bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thrombin Modulates PAR1-PAR4 Heterodimers
Authors: de la Fuente Maria , Arachiche Amal , Nieman Marvin T. 
Published: October 2019

Abstract
Abstract 2203
Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. There is structural evidence that GPCRs, as a class, function as dimers and that dimerization can alter signaling specificity. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1's ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). Similar to our previous studies with PAR4 homodimers, PAR1 homodimers were constitutive and did not require receptor activation. In contrast, PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. We next examined if PAR1-PAR4 heterodimers would be induced by stimulating PAR1 or PAR4 individually with their agonist peptides TFLLRN (100 μM) or AYPGKF (500 μM), respectively. The agonist peptides were unable to induce heterodimers when added to the cells individually or simultaneously. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation. To examine this further, we removed 37 amino acids from the C-terminus of PAR1, which disrupts the eighth helix. The truncated PAR1 was able to form constitutive heterodimers with PAR4 and these heterodimers were unaffected by thrombin. These data suggest that PAR1 is the allosteric modulator of the PAR1-PAR4 heterodimers. Finally, the stability of the constitutive PAR1 and PAR4 homodimers was unchanged in response to thrombin or the agonist peptides. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists.

Disclosures:
No relevant conflicts of interest to declare.
Novel Polyphosphate-Binding Proteins in Human Plasma and Platelet Releasates
Authors: Smith Stephanie A. , Collins Julie N. , Choi Sharon H. , Morrissey James H. 
Published: October 2019

Abstract
Abstract 2270

Introduction:
Inorganic polyphosphates (polyP) are negatively charged, linear phosphate polymers found throughout biology. PolyP is secreted in substantial amounts by activated human platelets and it also accumulates in many infectious microorganisms. We have shown that polyP is a potent hemostatic regulator, acting at multiple points in the clotting cascade; in particular, polyP triggers the contact pathway, enhances FXI activation by thrombin, accelerates factor V activation, and modulates fibrin clot structure. Since there may be other, as-yet undiscovered (patho)physiologic roles for polyP, we undertook a study to identify the major polyP-binding proteins in human plasma and in releasates from activated human platelets.

Methods:
PolyP was end-labeled by biotinylation, then bound to streptavidin coated onto magnetic beads (Dynabeads), polystyrene microtiter plates, or surface plasmon resonance sensorchips. PolyP-Dynabeads were exposed to plasma or to releasates from platelets activated by TRAP; the beads were then washed repeatedly, and polyP-binding proteins were eluted with 1 M LiCl. Eluted proteins were separated by 2-D gel electrophoresis and were identified by mass spectrometry. Some additional proteins known or suspected to bind to polyP were also analyzed in parallel. Binding affinities for selected proteins were quantified by microtiter plate assays or by surface plasmon resonance approaches.

Results:
Candidate poly-P binding proteins identified in plasma pull-downs included parvulin, factor H, complement C1q, complement C1q tumor necrosis factor-related protein, C4 binding protein, and apolipoprotein E. Candidate poly-P binding proteins identified in pull-downs of platelet-releasate included platelet factor 4, multimerin, thrombospondin, and cyclophilin. High affinity binding to polyP was also quantified for thrombin, FXI, FXIa, kallikrien, histones and the isolated polyphosphate-binding domain of E. coli exopolyphosphatase.

Conclusions:
PolyP binds with high affinity to certain proteins that participate in the coagulation cascade as well as several additional proteins. Some of these are involved in reactions known to be modulated by polyP, but several have yet to be evaluated. Further investigation is indicated into potential roles for polyP in modulation of the complement cascade, prolyl isomerase activity, angiogenesis, and platelet responses.

Disclosures:
Smith: University of Illinois: Patents & Royalties. Choi:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.
Generic Low Molecular Weight Heparins May Not Have the Same Safety –Efficacy Profile As the Branded Low Molecular Weight Heparins in Acute Coronary Syndrome
Authors: Hoppensteadt Debra , Jeske Walter , Walenga Jeanine M. , Lewis Bruce E , Fareed Jawed 
Published: October 2019

