bluuebunny/crossref_metadata_2025 huggingface dataset:
Translating Epigenetic Alterations Into Improvements In Patient Care
Authors: Reddy Pavan
Published: October 2019

Abstract SCI-24

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from the lysine residues of histone tails and non-histone proteins and play a key role in epigenetic modifications. HDAC inhibitors (HDACi) have emerged as epigenetic regulators with significant ant-tumor effects. Many of these are in cancer clinical trials. Their immuno-modulatory effects have largely been unrecognized. Several recent lines of investigation have demonstrated that targeting HDACs can potently modulate immune responses. We have shown that certain of HDAC inhibitors have important anti-inflammatory or immunosuppressive effects in experimental models of allogeneic bone marrow transplantation (BMT). Data generated by us demonstrate that HDACi regulate experimental acute graft-versus-host (GVHD), in part, through modulation of the functions of host antigen presenting cells (APCs) such as dendritic cells (DCs). HDACi regulated experimental GVHD in a STAT-3 acetylation dependent induction of indoleamine 2, 3 dioxygenase (IDO) by the host antigen APCs. The preclinical murine data were complemented by analyses of DCs from healthy humans and post-BMT samples. In an effort to translate these observations we have initiated a Phase II clinical trial that will determine whether addition of HDACi, suberoylinalide hydroxamic acid (SAHA, vorinostat) will reduce the rates of acute GVHD following matched related donor allogeneic BMT after reduced intensity conditioning (RIC). The study is ongoing and fifteen patients are so far evaluable for the primary end-point. The preclinical data, the rationale for the trial development and the clinical progress along with the ongoing correlative studies from the trial that are based on the preclinical data will be presented.

Disclosures:

No relevant conflicts of interest to declare.
Hemogenic Endothelium
Authors: Speck Nancy A. , Tober Joanna , Chen Michael , de Bruijn Marella , Chan Mun Chiang
Published: October 2019

Abstract SCI-44

Hematopoietic stem cells (HSCs) develop from endothelium in the midgestation mouse conceptus, starting at 10.5 days post coitus, through the formation of intra-aortic clusters. Studies in mouse, zebrafish, chick, and frog embryos established that Runx1 (AML1) is the earliest specific marker of the endothelium from which HSCs and definitive hematopoietic progenitors form. Conditional deletion of Runx1 in vascular endothelial cadherin (VEC) positive cells blocks the emergence of intra-aortic hematopoietic clusters, definitive hematopoietic progenitors (HPs), and HSCs, demonstrating that Runx1 is required in endothelial cells for HP and HSC formation. On the other hand, Runx1 deletion in fetal liver HPs and HSCs with Vav-Cre, or deletion in adult HSCs with Mx1-Cre, does not eliminate HP and HSC function, although it does affect homeostasis. These data (and others) demonstrated that the absolute requirement for Runx1 in HP and HSC formation is transient, and over by the time HPs and HSCs colonize the fetal liver. We are further defining the developmental windows during which Runx1, and the gene encoding its non-DNA binding partner CBFβ, are required for HP and HSC specification and commitment. We have taken two complementary approaches. One approach is to delete Runx1 at specific times and assess the effect on HPs. The second approach is to restore CBFβ function at specific times, or in specific cells in otherwise CBFβ deficient embryos and determine whether that rescues HP or HSC formation. From these experiments we can draw several conclusions: 1) Runx1 is absolutely required before the 40 somite pair stage in the AGM region, as deletion before then severely impairs HP formation or activity; 2) CBFβ activity is required before 9.5 dpc for HP and HSC formation; 3) CBFβ activity in endothelium alone is sufficient for HP formation, but insufficient for HSC formation. Together the data indicate that Runx1-CBFβ activity is required over a period of several days during embryogenesis to specific and commit HSCs.

Disclosures:

No relevant conflicts of interest to declare.
Improving Adoptive Immunotherapy Using Less-Differentiated T Cells
Authors: Restifo Nicholas P. , Sukumar Madhu , Gattinoni Luca
Published: October 2019

Abstract SCI-49

Recent findings in mice and humans indicate that current treatments based on the adoptive immunotherapy of cancer might be vastly improved with the use of “younger” cells. Analysis of human clinical trials conducted at the NCI reveals that poor clinical responses after ACT are associated with short telomeres and low expression of CD27, a costimulatory molecule, whose loss may indicate poor proliferative capacity. In a mouse model of adoptive immunotherapy developed in our laboratory (pmel-1), we found that transfer of less differentiated tumor-specific T cells results in substantially greater tumor regression compared with the transfer of terminally differentiated cells. Using progressively less-differentiated, Wnt-stimulated T cells, we have been able to reduce the number of tumor-specific T cells needed to induce tumor regression by almost 1,000-fold. Thus, reprogramming terminally-differentiated cells to less-differentiated T cells might substantially increase the success of adoptive immunotherapy. The possibility of cellular de-differentiation was vividly illustrated by groundbreaking work from Yamanaka showing that expressing transcription factors such as Oct-3/4, Sox2, Klf-4 and c-Myc could re-program the mouse somatic cells and induce pluripotent stem cells. Subsequently, human iPS cells were generated using a similar genetic approach. We seek to generate iPS-derived, tumor-specific lymphocytes that are robustly proliferative, have long telomeres and are unencumbered by the epigenetic changes associated with terminal differentiation, exhaustion and senescence. If significant safety concerns can be addressed preclinically, we plan to infuse iPS-dervied, tumor-specific T cells to treat patients with metastatic cancer that is refractory to all available treatments.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Characterization of Hemophilia and Implications for Novel Therapies
Authors: Lillicrap David
Published: October 2019

