxaparin); of the 35 drawn at an incorrect time, 33 had levels measured more than 5 hours after the previous enoxaparin dose. Fifty-four of the initial anti-Xa levels were not within the therapeutic range for the dosing interval of LMWH; of these, follow up action was taken for only 21 patients (39%), most frequently resulting in a dose increase or decrease.
Conclusions: In clinical practice, the use of anti-Xa monitoring is inconsistent and commonly done without a documented need. The majority of anti-Xa levels at our institution are drawn at the appropriate time after the previous dose of LMWH, but action is generally not taken in response to non-therapeutic levels. Additional guidance on the use of LMWH monitoring is needed.

Disclosures
No relevant conflicts of interest to declare.
The Erythroid Intron Retention Program Encompasses Developmentally Stable and Dynamic Networks and Regulates Diverse Gene Classes
Authors: Pimentel Harold , Parra Marilyn , Gee Sherry , Mohandas Narla , Pachter Lior , Conboy John G. 
Published: October 2019

Abstract
Computational analysis of RNA-seq data from highly purified human erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proerythroblasts, but elevated intron retention in orthochromatic erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues.

Disclosures
No relevant conflicts of interest to declare.
Identification of Polymorphic Gnpat As a Risk Factor for Porphyria Cutanea Tarda
Authors: Phillips John D , Farrell Colin P , Overbey Jessica , Naik Hetanshi , McLaren Gordon D. , McLaren Christine E , Zhou Luming , Parker Charles J. 
Published: October 2019

Abstract
Symptoms of porphyria cutanea tarda (PCT) resolve when iron stores are depleted by phlebotomy, and a sequence variant of HFE (C282Y) that increases iron absorption by reducing hepcidin expression is a risk factor for PCT. These observations suggest that PCT is an iron dependent disease and that factors that affect iron homeostasis influence the risk of developing the PCT. Recently, a polymorphic variant (D519G) of GNPAT was shown to be enriched in male patients with type I hereditary hemochromatosis (HFE C282Y homozygotes) who presented with a high iron phenotype [McLaren CE, et al. Hepatology Aug;62(2):429-39 2015]. Available evidence suggests that like HFE C282Y, GNPAT D519G increases iron absorption by reducing expression of hepcidin. Therefore, we investigated the prevalence GNPAT D519G in patients with PCT. The study population consisted of 247 patients with PCT. High-resolution DNA melting analysis and Taqman SNP assays were used to identify HFE (C282Y) and GNPAT (D519G) allelic variants, respectively and mutations in uroporphyrinogen decarboxylase (UROD) were identified by nucleotide sequencing. Patients with mutant UROD were categorized as familial PCT (F-PCT) (n=87, 36.0%) and those with wild-type UROD were categorized as sporadic PCT (S-PCT) (n=155, 64.0%). GNPAT D519G was significantly enriched in the patient population (prevalence 22.1%, p<0.001) compared to the general population (prevalence 13.6%) as was HFE C282Y (prevalence of 19.2% in the study population compared to 1.5% in the general population, p<0.001). The relative risk of GNPAT D519G was significantly increased in patients with F-PCT compared to those with S-PCT (odds ratio 1.81, CI 1.06-3.08) whereas the relative risk of HFE C282Y in the F-PCT was lower than in the S-PCT population (odds ratio 0.82, CI 0.46-1.47). Together, these observations suggest that GNPAT D519G, is more likely to require a cofactor (e. g., mutant UROD) to produce PCT whereas HFE C282Y functions as an independent PCT risk factor. This difference may be a consequence of the relative potency of GNPAT D519G (weak) and HFE C282Y (strong) with respect to effects on iron homeostasis. Our studies have identified a new risk factor (polymorphic GNPAT) for PCT that adds support for the involvement of aberrant iron metabolism in the pathobiology of PCT.