Abstract
Abstract 2308
Low molecular weight heparins such as enoxaparin and dalteparin are widely used for the management of Acute Coronary Syndrome (ACS). Recently, several generic versions of enoxaparin and dalteparin have been approved in various countries for all of the branded product's indications. However, no data on their clinical equivalence in ACS is available. Since generic versions of enoxaparin and dalteparin are manufactured by different processes and may use starting material from different sources, these drugs may differ in their pharmacological profile in simulated ACS settings. To compare the pharmacodynamic effects, a branded version of enoxaparin was compared with three generic versions in a primate model at a dosage of 1 mg/kg IV. Such pharmacokinetic parameters as TFPI release, TAFI modulation, vWF release, and TF mediated platelet activation were measured. Anticoagulant effects of these drugs were also measured after IV administration using iSTAT ACT and aPTT. Simulated catheter related thrombosis studies were carried out to differentiate each agent in contact, intrinsic and extrinsic clotting systems. The generic versions of enoxaparin namely, Cutenox (Gland Pharma), Fibrinox (Sandoz AG) and Versa (EuroPharma) exhibited product based pharmacodynamic differences compared with the branded product and the results are provided in the following table. Each of the generic products exhibited its own specific pharmacologic profile despite comparable molecular weight distributions and anti-Xa potencies. Significant differences were noted in the anticoagulant effects of each of these agents as compared with the branded products. Some differences were also noted in HIT antibody mediated aggregation studies. The differences between the branded and generic versions of LMWHs may be due to the higher dosages used and the IV administration which leads to higher circulating levels of these agents. These observations suggest the need for additional animal studies and clinical trials to validate the use of generic versions of LMWHs in such critical indications as ACS and related syndromes.

Disclosures:
No relevant conflicts of interest to declare.
Infusion of Recombinant Full-Length ADAMTS13 and Carboxyl-Terminal Truncated Variant Alters Composition of Ferric Chloride-Induced Arterial Thrombi in Adamts13−/− mice
Authors: Skipwith Christopher G. , Xiao Juan (Jenny) , Weisel John W. , Zheng X. Long 
Published: October 2019

Abstract
Abstract 2313
Proteolytic cleavage of ultra large von Willebrand factor (ULVWF) released from endothelial cells by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. However, the effect of infusing ADAMTS13 on thrombus composition remains poorly understood. In this study, we determined the morphology and composition of thrombi formed in carotid arteries after topical application of FeCl3 in Adamts13−/− mice receiving PBS, recombinant human full-length ADAMTS13 (FL) and carboxyl-terminal truncated variant after spacer domain (S), using scanning electron microscopy and quantitative image analysis. We showed that in Adamts13−/− mice 5 min after FeCl3 injury, formed arterial thrombi were comprised ∼39% platelets, ∼26% red blood cells, and ∼35% fibrin. The arterial thrombi in these mice were structurally deformed. An infusion of recombinant FL (final concentration of 10 nM) significantly reduced the accumulation of platelets (∼18%) but increased the fibrin network (57%) without affecting the composition of red blood cells (∼25%) in the arterial thrombi formed at the same time point after FeCl3 injury. Similar effects on the morphology and composition of FeCl3-induced arterial thrombi were observed after infusion of recombinant S (10 nM) into Adamts13−/− mice. Kinetic analysis showed that there was a decrease in platelet accumulation over the time of 30 min during thrombus formation with a slight increase in accumulation of red blood cells and formation of fibrin in Adamts13−/− mice. But, the infusion of recombinant FL and S into Adamts13−/− mice restored the kinetics of platelet/red blood cell accumulation and fibrin formation to those observed in wild-type mice. Our findings, revealing the apparent difference in thrombus composition, provide novel insight into the mechanism of ADAMTS13 function in vivo, which may shed more light on the pathogenesis of thrombotic thrombocytopenic purpura and other arterial thrombotic disorders associated with deficiency of plasma ADAMTS13 activity.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Nr4a1GFP Reporter System Reveals That Nr4a1 Expression Identifies Long-Term Hematopoietic Stem Cells
Authors: Land Ruben , Barlowe Trevor , Manjunath Shwetha , Eiger Sophie , Gross Matthew , Garrett Russell , Cunningham Nicole , Emerson Stephen G. , Punt Jenni A. 
Published: October 2019

Abstract
Abstract 2339
Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1GFP. Interestingly, circulating LSK's in the spleen express Nr4a1GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1GFP, 2) Nr4a1GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1GFP expression.