Abstract SCI-9

The genes encoding factor VIII (FVIII) and factor IX (FIX) were cloned 25 years ago. Since then, substantial translational application of this knowledge has been witnessed. First, genetic analysis of hemophilia has enabled a detailed understanding of the types and patterns of mutation responsible for these conditions. This knowledge has provided important biological insights into the mechanisms underlying mutation generation and has also formed the basis for precise molecular diagnosis for hemophilia carriers and potentially affected fetuses. In addition, the hemophilic genotype is the best-characterized and strongest predictor for the development of inhibitor generation in previously untreated patients. The second benefit deriving from the genetic characterization of the hemophilias has been the production of recombinant coagulation factor concentrates. Over the past 20 years, the use of recombinant factor concentrates has increased dramatically and we are currently witnessing a flurry of activity to develop second-generation, enhanced concentrates. The major objective of these projects is to facilitate clotting factor prophylaxis through the production of concentrates whose half-lives are extended, thus reducing the frequency of factor administration. These endeavors are utilizing a variety of approaches, but most are focused on either chemical modification of the factors through polymer conjugates or the generation of fusion proteins with immunoglobulin or albumin to take advantage of the recycling function of the neonatal Fc receptor. The first wave of these new products is now undergoing early phase clinical studies, and while substantial benefits for FIX half-life extension appear achievable, FVIII half-life modification may be more challenging. Finally, molecular genetic knowledge of the hemophilias has resulted in the pursuit of strategies to utilize genetic approaches to effect long-term “cures” of the disease. These initiatives have resulted in several small phase I/II trials of viral vector-based gene transfer but have also formed the basis of mutation-specific therapies such as a translational read-through approach being undertaken in patients with nonsense mutations. Currently, two phase I/II trials are in progress with liver-directed AAV FIX gene transfer in which two different AAV serotypes are being evaluated. These studies follow on from two previous AAV clinical trials in which the major obstacle to clinical benefit appears to have been the host immune response to the vector. There is considerable interest in the outcomes of these new studies.

Disclosures:

No relevant conflicts of interest to declare.
Proton Pump Inhibitor Use and the Ability to Replete Iron Stores
Authors: Huang Alina M , Ho Christine M , Reagan John L. , Winer Eric
Published: October 2019

Abstract 1039

Background:

Oral iron absorption occurs in the duodenum and requires increased gastric acidity to allow iron to remain in the more soluble, ferrous form. Many patients with iron deficiency anemia who require oral iron repletion are on proton pump inhibitors (PPIs) for management of various conditions that require acid suppression for treatment. In vitro studies suggest that inhibition of gastric acid secretion decreases the bioavailability of iron.

In patients with established iron deficiency anemia who are on PPIs, there is a paucity of data regarding the efficacy of iron replacement with concomitant PPI use. Sharma et al. published a case report on 2 patients who were iron deficient and on PPI therapy (Sharma et. al. Southern Medical Journal 2004). Their findings showed that discontinuation of the PPI resulted in improvement of iron deficiency anemia on the same dose of oral iron replacement. Additionally, Hutchinson et al. demonstrated that in individuals with hereditary hemachromatosis, those on PPIs had a significant reduction in phlebotomy requirements needed to keep serum ferritin ∼50μg (Hutchinson et. al. Gut 2007).

We conducted a retrospective study to assess whether a difference exists in terms of iron repletion between patients on a PPI versus those not on a PPI.

Methods:

Patients were selected from the medical primary care clinic from charts dating January 2000 until January 2009. Included patients were adults older than 18 years of age, a diagnosis of iron deficiency based on ferritin < 30 with at least one follow up ferritin value, and recipients of oral iron replacement. Excluded patients were those with chronic active bleeding and anemia of chronic disease. Indices examined include iron (Fe), total iron binding capacity (TIBC), ferritin, transferrin saturation, hemoglobin (Hb), hematocrit (Hct), and mean corpuscular volume (MCV). The primary endpoint of interest is repletion of iron stores defined by ferritin > 30. Those who were able to replete their iron stores are termed “responders” and those who were not are termed “nonresponders”.

Results:

Forty-five patients have been identified who have met the above criteria. Of these patients, 14 were on a PPI and 31 were not on a PPI. Of the patients not on a PPI, 38.7% responded to oral iron. Of the patients on a PPI, 50.0% responded to oral iron. The average initial and final values of indices of interest were compared between the groups (Table 1).

Discussion:

Our data suggests that patients both on and off PPIs show the ability to replete their iron stores. Furthermore, it appears that a slightly greater percentage of patients on a PPI compared with those not on a PPI are able to replete their iron stores, though the numbers remain small. Most importantly, these findings challenge the commonly held belief that PPI use impairs oral iron absorption. A prospective study is warranted to further confirm these observational data.