Disclosures
No relevant conflicts of interest to declare.
Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions
Authors: Pluskota Elzbieta , Szpak Dorota , Bialkowska Katarzyna , Bledzka Kamila , Plow Edward F. 
Published: October 2019

Abstract
Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain.
Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments.

Disclosures
No relevant conflicts of interest to declare.
Antibody Engineering of Anti-TFPI Bypass Therapeutic BAY 1093884: Isotype Selection and Sequence Optimization
Authors: Paz Pedro , Xie Jinger , Aswad Fred 
Published: October 2019

Abstract
Background: BAY 1093884 is a fully human monoclonal antibody against tissue factor pathway inhibitor (TFPI) developed as a bypass agent for patients with hemophilia with or without inhibitors. It restores insufficient thrombin burst, leading to stable clot formation in hemophilic conditions in vitro, and effectively stops bleeding in vivo.
Aims: Antibody targeting of TFPI provides the advantages of monoclonal antibodies, such as longer circulation half-life, and the possibility of subcutaneous administration. However, antibody therapeutics can present the challenge of delivering unwanted effects or activities, such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) that target healthy tissues in the patient. Additionally, antibody sequence engineering that optimizes binding to the target protein may introduce immunogenic epitopes that can induce antidrug antibodies and compromise efficacy.
Methods: In vitro assays used primary human umbilical vein endothelial cells and EA-hy926 cell lines as target cells. Total peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells isolated from healthy donors were used as effector cells for ADCC. Pooled human serum complement (Quidel®) was used for CDC assays. A proprietary immunogenicity scoring program for T-cell epitopes (Bayer HealthCare) was used to determine immunogenic potential of BAY 1093884 relative to antibodies in the market.
Results: In vitro ADCC assessment of BAY 1093884 using PBMCs and NK cells isolated from healthy donors showed no lytic activity against TFPI-expressing primary and immortalized cell lines. Furthermore, no CDC activity using pooled serum complement was detected with BAY 1093884. In silico scoring for T-cell epitopes using a human leukocyte antigen DR-weighted algorithm compared favorably to therapeutic antibodies that have been reported to have low immunogenicity in the clinic.
Conclusion: In vitro and in silico immunoprofiling has aided in the design of the bypass anti-TFPI immunoglobulin G2 antibody BAY 1093884 with low toxicity potential and immunogenicity in humans.

Disclosures
Paz: Bayer HealthCare: Employment. Xie:Bayer HealthCare: Employment. Aswad:Bayer HealthCare: Employment.
Recombinant FVIII and FVIIa Added to Flowing Hemophiliac Blood Co-Localize with Platelet Aggregates Adherent to a Collagen Surface, Promoting Local Fibrin Deposition in Wake-like Patterns Around the Aggregates
Authors: Grabowski Eric F. , Liu Bohan , Gerace Matthew , Grabowski Paul E. , Gunnarsson Peter , Ezban Mirella 
Published: October 2019

