bluuebunny/crossref_metadata_2025_split huggingface dataset:
Association of Protein Z Deficiency with Retinal Artery and Vein Occlusion.
Authors: Koren-Michowitz Maya , Eting Eva , Voltchek Yulia , Rahimi-Levene Naomi , Garach-Jehoshua Osnat , Kornberg Abraham 
Published: October 2019

Abstract
Central retinal vein occlusion (CRVO) and central retinal artery occlusion (CRAO) are common disorders mainly associated with traditional risk factors for atherosclerosis. There is no clear correlation between thrombophilia and CRVO. However it was suggested that hyperhomocysteinemia and the presence of antiphospholipid antibodies are risk factors for the development of CRAO. Protein Z is a vitamin K dependent cofactor for the inactivation of activated factor X (Xa) by the protein Z dependent protease inhibitor (ZPI). Protein Z deficiency presumably promotes thrombosis, in particular arterial thrombosis, as suggested by an association between low plasma protein Z levels and ischemic stroke. The similarity between risk factors for ischemic stroke and CRVO / CRAO and the fact that low protein Z levels are associated with an increased risk for ischemic stroke has led us to assess protein Z levels in patients with CRVO and CRAO. Plasma samples from 29 patients with CRVO and 7 patients with CRAO (total study group= 36, mean age 60±11) were screened for protein Z levels by ELISA. Study group was also screened for the presence of lupus anticoagulant (LAC) by the kaolin clotting time (KCT) index and the dilute Russells’ viper venum test (DRVVT), for activated protein C resistance (APCR) and anticardiolipin antibodies (ACA). Levels of protein Z were compared with a control group of 42 healthy individuals (mean age 43±11). Patients with CRAO and history of ischemic stroke or an embolic source were excluded. Thirty patients in the study group had traditional risk factors for atheroscalerosis including hypertension, diabetes mellitus, hyperlipidemia and smoking and 6 patients had none. There was no significant difference in protein Z levels between the whole study group patients and controls (1995±810 vs. 2010±603 ng/ml p=0.922). However, patients with no risk factors for CRVO/CRAO had significantly lower protein Z levels than controls (1379±682 vs. 2010±603 ng/ml p=0.022) and patients with risk factors (1379±682 vs. 2118±785 ng/ml p=0.04). In the study group three patients had abnormal APCR and 6 had positive LAC. There was no correlation between low protein Z levels and the presence of LAC, APCR or ACA, or a correlation between low protein Z levels and the specific diagnosis (CRVO or CRAO) or age. The data show that low protein Z levels may be an additional risk factor for CRVO and CRAO in patients without traditional risk factors for these disorders. The association should be explored in a larger group of patients.
Thrombopoietin Initiates Demethylation-Based Transcription of GP6 during Megakaryocyte Differentiation.
Authors: Kanaji Sachiko , Jacquelin Beatrice , Chang Mei , Nugent Diane J. , Komatsu Norio , Izuhara Kenji , Kunicki Thomas J. 
Published: October 2019

Abstract
Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-1, Sp1 and Fli-1. However, the mere presence of these is not sufficient to initiate transcription during megakaryocyte differentiation, and other mechanisms are suggested to be involved in the regulation of megakaryocyte-specific expression of GPVI. In this study, we show that GPVI expression during megakaryocytic differentiation is dependent on CpG demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI non-expressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human c-mpl, were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation which can be directly initiated by TPO. Our results establish, for the first time, a role for TPO in dynamic changes in CpG methylation status that are involved in the epigenetic regulation of megakaryocyte-specific gene expression.
A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.
Authors: Panicot-Dubois Laurence , Dubois Christophe , Furie Barbara C. , Furie Bruce , Lombardo Dominique 
Published: October 2019

Abstract
Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.
Human Platelets Contain and Release an Active ADAMTS-13-Like Metalloprotease.
Authors: Liu Li , Choi Huiwan , Aubrey Bernardo , Bergeron Angila , Nolasco Leticia , Moake Joel F. , Ruan Changgeng , Dong Jing-Fei 
Published: October 2019