Disclosures:

No relevant conflicts of interest to declare.
Dosed Deficiency of Iron Restricts Terminal Maturation and Enucleation of Cultured Human Erythroblasts
Authors: Byrnes Colleen , Lee Y.Terry , Meier Emily Riehm , Rabel Antoinette , Miller Jeffery L.
Published: October 2019

Abstract 1041

In addition to reduced hemoglobin production, iron deficiency causes anemia though poorly understood defects in erythropoiesis that reduce the number of erythroid cells released from the marrow space. To study the negative effects of iron deficiency upon erythropoiesis, CD34+ cells from three healthy human donors were cultured in serum-free media supplemented with transferrin-bound iron. Dosed titrations of holo-transferrin (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 mg/ml) were balanced by apo-transferrin supplementation to maintain total transferrin levels of 1.2 mg/ml in all cultures. Analyses were performed over the 21-day culture period that included assessment of proliferation, differentiation, enucleation and surface phosphatidylserine expression. Diminution of holo-transferrin in the culture medium caused a decrease in total cell counts and reduced cell divisions detected by CFSE analysis. During the first two weeks of culture, the progenitor cells differentiated into a glycophorin A positive population at all holo-transferrin doses. However, several iron-dependent changes were detected in the final stages of differentiation during culture days 14–21. Hemoglobin accumulation was reduced by 72% at the lowest holo-transferrin dose. Flow cytometry demonstrated a decrease in the loss of surface transferrin receptor in iron-deficient cultures. Thiazole orange analysis also demonstrated significantly lower levels of enucleation from 25.9±8.2% (1.2 mg/ml) to 4.4±4.2% (0.1mg/ml). Microscopic examination of the enucleated cells demonstrated a hypochromatic phenotype at lower holo-transferrin concentrations. Phosphatidylserine on the surface membrane (annexin V staining) increased with reduction of holo-transferrin from 6.3±2.6% (1.2 mg/ml) to 78.3±9.4% (0.1 mg/ml, p=0.003). The effects of iron on surface phosphatidylserine expression were limited to holo-transferrin concentrations of <0.6 mg/ml. Isocitrate supplemented at a concentration of 5.0 mM increased the cell counts in all concentrations of holo-transferrin, and partially rescued the iron deficient phenotype. These data suggest that iron deficiency ex vivo causes decreased proliferation, decreased loss of surface transferrin receptor, and decreased enucleation of normoblasts. There was also a significant increase in phosphatidylserine detected on the surface of the iron deficient normoblasts that provides a mechanism whereby macrophages can identify, phagocytose, and efficiently recycle iron from those cells to other normoblasts in the local marrow environment via erythroblastic islands.

Disclosures:

No relevant conflicts of interest to declare.
RNAi-Mediated Inhibition of Tmprss6 Elevates Hamp1 Expression and Reduces Serum Iron Levels in Mice
Authors: Toudjarska Ivanka , Cai Zuhua , Racie Tim , Milstein Stuart , Bettencourt Brian R , Hettinger Julia , Sah Dinah WY , Vaishnaw Akshay , Bumcrot David
Published: October 2019

Abstract 1045

The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality.

Disclosures:

Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.
Increased Incidence of Hematological Malignancies Among Californians with Sickle Cell Disease
Authors: Mahajan Anjlee , Brown Monica , Brunson Ann , Wun Ted
Published: October 2019

Abstract 1073

Background:

High cellular turnover and inflammation are associated with the development of malignancy. Both conditions exist in sickle cell disease (SCD). We used a population-based epidemiological approach to determine the incidence of hematological malignancy in patients with SCD.

Methods:

The California (CA) Office of Statewide Healthcare Planning and Development (OSHPD) operates a Patient Discharge Dataset (PDD) and Emergency Department Utilization (EDU) dataset that records the discharge diagnoses and procedures for all non-federal hospitalizations or ED visits in CA. Cases coded as SCD (282.60, 282.61, 282.62, 282.63, 282.68, 282.69, 282.41, 282.42) from 1991–2007 (PDD) or 2005–2007 (EDU) were found. Cases were excluded if the SCD code did not appear in the principal position and appeared in the secondary diagnosis position only once; or age < 2 years (lacked social security number for linkage). These cases were then linked to the California Cancer Registry (CCR) to determine cases with leukemia, myeloma, or lymphoma (including Hodgkin). Standardized incidence ratios (SIR) were calculated. Sensitivity analysis was also performed to determine the effect of doubling the CA SCD population on SIR.

Results:

There were 46 total cases of hematological malignancies identified: 25 females and 21 males. Fifty nine percent of the SCD patients with hematological malignancies were ages 30–59 years. The cohort was 78% Black, 11% White, 7% Hispanic, and 4% Asian.

The remaining cases had chronic myelogenous leukemia (n=4) and other leukemia not otherwise specified (n=5). Limiting analysis to only Non-Hispanic Blacks did not appreciably change the SIR estimates. Even if the SCD population was underestimated by half, sensitivity analysis showed the observed cases would yield an SIR for all hematopoietic malignancies of 19 (95% CI 14–25). The individual sub-type SIR would also still range from 13–24 and all would be statistically significant.