Disclosures:
No relevant conflicts of interest to declare.
Dual Role for CIB1 in Thrombopoiesis: CIB1 Suppresses Megakaryocyte Quantities but Supports Adhesion, Migration, and Proplatelet Formation
Authors: Kostyak John C , Naik Ulhas P 
Published: October 2019

Abstract
Abstract 2384
Megakaryocytes (MKs) are large polyploid cells that produce platelets through a process known as thrombopoiesis. Thrombopoietin (Tpo) is the major cytokine that regulates a variety of steps in this process, including hematopoietic stem cell (HSC) differentiation to MKs, proplatelet formation, and platelet release into the circulation. However, the molecular mechanism of thrombopoiesis is poorly understood. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in thrombopoiesis, we utilized a Cib1−/− mouse model. We observed that Cib1−/− mice have a slightly elevated number of platelets and bone marrow (BM)-derived MKs than wild-type (WT) controls (p<0.05). Rate of platelet clearance was comparable in Cib1−/− and WT mice, suggesting that the defective clearance is not the cause of the observed elevated platelet number. In order to determine if the HSC differentiation is dysregulated by the ablation of Cib1, we analyzed MK-colony forming unit production, which revealed an increase in the colony forming cells with Cib1 deletion compared to WT (p<0.05). Additionally, BM from Cib1−/− mice, cultured with Tpo for 24 hours, produced more highly polyploid MKs than WT BM (p<0.05). These results suggest that Cib1 may negatively regulate initial steps of megakaryopoiesis. Subsequent analysis of Tpo signaling revealed that activation of FAK, a known suppresser of Tpo signaling, is attenuated, as indicated by reduced FAKY925 phosphorylation in Cib1−/− BM-derived MKs treated with Tpo. Consequently, Akt and ERK1/2 activation downstream of Tpo was enhanced. These results suggested that Cib1 inhibits Tpo signaling by augmenting FAK activation. Interestingly, platelet recovery in Cib1−/− mice following platelet depletion by experimental immunothrombocytopenia was attenuated compared to WT (p<0.05). This could be due to impaired adhesion and migration of MKs on the extracellular matrix. Consistent with this notion, adhesion to fibrinogen and fibronectin and migration towards an SDF-1α gradient were significantly reduced in Cib1−/− MKs compared to WT (p<0.05). Additionally, Cib1−/− MKs formed fewer proplatelets compared to WT (p<0.05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in thrombopoiesis, initially by negatively regulating Tpo signaling, and later by supporting MK migration and proplatelet production.

Disclosures:
No relevant conflicts of interest to declare.
TET2 Favors Mesoderm and Hematopoietic Differentiation in Human Embryonic Stem Cells
Authors: Mor Barbara da Costa Reis Monte , Langlois Thierry , Droin Nathalie , Pronier Elodie , Couédic Jean-Pierre Le , Auger Nathalie , Almire Carole , Mercher Thomas , Delhommeau Francois , Najet Debili , Bernard Olivier , Solary Eric , Plo Isabelle , Vainchenker William 
Published: October 2019

Abstract
Abstract 2418
TET2 belongs to the TET family proteins that catalyze 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (hmc) and plays an important role in normal and malignant adult hematopoiesis. The role of TET2 in human hematopoietic development remains unknown. Here we show that TET2 is expressed at low level in human embryonic stem (hES) cell lines and that its expression increases during hematopoietic differentiation in three different hES. TET2 knockdown does not modify hmc level and pluripotent properties of ES cells. However TET2 depletion by two different shRNA skewed differentiation into neuroectoderm at the expense of endoderm and mesoderm. This was associated with a decrease or an increase in promoter methylation of neuroectoderm and meso/endoderm genes, respectively during hES specification. Subsequently, we observed a decrease in hematopoietic progenitors (CD34+CD43+) and their cloning capacities due to a marked increase in apoptosis. Alteration of hematopoietic differentiation was coupled with a profound alteration in gene expression with up and down regulated genes including the abnormal expression of neuronal genes in hematopoietic cells. Thus our results suggest that TET2 regulates embryonic development by inhibiting neuroectoderm specification and enabling hematopoietic differentiation in hES cells.

Disclosures:
No relevant conflicts of interest to declare.
Linking the Glycolytic Enzyme HK3 to Neutrophil Differentiation of APL Cells Via PU.1
Authors: Federzoni Elena A. , Valk Peter , Löwenberg Bob , Fey Martin F. , Tschan Mario P. 
Published: October 2019

Abstract
Abstract 2425

Objectives:
Identification and characterization of hexokinase 3 (HK3), as novel transcriptional target of the myeloid master regulator PU.1, with a role in APL cell differentiation and survival.

Materials:
HK3 and PU.1 quantitative RT-PCR in primary AML patient samples (n=170) and granulocytes isolated from healthy donors (n=20). Gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Generation of NB4 and HT93 PU.1 as well as HK3 knockdown cell lines. ATRA-induced differentiation of these cell lines was assessed by CD11b and CEBPE expression. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was shown by ChIP assays. Viability of NB4 cell lines was assessed by Alamar Blue staining.

