bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impaired Platelet Responses to Thromboxane a2 and Thrombin in Mice Lacking Receptor-Interacting Protein Kinase 3
Authors: Zhang Yiwen , Zhang Jian , Yan Rong , Zhang Jie , Chen Mengxing , Jiang Miao , Li Zhenyu , Ruan Changgeng , He Sudan , Dai Kesheng 
Published: November 2019

Abstract
Objective: Receptor-interacting protein 3 (RIP3) is a member of RIP family with a Ser/Thr protein kinase domain in its amino-terminus which is essential for kinase activity and autophosphorylation. The roles of RIP3 in embryonic development and different disease pathologies, such as inflammation and infections, have been reported in recent years. However, the role of RIP3 in thrombosis and hemostasis remains unknown.
Methods: Hematologic analysis was performed and tail bleeding time was monitored. Mouse platelets were isolated from anti-coagulated whole blood. Platelet aggregation and secretion were recorded at real time. Platelet P-selectin exposure and specific fibrinogen binding were detected by flow cytometry. TXA2 generation was measured with enzyme immunoassay (EIA) kit. Protein phosphorylations were detected by western blotting.
Result: RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited delayed thrombus formation, fewer accumulated platelets, smaller thrombi, and prolonged occlusion times. RIP3 was expressed in both human and mouse platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619. The defect in ADP secretion appears responsible for the impaired platelet aggregation, because addition of exogenous ADP rescued the reduced platelet aggregation. Although TXA2 generation and α-granule secretion were not impaired, integrin αIIbβ3 activation was attenuated in RIP3-/- platelets. Moreover, phosphorylation of Akt induced by U46619 or thrombin was markedly reduced in the absence of RIP3. Activation of Akt signaling restored the impaired aggregation of RIP3-/- platelets. ERK and p38 phosphorylation elicited by either U46619 or thrombin was attenuated in RIP3-/- platelets. In contrast, U46619- and thrombin-induced activation of PTEN, PDK1, or Src was not impaired in RIP3-/- platelets.
Conclusion: Our data demonstrate a novel role for RIP3 in amplifying U46619- and thrombin-induced platelet activation by mediating Akt-dependent ADP secretion, and in supporting hemostasis and thrombus formation in vivo. RIP3 may represent a novel target to modulate PARs and TP signaling and a potential new target for antithrombotic strategy.

Disclosures
No relevant conflicts of interest to declare.
Pharmacological and Genetic Inhibition of Autophagy Decreases the Secretion of High Molecular Weight Von Willebrand Factor from Endothelial Cells in Vitro and in Vivo
Authors: Hanby Hayley A. , Zheng X. Long 
Published: November 2019

The regulation of von Willebrand factor (VWF) processing, packaging, and secretion is important for primary hemostasis. Recently, autophagy has been implicated in modulating VWF maturation and Weibel-Palade body (WPB) morphology. Additionally, treatment of mice and humans with chloroquine (CQ), an anti-malarial agent and pharmacological inhibitor of autophagy, is shown to increase bleeding time. Therefore, we hypothesize that targeting autophagic flux may be therapeutic for arterial thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP). To test this hypothesis, we first treated human umbilical vein endothelial cells (HUVECs) with CQ in culture and showed that CQ dose-dependently decreased VWF antigen levels and multimer sizes in the conditioned medium of histamine-stimulated cells (not shown). Knockdown of an autophagy-related protein (Atg7) with shRNA in HUVECs had similar effect to CQ treatment in VWF secretion and multimer distribution (not shown). More interestingly, daily injections (i.p.) of CQ at 60 mg/kg into Adamts13-/- mice (CAST/Ei strain) for 7 days reduced plasma VWF concentration by more than 60% compared to vehicle control (Fig. 1). Interestingly, VWF secreted from CQ-treated mice remained functional as collagen binding activity/antigen ratio was comparable to vehicle control mice. However, multimer analysis demonstrated the selective lack of ultra large VWF in plasma of Adamts13-/- mice treated with CQ as compared with those treated with vehicle alone (Fig. 2). These results indicate that targeting autophagy pathway with a pharmacological agent, such as CQ, may modulate VWF secretion and, thereby, arterial thrombosis. Our ongoing experiments are to determine the therapeutic efficacy of CQ and other autophagy inhibitors in murine models of arterial thrombosis and TTP.
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Association Between a Pro-Inflammatory Axis Comprised By TSP1-TGFβ-CTGF with Three microRNAs, Mir-19a, miR122 and Mir-133a in the Pathophysiology of Diabetic Retinopathy: A Therapeutic Novel Modality
Authors: Dela Cadena Raul A , Lester Melissa , Del Carpio-Cano Fabiola , Shevchenko Alina , St. Angelo John , Boakye Jeffery , Deutsch Leonardo , Alvarez de la Cadena Philippe , Foster William 
Published: November 2019