Conclusions:

The CA SCD cohort had elevated SIR for hematological malignancies suggesting higher incidence in this population. Limitations include those inherent in query of any large administrative dataset. However, our SCD population estimate is consistent with that of others and the CCR has validated robust tumor data. Current efforts to develop a US registry for SCD patients should include tracking for hematological, and perhaps all malignancies. Further studies using independent datasets are important to confirm these provocative findings.

Disclosures:

Wun: Glycomimetics, Inc.: Research Funding; Eli Lilly, Inc.: Research Funding.
Does Quantitative Heterogeneity of Human Fetal Hemoglobin (Hb F) Reveal Friends or Foes of KLF1 in Globin Gene Switching ?
Authors: Felice Alexander E. , Galdies Ruth , Borg Joseph , Grech Godfrey , Cassar Wilma , Scerri Christian A , Patrinos George P , Philipsen Sjaak
Published: October 2019

Abstract 1092

The chemical heterogeneity of fetal hemoglobin (Hb F) due to variable ratios of the Gγ and Aγ globin subunits reflects genetic complexity because of common dimorphisms such as Hb F Sardegna (or Aγ75(E19) Ile>Thr; also known as AγT) in Caucasians, and common variants such the Gγ globin variant, Hb F Malta I (or Gγ117(G19) His>Arg) that is in tight linkage disequilibrium with the β globin variant Hb Valletta (or β87(F3) Thr>Pro) and is found in 1.8% of neonates from Malta. Comprehensive and integrated maternal and neonatal testing has led to the finding of triple compound heterozygotes with Hb F Malta I in association with Hb F Sardegna and Hb Valletta in whom all globin genes that are functional in the transition from fetal to adult Hemoglobin profiles are genetically tagged, and quantifiable with High Performance Liquid Chromatography in the neonate or mRNA in the adult in the context of diverse XMNI −158 C>T Gγ globin and (AT)XTY −540 β globin haplotypes. The interaction between XMNI and (AT)XTY revealed “conditional” cis-trans interplay that appeared to be under developmental control. A family with members carrying Hb F Malta I in association with a rare form of the Hereditary Persistence of Fetal Hemoglobin has also been found. The genetic cause has been traced to haplo-insufficiency of the putative erythroid master regulator KLF1, and, as confirmed by functional assays in vitro. However, levels of Hb F expression varied considerably (3.3% – 19.5%) while a second family from Malta with the same KLF1 mutation (p.K288X) had normal Hb F indicating interplay of KLF1 with modifying genes. These data, together with comparative expression profiling of human erythroid progenitors, indicated a small set of additional gene products that may interact positively (friends) or negatively (foes) at the level of commitment or expression in globin gene switching with significant effects on the Mean Corpuscular - Hb F. Whole genome sequencing on critically informative family members currently in progress may further uncover the complex genetic interactions in developmental globin gene control.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Sialic Acid Loss Greatly Improves Survival of Refrigerated Platelets
Authors: Jansen Gerard , Josefsson Emma C , Liu Qiyong Peter , Rumjantseva Viktoria , Falet Herve , Grozovsky Renata , Bergmeier Wolfgang , Wagner Denisa D. , Hartwig John H. , Hoffmeister Karin M.
Published: October 2019

Abstract 1133

Platelets have the shortest shelf life of all major blood components and are the most difficult to store, a fact that complicates platelet transfusion practices. Platelet refrigeration could slow bacterial growth and possibly retard the loss of platelet function following storage. However, in contrast to other blood components, platelets do not tolerate refrigeration and are rapidly cleared from the circulation. We demonstrated that two distinct pathways recognizing GPIba remove refrigerated platelets in recipient's livers: 1) αMβ2 integrins (Mac-1) on hepatic resident macrophages (Kupffer cells) selectively recognize irreversibly clustered b-N-acetylglucosamine (β-GlcNAc)–terminated glycans on GPIbα, and 2) hepatic Asialoglycoprotein (Asg) receptors (Ashwell Morell receptors) recognize desialylated GPIba. We here investigated the mechanism of sialic acid loss during refrigeration. We show, that when refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1 that remove sialic acid from platelet receptors, specifically from GPIbα. Platelets also express Neu3 on their surfaces, however Neu3 expression appears to be unaffected by platelet refrigeration. Importantly, the recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of the competitive sialidase inhibitor N-Acetylneuraminic Acid, 2,3-Dehydro-2-deoxy-Sodium Salt (DANA). Desialylated von Willebrand receptor (vWfR) complex is also a target for metalloproteinases (MMPs), as GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the metalloproteinase inhibitor GM6001 and does not occur in ADAM17ΔZn/ΔZn platelets expressing inactive ADAM17. Critically, desialylation in the absence of metalloproteinase-mediated receptor shedding is sufficient to induce the rapid clearance of platelets from circulation. Desialylation of platelet vWfR therefore triggers platelet clearance, and primes GPIbα and GPV for metalloproteinase-dependent cleavage. We conclude that desialylation of platelets is caused by increased surface sialidase activity following refrigeration and desialylation of glycoproteins, specifically of GPIbα, promotes receptor cleavage by MMPs.