Results:
We identified HK3 as putative transcriptional target of PU.1 by gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Unlike the other three hexokinase family members (HK1, HK2 and HK4), HK3 expression is limited to hematopoietic cells, where it is mainly expressed in the myeloid lineage. We found 50-times lower HK3 mRNA levels in primary non-APL patients as compared to granulocytes from healthy donors. Most interestingly, we found that HK3 expression in APL patients with the t(15;17) translocation was 400-times lower (p<0.0001) than compared to granulocytes. Given the particularly low HK3 and PU.1 expression in APL patients, we decided to investigate the role of HK3 and its possible regulation by PU.1 during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HT93 APL cell lines. Firstly, we observed a 5500- and 1100-fold increase of HK3 mRNA levels during neutrophil differentiation of NB4 and HT93, respectively. This was paralleled by markedly increased HK3 protein levels. No increased HK3 levels were observed in ATRA-resistant NB4-R2 cell lines. HK1 and HK2 mRNA expression was not markedly increased during neutrophil differentiation pointing to a particular role for HK3 in neutrophil differentiation. Regulation of the liver specific HK4 was not investigated. Next, we observed significantly decreased HK3 expression, both at the mRNA and protein levels, in all PU.1 knockdown cell lines upon ATRA-induced differentiation. In vivo PU.1 binding to three different regions of the HK3 promoter was found. Given our findings in primary APL patients, we decided to test if PML-RARa binds to the HK3 promoter, and indeed, PML-RARA binds to the same promoter regions as PU.1. Knocking down HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation as measured by CD11b and CEBPE expression. Also, we observed that NB4 HK3 knockdown cell lines treated with ATRA were less viable compared to control cells.

Conclusions:
Our results strongly suggest that HK3 is a novel PU.1 transcriptional target functionally involved in neutrophil differentiation and cell viability of APL cells.

Disclosures:
No relevant conflicts of interest to declare.
Bortezomib Restores Defective Apoptosis by Upregulation of Noxa in Enteropathy-Associated T-Cell Lymphoma
Authors: de Baaij Laura R , Radersma Marijn , van de Water Jolanda MW , Groot Kim BJ , Hijmering Nathalie J , Moesbergen Laura M , Visser Otto J , Oudejans Joost J , Mulder Chris JJ , Meijer Chris JLM , Cillessen Saskia AGM 
Published: October 2019

Abstract
Abstract 2722
Enteropathy-associated T-cell lymphoma (EATL) is a rare intestinal lymphoma that arises from intraepithelial lymphocytes. Clinical outcome of patients with EATL is very poor, due to chemotherapy-resistance and high relapse rates. Therefore, new therapeutic options for EATL are urgently needed. Studies in other types of lymphoma have shown that inhibition of apoptosis may cause chemotherapy-resistance and that restoration of defective apoptosis can induce cell death in these lymphomas. Preliminary data in EATL samples have demonstrated an increased expression of a fraction of NF-κB target genes, suggesting upregulation of NF-κB activity in EATL tumor cells. NF-κB activity can be inhibited by the proteasome inhibitor bortezomib resulting in induction of apoptosis. In the present study, we evaluated if apoptosis is inhibited in EATL cells and if Bortezomib can restore apoptosis in EATL cells.
Laser-capture microdissection was applied to 16 fresh frozen EATL samples to obtain purified tumor cells for RNA isolation. Intraepithelial lymphocytes (IEL) of healthy controls were obtained from fresh duodenal biopsies and isolated by cell sorting. RT-MLPA analysis revealed that the pro-apoptotic BH3-only gene Noxa was significantly downregulated in most EATL samples compared to healthy donor IEL. Induction with etoposide resulted in caspase-9 mediated apoptosis in EATL cells with relatively high Noxa expression, whereas in EATL cells with low Noxa expression no apoptosis was induced, suggesting an inhibition in the intrinsic apoptosis pathway. Treatment with Bortezomib resulted in induction of apoptosis in EATL cells. The lethal dose (LD50) varied between 7.5 nM and 15 nM. Bortezomib induced cell death in EATL cells was caspase-9 mediated. mRNA and protein expression analysis showed upregulation of Noxa after incubation with bortezomib.
In conclusion, our study showed that bortezomib induces apoptosis by upregulation of Noxa in EATL cells. Bortezomib therefore may be a potential drug in the treatment of patients with EATL.

Disclosures:
No relevant conflicts of interest to declare.