Abstract
Introduction: Diabetic retinopathy (DR) is a severe ocular complication of type 2 diabetes (T2DM). Our laboratory has been studying the role of a pro-inflammatory axis comprised by TSP1-TGFb and CTGF in T2DM. The axis is differentially expressed in patients with T2DM and non-proliferative DR (NPDR) when compared with patients with T2DM and proliferative DR (PDR). The differential expression of such axis is accompanied by interesting cytokine profiles as well. The purpose of this study was to evaluate potential microRNAs associated with the above changes in order to understand better the pathophysiology of T2DM. Recent studies indicate that miRNAs transported to exosomes or HDL can be transferred in an active form to recipients cells potentially involving them in cell-to-cell communication.
Methods: Institutional approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals afflicted by T2DM (n=20 NPDR and n=20 PDR) from the Ophthalmology Department Diabetes Clinic at Temple Hospital, normal controls (n=20) were recruited from the Sol Sherry Thrombosis Research Center at Temple. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been documented in vitreous fluid to be angiogenic. Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4 and MIP-1b. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFb and CTGF. Total RNA was extracted from human plasma using the miRCURY TM RNA isolation kit by Exiqon at their facility in Denmark.
Results and Discussion: TSP1, TGFb, CTGF, IL4, MIP-1b were significantly elevated in NPDR patients when compared to PDR patients. Fragments of CTGF were identified more abundant in patients with PDR when compared to NPDR. Only three microRNAs were differentially expressed in the normal group when compared with either NPDR (decreased), namely miR-133a (p<0.005), miR-19a (p<0.008), miR-122 (p<0.02) or PDR (decreased), miR-19a p<0.000096) and miR-122 (p<0.005). miR19a has been implicated with expression of TSP1 and CTGF, glucose levels, insulin resistance and diabetes-associated pancreatic cancer. miR-133a has been associated with cardiac hyperthrophy, fibrosis and heart failure in diabetes. miR-122 has been identified as a potential biomarker for non-alcoholic fatty liver. In summary our results link for the first time in the literature three circulating microRNAs with a the above axis in the pathophysiology of NPDR and PDR.

Disclosures
No relevant conflicts of interest to declare.
Maternal Anti-Platelet β3 Integrin Antibodies Impair Angiogenesis and Cause Intracranial Hemorrhage in Fetal and Neonatal Alloimmune Thrombocytopenia
Authors: Issaka Yougbare , Lang Sean , Yang Hong , Chen Pingguo , Zhao Xu , Tai Wei-She , Zdravic Darko , Vadasz Brian , Li Conglei , Piran Siavash , Marshall Alexandra H. , Zhu Guangheng , Tiller Heidi , Killie Mette Kjaer , Boyd Shelley , Leong-Poi Howard , Skogen Bjorn , Adamson S. Lee , Freedman John , Ni Heyu 
Published: November 2019

Abstract
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia caused by maternal antibodies against β3 integrin and occasionally against other platelet antigens (e.g. GPIbα) has long been assumed to be the cause of bleeding, the mechanism of ICH has never been adequately explored. Utilizing murine models of FNAIT and a high frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin- but not anti-GPIbα-mediated FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signalling, and increased endothelial cell apoptosis; which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis was further reproduced in neonates by injection of anti-β3 integrin- but not anti-GPIbα-antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and Akt phosphorylation were inhibited only by murine anti-β3 integrin-antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest fetal hemostasis is unique in that impairment of angiogenesis rather than thrombocytopenia is likely the cause of ICH; importantly maternal IVIG therapy can effectively prevent this devastating disorder.

Disclosures
No relevant conflicts of interest to declare.
Von Willebrand Factor-Dependent Inflammatory Responses in Mouse Septic Model By Cecal Ligation and Puncture
Authors: Kasuda Shogo , Matsui Hideto , Ono Shiro , Matsunari Yasunori , Nishio Kenji , Shima Midori , Hatake Katsuhiko , Sugimoto Mitsuhiko 
Published: November 2019