Disclosures:

Liu: Velicomedical, Inc: Employment.
Noval Mutation in Four Saudi Families with Glanzmann Thrombasthenia
Authors: Owaidah Tarek , Abalkhail Hala , Musa Abdulrahman Al , Mosmali Hasan , Abdulmajeed Albanyan , Jefri Abdullah Al , alNounou Randa , Zahrani Hazzaa Al , Saleh Mahasen
Published: October 2019

Abstract 1136

Introduction:

Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation and variable bleeding tendency. Inherited genetic mutations in integrin alpha IIb and beta3 (ITGA2B, ITGB3) result in a heterogeneity of the thrombasthenia phenotypes. It is phenotypically expressed in homozygotes or compound heterozygotes, given that 50% of normal aIIbb3 is sufficient to guarantee unimpaired platelet function that result in asymptomatic carriers. Defects in ITGB3 result in failure of binding of B3 and alpha IIb. These defects had been reported in Arabs (Iraqi Jews). We are reporting some results of Saudi GT genotype project.

Materials & Methods:

In this study, we analyzed the entire coding region ITGB3 gene using polymerase chain reaction (PCR) and direct sequencing with primers specifically designed to amplify the coding region of exon 1–15 and exon /Intron boundaries in a cohort of 51 GT patients diagnosed and treated in our institute.

Results:

Out of 51 cases from 20 families had mutational screening of the ITGB3 gene with the aim to detect the causative pathogenic mutations to enable the pre-symptomatic diagnosis in at risk family members. In this study we detect 1 novel germline mutation c.2190delC (p.Ser703fs) in exon 13. The mutation is predicted to result in premature stop codon and protein truncation. The mutation was detected in 6 patients in homozygous stat (3 males and 3 females). Three tested samples from the patients family members detected the mutation in heterozygous state and all of them were asymptomatic with normal PFA and Intact expression of Platelet Glycoprotiens CD41(Gpllb), CD42a(GPIX), CD42b(GPlb), and CD61(Gpllla). All the GT patients with this mutation were type I GT with Prolonged PFA and complete absence of CD41(Gpllb) and CD61(Gpllla) glycoprotein.

Conclusion:

The result of this study represents the first Molecular analysis of ITGB3 gene in Saudi Arabia and displays the existence of novel pathogenic and possibly a founder effect in Saudi families.

Disclosures:

No relevant conflicts of interest to declare.
Protein S Regulates Factor IXa in the Absence and Presence of Factor VIIIa Independently of Activated Protein C
Authors: Majumder Rinku , Chattopadhyay Rima , Sengupta Tanusree
Published: October 2019

Abstract 1197

Coagulation is a finely tuned process. During thrombin formation, several anticoagulant reactions are initiated to prevent systematic activation of coagulation, and impairment of anticoagulant activity causes an increased risk of venous thrombosis. One such anticoagulant factor is protein S, deficiencies of which have been linked to venous and arterial thrombosis. While protein S has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear.

Protein S is a vitamin K-dependent plasma protein that functions in feedback regulation of thrombin generation. Protein S was initially identified as a cofactor for activated protein C (APC) but later it was observed that there is only a 3–10 fold increase in APC activity in the presence of protein S. Plasma coagulation assays in the absence of APC suggest that protein S may have other anticoagulant role(s).

We report here an anticoagulant activity of Protein S mediated by inhibition of fIXa in the absence and presence of fVIIIa independent of APC. Although an APC-independent anticoagulant activity has been reported for protein S interacting with fVIIIa, no study has shown that the inhibitory effect of protein S is mediated through its interaction with fIXa, thus making our observations novel and significant. Moreover, previous studies that reported an interaction between fVIIIa and protein S were performed with low amounts of phospholipid, a condition that produces activity measurement artifacts due to the presence of active protein S multimers.

We used both ex vivo (plasma studies) and in vitro methods at high phospholipid (100–200 micro molar) concentration to determine whether and how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: 1) activated partial thromboplastin time (aPTT) assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time, and a normal clotting time was restored with addition of anti-protein S antibody, 2) a modified aPPT assay with fIX-deficient plasma confirmed that protein S affects fIX-initiated clotting time, 3) thrombin generation assay through fIXa/fVIIIa pathway, initiated with a limiting amount of tissue factor (TF), was regulated by protein S, 4) in vitro studies with fIXa/fVIIIa and protein S in the presence of phosphatidylserine (PS) vesicles showed ∼40% and ∼65% inhibition in the activity of fIXa in the absence and presence of fVIIIa, respectively, and 5) protein S altered only the KM for fX activation by fIXa but altered both kcat and KM for fX activation by fIXa and fVIIIa. Our findings underscore the central role of protein S in regulation of coagulation. We anticipate these results will unravel important implications for the evaluation of thrombotic risk associated with protein S-deficiency.