Abstract
The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. These cells release VWF in response to similar stimulations. However, a longstanding observation stated that, unlike endothelial cells, platelets do not release the ultra-large form (UL) of VWF upon activation. The lack of ULVWF release may be attributed to the difference in multimerization process in the storage granules or the intrinsic proteolysis of the hyperreactive ULVWF. To examine these possibilities, we analyzed the multimer compositions of VWF stored in and released from platelets. We found that ULVWF was detected by immunobloting of washed platelet lysates in the presence, but not in the absence of 5 mM EDTA. Similarly, when stimulated with 5 μM of thrombin receptor-activating peptide (TRAP), platelets released only VWF that was similar in sizes to plasma VWF, suggesting that platelet ULVWF may have been cleaved before or during its release. In support of this hypothesis, we found that platelets expressed ADAMTS-13-like molecule by western blot of platelet lysates and flow cytometry using two different ADAMTS-13 antibodies. The surface expression of ADAMTS-13-like molecule increased significantly upon platelet activation by TRAP and ADP. Consistent with these observations, the Triton-X-100 lysates of washed platelets and supernatant of washed platelets activated by TRAP contained VWF-cleaving activity, as demonstrated by the cleavage of ULVWF strings formed on histamine-stimulated endothelial cells under fluid shear stress. Our finding is consistent with a previous report, but further showed that this ADAMTS-13-like molecule exists not only in cytoplasma, but also on the surface of platelets. More importantly, the ADAMTS-13-like molecule increased its expression on platelet surface and was released upon platelet activation. The ADAMTS-13-like molecule either in platelets or released upon activation was active in cleaving ULVWF. This intrinsic mechanism for ULVWF proteolysis may be physiologically important. It may prevent the sudden release of hyper-reactive ULVWF from platelets and serve as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells during conditions such as severe infection. The role of the platelet membrane-bound ADAMTS-13 in hemostasis remains to be further determined.
Mechanisms for Thrombopoietin-Induced Potentiation of Platelet Aggregation.
Authors: Lova Paolo , Campus Francesca , Bertoni Alessandra , Sinigaglia Fabiola , Balduini Cesare , Torti Mauro 
Published: October 2019

Abstract
Thrombopoietin (TPO) is the major cytokine regulating megakaryocytes proliferation and thrombopoiesis. Binding of TPO to the c-Mpl receptor expressed on human platelets promotes tyrosine phosphorylation of a number of proteins and potentiates platelet aggregation induced by low concentrations of a variety of agonists including ADP. Nevertheless, TPO alone is not able to induce any platelet functional response, such as shape change, granule secretion, or aggregation. It is now clear that platelet aggregation induced by many different agonists results from the concomitant activation of both Gi-and Gq-dependent signaling pathways. In particular, activation of a Gi-dependent pathway is essential, albeit not sufficient, to elicit complete platelet aggregation. Recently, it has been proposed that the small GTPase Rap1B is an important element in the Gi-dependent pathway for platelet aggregation. ADP activates Gq and Gi-dependent pathways by binding to the P2Y1 and the P2Y12 receptors, respectively. In this study we investigated the contribution of TPO in platelet activation induced by ADP. We found that TPO could restore ADP-induced platelet aggregation when the Gq-coupled receptor was blocked, but failed to restore ADP-induced aggregation in absence of Gi stimulation. Similarly, TPO was unable to restore U46619-induced platelet aggregation, when the Gi-coupled P2Y12 receptor for ADP was blocked, but caused irreversible platelet aggregation when added in combination with epinephrine, which binds to a Gi-coupled receptor. TPO did not cause any detectable calcium movements or pleckstrin phosphorylation in human platelets. Moreover, TPO failed to restore ADP-induced cytosolic calcium incresases and pleckstrin phosphorylation when the Gq-coupled P2Y1 receptor was blocked, although aggregation was completely restored. Moreover, we found that TPO induced activation of Rap1B in platelets, and could completely restore ADP-induced Rap1B activation, which was partially reduced upon blockade of the P2Y1 receptor. Finally, ADP-induced binding of both fibrinogen and PAC-1 monoclonal antibody to integrin αIIbβ3 was prevented by the blockade of the Gq-coupled P2Y1 receptor, but completely restored by the simultaneous addition of TPO. These findings indicate that TPO can complement Gi-, but not Gq-dependent pathways for integrin αIIbβ3 activation and platelet aggregation, through a new mechanism that does not involve activation of phospholipase C, but may involve the small GTPase Rap1B.
Phosphoinositide3-Kinase Independent Regulation of Bruton’s Tyrosine Kinase in Platelet Immunoreceptor Signaling.
Authors: Watanabe Naohide , Nakajima Hideaki , Oda Atsushi , Ikeda Yasuo , Handa Makoto 
Published: October 2019