Abstract
Sepsis is a serious inflammatory response syndrome, in which systemic activation of both inflammatory and coagulation pathways are provoked by severe microbial infection. In addition to the known hemostatic functions, von Willebrand factor (VWF) is recently assumed to participate in a cross-talk between inflammation and thrombosis. Indeed, recent mouse model studies demonstrated that proper functional regulation of VWF-dependent inflammatory responses by ADAMTS13 considerably improved the disease progression of brain stroke or myocardial infarction. However, little is known about the detailed mechanisms or relevant role of VWF functions in inflammation. We therefore studied the physiologic relevance of VWF-dependent inflammatory responses in a mouse model of experimental sepsis by cecal ligation and puncture (CLP). The mouse CLP was performed according to the established standard protocol (Rittirsch D, et al., Nat Protoc, 2009). Briefly, mouse cecum was ligated distal to the ileocecal valve under laparotomy, punctured with 18 gauge needle and gently pressed until a small drop of stool appeared. The cecum was returned to the peritoneal cavity and 200 μL of saline was injected into the cavity to avoid dehydration before body wall and skin incision were closed with a 4-0 Sofsilk. We compared 17 wild-type (WT) and 17 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 10-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all (WT or KO) mice during the CLP operation. Kaplan-Meier analysis revealed the significantly (p < 0.05) lower survival rate of KO mice than that of WT (KO; 23.5% vs. WT; 58.8% at the Day 7 of CLP). The impaired survival rate of KO mice was restored by the bolus administration of human VWF (n=21, 2.5 U/mouse) to an extent comparable to that of WT. Peripheral blood analysis of KO mice at 24 hours after CLP showed the severe leukocytopenia with sharp decrease of neutrophils in blood, as compared to WT. In addition, formation of walled-off abscess was confirmed at the peri-cecal space in all the WT and KO mice alive even at the Day 7 of CLP, while such focal abscess formation was not found in mice died before the Day 3 of CLP. Thus, our results altogether indicate that VWF could play a role on the recruitment or accumulation of neutrophils for microbial killing at the local inflammatory focus. VWF-mediated platelet aggregate formation in peripheral capillary vessels then could shut down the local microcirculation, thereby blocking systemic microbial expansion as a crucial biological defense mechanism.

Disclosures
Shima: Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Bilirubin Directly Inhibits Proteolytic Cleavage of a Fluorogenic VWF73 Peptide and Plasma Von Willebrand Factor
Authors: Lu Ruinan , Zheng X. Long 
Published: November 2019

Abstract
Bilirubin is a yellow breakdown product of heme catabolism. Increased levels of plasma bilirubin are a condition commonly seen in neonates and adults with hemolysis. The unconjugated bilirubin consists of an open chain of four pyrrole-like rings. Previous studies have shown that bilirubin decreases the cleavage of a commercially available FRETS­­­­­-VWF73 assay, resulting in falsely low plasma ADAMTS13 activity. However, the mechanism underlying such a reduction of ­­­­ADAMTS13 activity is not fully understood. Plasma ADAMTS13 activity is critical for differential diagnosis of thrombotic microangiopathy in which low ADAMTS13 activity (less than 5-10%) is more consistent with thrombotic thrombocytopenic purpura (TTP), while normal to mildly reduced ADAMTS13 activity (greater than 10-20%) is the common findings of hemolytic uremic syndrome (HUS). Here, we show that bilirubin directly inhibits the proteolytic cleavage of a fluorogenic VWF73 peptides labeled with fluorescein-5-maleimide (FL485-VWF73) (ext. 485nm/emi.530nm) or DyLight-633-maleimide (DL633-VWF73) (ext.638nm/emi.658 nm) in a concentration-dependent manner (Fig. 1). The IC50 for inhibiting the cleavage of FL485-VWF73 and DL633-VWF73 was estimated to be 13 micro mol/L and 70 micro mol/L, respectively. To confirm the direct inhibitory activity, bilirubin at various concentrations was incubated with plasma-derived multimeric VWF and recombinant ADAMTS13 in the presence of 1.5 M urea, 5 mM Tris-HCl, pH 8.0 for four hours. The cleavage of multimeric VWF was determined by 1% agarose and SDS-polyacrylamide gel electrophoreses, followed by Western blotting with rabbit anti-VWF IgG. The results demonstrated that bilirubin also inhibited recombinant ADAMTS13-mediated cleavage of VWF in a concentration-dependent manner with an IC50 ~0.43 milli mol/L (Fig. 2). The inhibitory activity of bilirubin on the cleavage of a fluorogenic peptide or a multimeric VWF substrate was largely eliminated by addition of bilirubin oxidase that converts bilirubin to biliverdin (not shown). Our results demonstrate for the first time that unconjugated bilirubin directly inhibits plasma and recombinant ADAMTS13 activity in addition to its known interference with certain fluorogenic assays for ADAMTS13 activity. These findings are clinically important concerning diagnosis and differential diagnosis of thrombotic microangiopathy. Additionally, our findings may shed new light on the mechanism of thrombotic complications pertinent to acute hemolysis and liver diseases.
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
β7 Integrin Regulates Intra-Marrow Trafficking of Hematopoietic Stem Cells
Authors: Murakami Jodi , Xu Baohui , Franco Christopher B. , Hu Xingbin , Galli Stephen J. , Weissman Irving L , Chen Ching-Cheng 
Published: November 2019