Disclosures:

No relevant conflicts of interest to declare.
Probing Anticoagulant Fondaparinux for Interference with Prothrombotic B2GPI/Anti-B2GPI Antibody Complexes
Authors: Kolyada Alexey , De Biasio Alfredo , Beglova Natalia
Published: October 2019

Abstract 1209

Background:

The presence of autoimmune antibodies directed to beta2-glycoprotein-I (B2GPI) often leads to thrombosis in antiphospholipid syndrome (APS). Heparin, low molecular weight heparin (LMWH) and fondaparinux are commonly used for prophylaxis and treatment of thromboses in APS. These drugs bind and activate antithrombin III to inactivate blood clotting proteases. Fondaparinux is a synthetic pentasaccharide matching a specific sequence within heparin interacting with antithrombin. Aim: We investigated if fondaparinux can bind B2GPI and ameliorate prothrombotic properties of B2GPI/anti-B2GPI antibody complexes. Results: We found that fondaparinux interacts with B2GPI and that the binding is dominated by electrostatic interactions. We measured the binding affinity by monitoring changes in the intrinsic fluorescence of domain V of B2GPI (B2GPI-DV) upon titration with fondaparinux. In the presence of 100 mM NaCl, the binding affinity was about 1.5 uM and stoichiometry of the binding is 1:1. Using solution NMR spectroscopy, we determined that the binding interface of the complex is centered on Lys251 of B2GPI-DV. This observation was confirmed by site-directed mutagenesis. The Lys251/Asp mutant fails to bind B2GPI-DV. Interestingly, the binding site for fondaparinux on B2GPI does not overlap with the major binding site for heparin. Cellular activation by the binding of B2GPI/anti-B2GPI antibody complexes with cell-surface receptors (among them ApoER2, a lipoprotein receptor from the LDLR family) and interference with the protective function of annexin V on anionic phospholipids expressed on the surfaces of activated cells are two potential prothrombotic mechanisms of B2GPI/antibody complexes. We found that fondaparinux does not prevent the association of the ligand-binding modules from ApoER2 with B2GPI-DV. Therefore, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes with lipoprotein receptors. Neither fondaparinux, nor heparin and LMWH were effective in inhibiting the binding of B2GPI/anti-B2GPI antibody complexes to cardiolipin-coated plates suggesting that these drugs do not prevent the destructive effect of B2GPI/antibody complexes on antithrombotic function of annexin V. Conclusions: At therapeutic concentrations, fondaparinux forms only small number of complexes with B2GPI, given that the binding affinity of the complex is in a micromolar range. When bound to B2GPI, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes to lipoprotein receptors and anionic phospholipids.

Disclosures:

No relevant conflicts of interest to declare.
Retrospective Comparison of Recombinant Thrombomodulin and Conventional Drugs in a Treatment of DIC Caused by Acute Leukemia
Authors: Yokoyama Hisayuki , Itoh Toshihiro , Harigae Hideo
Published: October 2019

Abstract 1220

Acute leukemia (AL) is often complicated with DIC, and its control is important for preventing severe adverse events. Here, we evaluated the efficacy of rTM compared to conventional drugs, such as Nafamostat, Gabexate and Dalteparin, in the treatment of DIC caused by AL. Between 2006 and 2011, 27 cases of AL were complicated with DIC. Eleven cases were APL and 16 were other leukemia. Among APL, 5 cases were treated with rTM and 6 were treated with conventional drugs. Patient characteristics were similar except the high level of FDP (149 vs 27.4 ng/ml, p=0.028), and D-dimer (DD) (102 vs 26.6ng/ml, p=0.032) in rTM group. To evaluate the efficacy of drugs, we investigated the number of days to reach less than 20ng/ml of FDP, or 10ng/ml of DD. Median days to reach the level (12 vs 11 days) and cumulative curve of achievement rate (HR 1.0, 95%CI 0.2–5.2) was similar. Considering the high level of FDP and DD in rTM group at the beginning of treatment, rTM might quickly improve DIC compared to conventional drugs. Next, we also evaluated the efficacy in AL other than APL. 8 out were treated with rTM and 8 were treated with conventional drugs. Patient characteristics were similar in each group. As for DIC improvement, median days of achievement was significantly short in rTM group (7 vs 13days, p=0.02). Cumulative rate also showed fast improvement of DIC in rTM group (HR 5.4, 95%CI 1.3–22.4) (figure). These results suggest that rTM could rapidly improve DIC complicated with AL compared to conventional drugs, and thus might have a benefit to prevent further severe complications. In addition high cost is a drawback of this drug, and hence appropriate use should be considered.

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Cleavage of Recombinant VWF Upon Intravenous Administration in Preclinical and Clinical Settings
Authors: Varadi Katalin , Rottensteiner Hanspeter , Schreiner Jutta , Gritsch Herbert , Ehrlich Hartmut J. , Scheiflinger Friedrich , Turecek Peter L
Published: October 2019

Abstract 1222

Von Willebrand factor (VWF) is composed of a series of multimers with a molecular size ranging from 600 to 20,000 kDa. The ultra-high molecular weight (UHMW) portion of VWF multimers is more efficient in platelet interactions than the lower molecular weight portion, and as such it might have a thrombogenic potential under certain pathologic conditions. The concentration of UHMW multimers in healthy individuals' blood is low because the circulating specific metalloprotease ADAMTS13 rapidly cleaves UHMW VWF upon its release into the circulation.

