bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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.
Scl and Notch Act Downstream of Hedgehog Signaling to Promote Embryonic Hematopoiesis From Endothelial Cells
Authors: Kim Peter G , Albacker Colleen E , Jang Il-ho , Heffner Garrett C , Lim Yoowon , Bowman Teresa V. , Lin Michelle I , Zon Leonard I. , Loewer Sabine , Daley George Q. 
Published: October 2019

Abstract
Abstract 1283
Adult hematopoietic cells transition through a hemogenic endothelial (HE) intermediate during development, but the signaling pathways modulating this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in blood and endothelial cell formation, early embryonic lethality of mice lacking Hedgehog signaling precludes such analysis. To determine a role for Hh signaling in HE patterning, we assessed the effect of altered Hh signaling in differentiating mouse embryonic stem cells (mESCs), cultured embryonic day 9.5 mouse embryos, and developing zebrafish embryos. In differentiating mESCs, addition of Indian Hh ligand (IHH) increased the number of CD41+c-Kit+ hematopoietic progenitors, whereas chemical inhibition of Hh signaling led to a decrease without affecting primitive-streak mesoderm gene expression. In the setting of Hh inhibition, Notch induction rescued hemogenic VE-cadherin+ cells, demonstrating that Notch expands HE. Scl/Tal1 (stem cell leukemia/T-cell associated leukemia 1) induction rescued VE-cadherin+CD41+ cells, demonstrating that Scl/Tal1 converts endothelial cells to hematopoietic tissue. Similar experiments using cultured mouse yolk sacs demonstrated that signaling pathways are conserved in vivo. Moreover, VE-cadherin+ cells isolated from the mouse yolk sac or paraaortic splanchnopleura, when virally transduced with Notch signaling or Scl, had increased hematopoietic colony-forming activity. Finally, ectopic Notch or Scl induction in zebrafish embryos rescued the expression of the prototypical hemogenic endothelium marker Runx1 in the absence of Hh signalling. Together, our results reveal that the Hh-Notch-Scl axis promotes embryonic hematopoiesis through endothelial-to-hematopoietic transition.

Disclosures:
Zon: Fate Therapeutics: Consultancy; Stemgent: Consultancy. Daley:iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.