Abstract
Mural platelet thrombi are considered to facilitate hemostasis in hemophilia via rFVIII/ rFVIIa binding to platelet membranes with enhancement of local fibrin deposition. We therefore sought to show that Factors VIII and VIIa co-localize to platelet membranes of adherent platelet aggregates and promote local deposition of fibrin. We collected blood from 9 patients with severe hemophilia A just prior to a prophylactic dose of rFVIII, and from 8 normals, into a plastic tube containing 5 U/ml FC low MW heparin, 1.75 µg/ml of the Tab (anti-CD41) mo antibody, and 1.0 μg/ml of ALEXA 555-conjugated rabbit anti-mouse secondary antibody, plus 0, 11, 33 or 100 ng/ml rFVIII-Oregon green. Similar approaches were used for rFVIIa or fibrin. Fibrin was detected using the monoclonal antibody 59D8 directed against human β fibrin, together with the above Alexa555-conjugated rabbit anti-mouse secondary antibody, and co-localized to platelet aggregate surfaces using 10 µM quinicrine dihydrochloride. Blood at 37° C was withdrawn at 270 sec-1 through a flow chamber, over 150 µm glass cover slips with preadsorbed microfibrillar collagen, using epifluorescence video microscopy and digital image analysis (MetaMorph Premier software). Added rFVIII or rFVIIa co-localized (Fig. 1) with platelet membranes and increased with rFVIII concentration. With hemophiliac blood and 100 ng/ml rFVIII, percent co-localization at 7 min was 52.6 ± 7.7 (N=4). Platelet aggregate growth rate, image pixels/min from time 3 to 9 min, was 26% lower in hemophiliac blood, but increased (p = 0.0001) 2.34 ± 0.62 (6)-fold with rFVIII, and (p = 0.0005) 1.91 ± 0.56 (6)-fold with 30 nM rFVIIa (Fig. 2). These increases essentially restored (or only slightly augmented) platelet deposition to levels observed in normal blood. In contrast, the effect on local fibrin deposition was profound: fibrin deposition was delayed by 3-5 min in normal blood, but by >13 min in hemophiliac blood. Addition of rFVIII or rFVIIa shortened the latter time to 7-9 min. Increases in platelet aggregate growth rate, following a lag phase in growth rate, paralleled the onset of fibrin formation. Fibrin appeared as comet-shaped deposits (comet head 20-25 µm in diameter) centered over each platelet aggregate (3-10 µm), the comet tail trailing 60-75 µm downstream (Fig. 3). In flowing whole blood in real time, these data show that rFVIII and rFVIIa co-localize with platelet membranes of activated, surface-adherent platelet aggregates, and support fibrin deposition in the slow-flow wakes immediately surrounding the platelet aggregates.

Disclosures
Grabowski: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk, Denmark: Research Funding. Gunnarsson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment.
Novel Association of a F11 Variant with Venous Thromboembolism in a Chinese Han Population
Authors: Wu Ying Ying , Tang Liang , Hu Yu , Zeng Wei , Liu Han 
Published: October 2019

Abstract
Background: Coagulation factor XI (FXI) is essential for normal function of the intrinsic pathway of blood coagulation. Nevertheless, high coagulation factor XI (FXI) levels were related to an increase in the risk of venous thromboembosis. The aim of the present study was to investigate whether any genetic variant in the factor XI gene (F11) was associated with susceptibility to venous thromboembosis in the Chinese Han population.
Methods: All exons and the 5'- and 3'-untranslated regions of F11 were initially sequenced to identify informative variants. Potential abnormal variants were analyzed in a population of 1531 venous thrombosis patients and 1343 healthy controls. Finally, functional studies were performed to evaluate the effects of the variant.
Results: A mutation c.539A>G(rs190283743) was identified and harbored a high predisposition to venous thrombosis (adjusted OR = 1.861, 95%CI = 1.221-2.836). The plasma factor XI activity and antigen levels in heterozygotes were increased.
Conclusions: Our results suggested that the novel F11variant is a possible genetic determinant of an increased risk venous thrombosis of and increased coagulant activity of factor XI.

Disclosures
No relevant conflicts of interest to declare.
Increased D-Dimer Levels and Residual Venous Thrombosis Are Associated with Late Recurrence of Deep Venous Thrombosis
Authors: Mazetto Bruna Moraes , Orsi Fernanda Andrade , Flores-Nascimento Mariane , Silveira Sandra , Bittar Luis Fernando , Zapponi Kiara , Colella Marina Pereira , De Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria 
Published: October 2019