Abstract
Phosphoinositide 3-kinase (PI3K)-dependent activation of Bruton’s tyrosine kinase (Btk) is an indispensable step of B cell antigen receptor (BCR)-mediated signaling leading to cell development and function. Btk is a cytosolic tyrosine kinase and its recruitment to the plasma membrane is a necessary step for its function. In the BCR pathway, class 1A PI3K is though to play a major role in Btk recruitment by generating the D3 phosphoinositide as a docking site for the pleckstrin homology (PH) domain of this effecter kinase. This widely accepted hypothesis has been tested in platelets from gene knockout or mutant mice, since the cells utilize sets of transducers in collagen-induced GP VI signaling similar to those involved in immunoreceptor tyrosine-based activation motif-mediated signaling cascades activated by BCR and T cell antigen receptor (TCR) ligation. GP VI stimulation by collagen or collagen related peptide induces cellular responses including aggregation, granular secretion and adhesion, and Btk/phospholipase C (PLC) γ2 activation. As compared with control mice, these cellular responses and PLCγ2 tyrosine phosphorylation of either Btk or PI3K p85α−/− platelets were readily impaired, but the defect was greater in Btk−/− than p85α−/− platelets. Most strikingly, platelets from double-deficiency mice showed a most severely compromised phenotype implying the existence of a PI3K-independent pathway for Btk activation. Moreover, unlike B cells, as compared with Btk−/− platelets, only subtle functional defect was observed in X-linked immunodeficiency (Xid) platelets in which PI3K-dependent Btk activation is selectively lacking due to a naturally occurring point mutation of the gene encoding the PH domain of the kinase. In the TCR pathway, an adaptor complex formed by LAT, Gads and SLP-76 proteins that is membrane-bound via LAT palmitoylation readily recruits Itk, which is a counterpart Btk/Tec family kinase specific for TCR. Indeed, Btk was found to be associated with LAT/Gads/SLP-76 complex in platelets in a GP VI-stimulation dependent manner, and this phenomenon was unaffected by either PI3K defect or PI3K inhibitor. These results indicate that in platelet immunoreceptor signaling, Btk function is under control, at least in part, by a mechanism independent of PI3K engagement.
Reversible Exposure of Phosphatidylserine on Thrombin-Stimulated Platelets Detected by Binding of Lactadherin.
Authors: Shi Jialan , Rasmussen Jan T. , Heegaard Christian W. , Gilbert Gary E. 
Published: October 2019