A recombinant VWF (rVWF) expressed in CHO cells has been developed as a new drug candidate for treating patients with VWF deficiency. Since this protein is not cleaved by ADAMTS13 during the expression phase, it has a high portion of UHMW multimers, similar to the human VWF stored in Weibel-Palade bodies of endothelial cells.

During the development phase of rVWF in vitro experiments showed that when treated under mild denaturing conditions or under shear, the molecule was susceptible to ADAMTS13 of human or animal origin.

Here we present data from preclinical studies where analyzing the ex vivo samples showed the ADAMTS13-mediated cleavage of intravenously applied UHMW-containing rVWF. In good correlation with the in vitro results, studies in rabbits and cynomolgus monkeys showed that the ADAMTS13-specific cleavage fragments appeared as soon as 15 minutes after application, as visualized by immunoblot analysis. The appearance of cleavage bands coincided with the decrease in multimer numbers, indicating that rVWF had been specifically cleaved by endogenous ADAMTS13 in the circulation. The effect was species-dependent, only a low or minimal cleavage was observed in different mice strains.

A recently completed Phase I clinical study clearly demonstrated that rVWF administered intravenously to severe VWD patients in a dose-range of 7.5 to 50 VWF:RCo U/kg body weight was rapidly processed in the circulation, resulting in the disappearance of the UHMW multimers with visible cleavage fragments, as shown by immunoblot and high resolution multimer analysis. No thrombotic adverse events were seen during the study, underlying the suitability of the UHMW-containing rVWF for treating VWD patients.

Disclosures:

Varadi: Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schreiner:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
Product Associated Variation in Generic Enoxaparin. Potential Clinical Implications
Authors: Fareed Jawed , Hoppensteadt Debra , Ottman Albara , Iqbal Omer , Jeske Walter , Gray Angel , Walenga Jeanine M. , Kalodiki Evi , Thethi Indermohan , Wahi Rakesh
Published: October 2019

Abstract 1249

Recently the generic versions of enoxaparin brand (Sanofi-Aventis) have widely become available. Since enoxaparin is a biological product, minor compositional changes in these agents may potentially have clinical implications. Generic enoxaparins are manufactured by several companies and marketed under specific trade names. The purpose of this investigation was to determine the differences between generic products and within product variations in the same generic product. Eight generic products from India (Cutenox, Markaparin,

Clenox, Lomoparin, Lupenox, Lomorin-NX, Lomoh-40 and Troynoxa-40), four generic products from South America (Enoxalow, Versa, Heptron and Endocris), and one generic product from the USA (Enoxaparin sodium) were studied. The molecular weight profile of generic products showed relatively similar distribution profile with a molecular weight range of 3.9–4.9 kDa. The anti-FXa activity ranged from 87–110 U/mg, whereas wider variations were noted in anti-FIIa activity (28–41 U/mg). The anti-FXa:anti-FIIa ratio ranged from 3.1–4.2. Much wider variations (up to 60%) were noted in the anticoagulant assays (ACT, aPTT, Heptest, and thrombin time). Additionally the generic products also showed variations in the thrombin generation assay. Protamine and PF4 titration profile also showed variations. AT mediated inhibition of thrombin and FXa in the biochemical assays showed wider differences in IC50 values. Heparinase digestion profile showed variable oligosaccharide composition and some of the products contained heparinase resistant glycosaminoglycans. A comparison of 5 batches of the branded enoxaparin (Clexane) and 5 batches of a generic enoxaparin showed that the generic product batches exhibited greater variation in the biologic assays despite a comparable anti-FXa profile. These observations suggest that despite the current regulatory requirements, product based differences in the generic enoxaparins exist, which may impact their safety and efficacy profile in cardiovascular and thrombotic indications for which the branded enoxaparin is approved. Additional pharmacodynamic studies and clinical validations of the safety and efficacy of each of the generic products maybe helpful in assuring a clinical equivalence of the generic products with the branded enoxaparin.

Disclosures:

No relevant conflicts of interest to declare.
Determination of Rivaroxaban in Serum and Urine and As Point of Care Method in Urine
Authors: Harenberg Job , Hagedorn Antje , Giese Christina , Kraemer Roland
Published: October 2019

Abstract 1253

The oral direct factor Xa inhibitor rivaroxaban effectively prevents postoperative venous thromboembolism following elective knee and hip replacement surgery. Rivaroxaban is determined in plasma by coagulation and chromogenic methods. It is eliminated from blood to about 20% by kidneys. We aimed to determine rivaroxaban in urine by means of a quantitative measurement in serum and urine and as qualitative determination in urine as point of care method.

Rivaroxaban were extracted from commercially available drug. Identity and purity of the isolated compound were confirmed by mass spectrometry, elemental analysis (carbon, hydrogen, nitrogen content) and 1H-NMR spectroscopy. Serum and urine (patent pending) from healthy donors were spiked with rivaroxaban (50ng/ml to 1000 ng). Factor Xa inhibition was performed by means of a chromogenic substrate method comparing the results obtained from plasma. The reagents were lyophilized on mini-strips and incubated with urine (patent pending). The concentration of rivaroxaban was determined from plasma, serum, and urine and qualitatively by the mini-strips from 10 patients on treatment with rivaroxaban.