Abstract
Background: Many patients present new thrombotic events even long-time after the first thrombotic episode and risk factors for recurrence in these cases are not fully elucidated.
Aim: To evaluate the risk of late recurrence of thrombosis after a first DVT episode.
Methods: This is a prospective observational study. We evaluated the association of D-dimer (DD) levels and persistent residual venous thrombosis (RVT), by Doppler ultrasound, with the occurrence of new thrombotic events long-time after the acute DVT episode.
Results: Fifty-six patients were enrolled for the study. For all patients, DVT episode occurred more than 12 months apart from the enrollment day. Median follow-up was 28 months. During the follow-up time, 10 patients presented thrombosis recurrence. Patients who suffered DVT recurrence had higher DD levels than those who did not had recurrence (median= 0.99 vs 0.40, respectively). The best cut-off value to discriminate those at risk for recurrence was DD > 0.63mg/L (area= 0.7380 95%CI= 0.5800 to 0.8961, P=0.01). Recurrence occurred in 27.7% of patients with previous DD>0.63mg/L and in 5.9% of patients with previous DD< 0.63mg/L (relative risk = 6.46; 95%CI 1.36- 30.52, P=0.007). New thrombosis events occurred only in patients with previously documented RVT, mainly in patients with hypoechoic RVT. During follow-up, new thrombotic events were diagnosed in 75% of patients with hypoechoic RVT. The relative risk for recurrence according to the presence of RVT was 9.129 (95% CI= 2.60-32.02, P<0.001). The presence of RVT also modified the effect of DD on the recurrence risk. Patients with DD > 0,63mg/L but without RVT had similar risk of recurrence than those with DD< 0.63mg/L, whereas the risk for recurrence increased in 14-15 times in patients with RVT.
Conclusion: These results suggests that the persistence of residual thrombosis combined with high levels of DD, long-time after the acute DVT, are risk factors associated with late thrombosis recurrence.

Disclosures
No relevant conflicts of interest to declare.
Myelodysplastic Syndrome Caused By Activating Kras Mutation in the Non-Hematopoietic Bone Marrow Microenvironment
Authors: Osswald Lena , Miething Cornelius , Duyster Justus , Brummer Tilman , Zeiser Robert 
Published: October 2019

Abstract
Oncogenic Ras mutations occur frequently in myelodysplastic and myeloproliferative syndromes as juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) as well as in acute myeloid leukemia. However in these reports the mutations were in the hematopoietic cells. Here, we show that an activating mutation of Kras in the non-hematopoietic system leads to hematologic disorder resembling human myelodysplastic syndrome (MDS).
Rosa26CreERT2;LSL-KrasG12D mice (CD45.2) were lethally irradiated and transplanted with wild-type bone marrow (CD45.1). After control of engraftment efficiency (above 99.6%), the mice were treated with Tamoxifen to induce the expression of KrasG12D in non-hematopoietic cells.
6-8 weeks after Tamoxifen treatment, the mice developed anemia, leukocytopenia and thrombocytopenia and had a highly increased percentage of myeloid cells in peripheral blood, spleen and bone marrow. FACS-analysis confirmed that these cells were donor-derived and therefore of wild-type origin. The frequency of immature myeloid progenitors (CD11b+ c-kit+) was increased in bone marrow of Rosa26CreERT2;LSL-KrasG12D mice compared to littermate controls suggesting a disturbed differentiation. Morphological analysis of blood smears and bone marrow revealed a high number of dysplastic hypersegmented neutrophils as well as the occurrence of myeloid blasts.
Additionally, a significant decrease of B-lymphocytes was observed in the bone marrow of KrasG12D recipient mice which has also been described in human MDS. Osteoblasts have been shown to contribute to B-cell lymphopoiesis which implicates that decreased B-cell lymphopoiesis in this study may be a result of oncogenic Kras expression in osteoblasts.
All these data indicate that a single mutation in the hematopoietic microenvironment can initiate a severe hematologic disorder. The expression of oncogenic Kras in bone marrow stroma cells leads to a shift to myeloid differentiation, severe anemia and thrombocytopenia as well as reduced B-cell counts recapitulating main signs of human myelodysplastic syndrome.

Disclosures
No relevant conflicts of interest to declare.