Abstract
α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis remains controversial. To investigate this, we conducted studies using a mouse model in which β7 integrin is absent. Hematopoietic stem cells (HSCs) that lacked β7 integrin (β7KO) had significantly reduced engraftment potential. Intriguingly, the survival of β7KO mice was enhanced and their hematopoietic recovery after 5-fluorouracil-induced myeloablative stress was better compared to wild type (WT) mice, indicating that the decreased engraftment of β7KO HSCs was not caused by a defect in HSC hematopoietic activity. Next we examined the homing abilities of HSCs and we observed that β7KO HSCs had impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand - mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on bone marrow (BM) endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment. Interestingly, we also found that β7KO HSCs were retained in the BM, suggesting that β7 integrin may influence the localization of HSCs within different stem cell niches through interaction with MAdCAM-1. To examine the localization of HSCs within the BM, we used the hypoxyprobe pimonidazole to correlate oxygen status with niche localization. We observed that both β7KO and MAdCAM-1KO HSCs were more hypoxic compared to WT HSCs, demonstrating that the absence of either β7 integrin or MAdCAM-1 in mice causes HSCs to be localized in a more hypoxic region of the BM. To confirm these findings, we performed single-cell RT-PCR using Fluidigm Dynamic Array Chips and we discovered that β7KO HSCs differentially expressed genes associated with niche localization and cell cycle status compared to WT HSCs. Since hypoxia correlates with quiescence, we next assessed the cell cycle status of HSCs using Ki67 staining and in vivo BrdU assay and we found that β7KO HSCs may have reduced cell cycle activity. Collectively, these studies suggest that expression of β7 integrin on HSCs may promote exit from quiescence and influence HSC localization within the BM niche.

Disclosures
No relevant conflicts of interest to declare.
RPS19 Deficiency Leads to GATA1 Downregulation through TNF-Mediated p38 MAPK Activation
Authors: Youn Minyoung , Bibikova Elena , Danilova Nadia , Ono-Uruga Yukako , Konto-Ghiorghi Yoan , Ochoa Rachel , Narla Anupama , Glader Bertil , Lin Shuo , Sakamoto Kathleen 
Published: November 2019

Abstract
Diamond-Blackfan Anemia (DBA) is a rare inherited bone marrow failure disorder, characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. While several studies have found that the tumor suppressor protein p53 contributes to DBA pathogenesis, and that certain GATA1 mutations can give rise to DBA, the link between ribosomal protein mutations and erythroid defects is not well understood.
To investigate the molecular pathways downstream of RPS19 deficiency, we infected human cord blood CD34+ cells with RPS19 shRNA lentivirus and observed that RPS19 knockdown resulted in decreased GATA1 mRNA and protein expression, resulting in defective erythropoiesis. We also detected increased TNF-α levels in RPS19 knockdown cells, and in zebrafish that have been treated with RPS19 morpholino. TNF-α was primarily upregulated in the non-erythroid population of RPS19 deficient cells, while its receptor, TNFR1, showed increased expression on the surface of erythroid cells.
To understand the relationship between GATA1 and TNF-α, we treated primary hematopoietic CD34+ cells with TNF-α or its inhibitor etanercept. Our results demonstrated that TNF-α treatment reduced GATA1 expression, and that this effect could be rescued by the addition of etanercept. Treatment of RPS19 deficient cells with etanercept improved their erythroid differentiation, and increased GATA1 expression. Additionally, etanercept treatment successfully reversed the anemia phenotype observed in RPS19 deficient zebrafish.
To study pathways downstream of TNF-α, we examined phosphorylation of signaling pathways such as p38 MAPK, NFκB, ERK, STAT1, and STAT5 in RPS19 deficient erythroid cells using phospho-flow cytometry. Among these pathways, we found a significant increase in phosphorylation of p38 MAPK, but not ERK, NFκB, STAT1, or STAT5, suggesting that p38 MAPK activation by TNF-α contributes to decreased GATA1 expression in RPS19 cells. We suggest a novel mechanism for the erythroid defects observed in DBA, in which RPS19 deficiency leads to increased TNF-α production in non-erythroid cells, and activation of p38 MAPK, followed by decreased GATA1 expression, in erythroid cells. Our data also suggest that TNF-α inhibitors, such as etanercept, may be beneficial in treating patients with DBA.

Disclosures
No relevant conflicts of interest to declare.