The concentrations of rivaroxaban spiked to plasma, serum and urine were independent of the medium (correlation between r=0.888 and 0.970). Patients on treatment with rivaroxaban showed a high correlation of plasma and serum values (r=0.9209). The correlation to urine samples was lower do to the time dependence of drug intake. The qualitative mini-strips detected rivaroxaban in all samples independently of drug intake. There were no false positive or false negative detections of rivaroxaban by the mini-strips.

Detection of rivaroxaban in serum avoids separate blood sampling to obtain plasma. Detection in urine avoids blood sampling at all and allows point of care testing also by the patient him/herself. Point of care detection of rivaroxaban in urine samples using ministrips may improve patient care.

Disclosures:

No relevant conflicts of interest to declare.
Activated Recombinant Factor VIIa for Intractable Extra-Cranial Hemorrhage in 55 Consecutive Patients
Authors: Vidarsson Brynjar , Palmason Robert , Gudbjartsson Tomas , Onundarson Pall T.
Published: October 2019

Abstract 1264

Background:

Recombinant activated coagulation factor VII (rFVIIa) has been increasingly used in non-hemophiliac patients for unapproved indications. We undertook a 10 year nation-wide retrospective survey in order to evaluate the use of rFVIIa in Iceland in patients with intractable extra-cranial hemorrhage.

Material and methods:

Hospital charts of all patients that received rFVIIa between 1999–2008 at Landspitali, the single institution administering rFVIIa in Iceland, were reviewed and indications, coagulation profiles and clinical outcome were evaluated. This analysis focuses on patients treated off-label for desperate (intractable) extra-cranial hemorrhage (IH), ie only patients in who all other measures were considered to have failed to stop hemorrhage.

Results:

rFVIIa was used for IH in 55 patients (median age 53 yrs, range; 0–84, 51% males). This included IH in open heart surgery (n=23), non-cardiac surgery (n=7), post-partum hemorrhage (n=9), multitrauma (n=8) and miscellaneous (n=8). A single rFVIIa dose was used in 86% of cases and hemorrhage was considered effectively contained by immediate clinical response to rFVIIa in 81% of the IH cases. Survival at 30 days for all patients was 68%. Out of 55 patients with IH, six died within 24 hours of administration of rFVIIa (five assessed clinically as rFVIIa non-responders) and 11 other patients (total 17 patients, 31%) died within 30 days (six non-responders). The 24-hour mortality in rFVIIa clinical responders and non-responders was 2% and 50% respectively (p=0.0004) and the 30 day mortality was 25% and 60% respectively (p=0.05). The need for transfusion of blood products was significantly decreased (p<0.01) and also the prothrombin time (20.0 to 13.3 sec, p<0.0001).

Conclusion:

The majority of unselected consecutive patients receiving rFVIIa as last resort treatment for IH were considered to have favorable immediate clinical response as well as reduced transfusion requirements and normalisation of coagulation parameters. Also, patients deemed with clinical response to rFVIIa may have had lower mortality.

Disclosures:

Off Label Use: Activated recombinant factor VII for use in desperately bleeding patients.
Ezrin Regulates Hematopoietic Stem/Progenitor Cell Motility
Authors: Zeng Yi , Staser Karl , Menon Keshav Mohan , Park Su-jung , Mwanthi Muithi , Jiang Li , Clapp D. Wade
Published: October 2019

Abstract 1282

Ezrin is a member of the ERM (ezrin, moesin and radixin) protein family that links plasma membrane proteins to the actin cytoskeleton. Ezrin in other in vitro cell systems has been hypothesized to participate in cell-cell contact and could have a role in stem/ progenitor cell mobilization and adhesion. To test this hypothesis, we crossed ezrinflox/flox mice with Mx1 cre transgenic mice to generate an inducible ezrin knock out mouse model. Inducible disruption of the ezrin gene in hematopoietic cells was achieved by the administration of polyIC. Ezrin knock out HSPCs exhibited a 30–40% decrease in baseline and chemokine stromal cell-derived factor-1 (SDF-1) stimulated motility in transwell migration assays in vitro. In addition, loss of ezrin led to a 60% decrease in the homing capacity of HSPCs in lethally irradiated recipient mice following transplantation. There was a 40–55% decrease in colony forming cells in peripheral blood and spleen of the mice following ezrin knock out, suggesting that ezrin knock out HSPCs may be deficient in egressing out of the bone marrow. To further understand the cause of the impaired motility of ezrin knock out HSPCs, we examined F-actin level of HSPCs at baseline and in response to SDF-1. Ezrin knock out HSPCs displayed 1.5 to 2 fold higher level of F-actin at baseline when compared with wild type cells. Following stimulation with SDF-1, wild type HSPCs that migrated to the bottom compartment of the transwell demonstrated a 2 time greater decrease in F-actin level when compared with ezrin knock out cells, suggesting that ezrin may participate in the regulation of F-actin depolymerization in HSPCs. In summary, we demonstrate that ezrin modulates HSPC migration and homing likely through its regulation on F-actin organization.

Disclosures:

No relevant conflicts of interest to declare.