Abstract
Platelets stimulated by thrombin support activity of the phosphatidylserine (PS)-dependent prothrombinase and factor Xase complexes. However, annexin V, a PS-detecting protein, does not bind to thrombin-stimulated platelets. Thus, the exposure of PS and its necessity for the prothrombinase and factor Xase complexes on platelets has been questioned. We hypothesized that platelets expose limited quantities of plasma membrane PS at levels that remain insufficient to support annexin V binding. We tested the hypothesis by investigating the capacity of platelets to support binding of lactadherin, a PS-binding milk protein. Binding of fluorescein-labeled lactadherin vs. fluorescence-labeled annexin V to synthetic membranes and to platelets was measured by flow cytometry. Lactadherin binding was proportional to PS content over the 0–4% range while annexin V required a threshold of approx. 2.5% PS. Platelets stimulated with 0.1–0.5 μM A23187 bound lactadherin but not annexin V. Binding of lactadherin was Ca++-independent, inhibited by PS-containing phospholipid vesicles and by antibodies to the C2 domain of lactadherin indicating that binding occurs via the phospholipid-binding rather than the integrin-binding motif of lactadherin. Platelets stimulated by thrombin or via the PAR-1 thrombin receptor also bound lactadherin but not annexin V. The KD was 84 ± 1 nM and there were 380 binding sites/platelet. Experiments in which platelets were labeled with fluorescent NBD-PS indicated that PS exposure occurred over 2 min and involved less than 15% of platelet PS. Exposed PS subsequently decreased, with more than 75% loss over 2 hr. Lactadherin inhibited greater than 98% of prothrombinase and factor Xase activity on platelets stimulated by A23187 or by thrombin. These results indicate that thrombin-stimulated platelets support the prothrombinase and factor Xase complexes through reversible exposure of a small fraction of membrane PS. Exposure of PS can be detected with Ca++-independent lactadherin binding in dilute platelet rich plasma, a method suitable for clinical studies.
CpG Binding Protein Is Required for Hematopoiesis in Zebrafish.
Authors: Young Suzanne R.L. , Doyle Christine , Marrs James , Skalnik David G. 
Published: October 2019

Abstract
CpG binding protein (CGBP) is a transcriptional activator that binds unmethylated CpG motifs. CGBP is ubiquitously expressed, and deletion of the CGBP gene in mice results in an embryonic lethal phenotype (E3.5–E6) indicating that CGBP is required for embryonic mammalian development. CGBP-null murine embryonic stem (ES) cells are viable but unable to differentiate and have a 60% decrease in CpG methylation compared to wild type ES cells. Based upon these results, it is hypothesized that CGBP is required for normal stem cell function. RT-PCR was used to identify a homologue of CGBP in zebrafish which is ~70% identical to the mouse CGBP. It was determined that CGBP is expressed in the zebrafish as early as 2 hpf. Morpholino oligonucleotides were designed to the zebrafish CGBP and injected into 1–2 cell embryos. Approximately 80% of the zebrafish treated with the CGBP morpholino oligonucleotides had little or no circulating red blood cells and had abnormal yolk sac morphology at 48 hpf. More than half of the CGBP treated zebrafish also exhibited cardiac edema, and ~14% were dead at 24 hpf. A similar phenotype was observed with a second morpholino designed to an independent site of the zebrafish CGBP cDNA. Zebrafish treated with a control morpholino oligonucleotide at the 1–2 cell stage exhibited normal yolk sac morphology and normal red blood cell circulation at 48 hpf. The control treated zebrafish did not have cardiac edema, and only 2–3% of these fish were dead at 24 hpf. The specificity of phenotypes observed following treatment with the CGBP morpholino oligonucleotide was assessed by co-injection of mouse CGBP mRNA with the CGBP morpholino oligonucleotide at the 1–2 cell stage. Only 37% of these fish exhibited a decrease in circulating red blood cells compared to 80% of zebrafish treated with CGBP morpholino alone. While 14% of the CGBP morpholino treated zebrafish were dead at 24 hpf, only 4% of the co-injected zebrafish were dead at 24 hpf. This experiment demonstrates that the mouse CGBP is able to rescue the phenotypes induced by a reduction of the endogenous zebrafish CGBP. These data indicate that CGBP is essential for normal zebrafish hematopoiesis and establishes the importance of CGBP in post-gastrulation development.
Carrier-Independent Nuclear Import of the Transcription Factor PU.1.
Authors: Yaseen Nabeel R. , Takeda Akiko , Nazari Reza , Shio Helen , Blobel Gunter , Zhong Hualin 
Published: October 2019

Abstract
PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using GFP-PU.1 fusions, we show that the Ets DNA-binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted with the FG repeats of nucleoporins Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, dramatically increased in the presence of RanGMPPNP, indicating the formation of a PU.1/RanGTP/Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Since Nup62 is located close to the midplane of the nuclear pore complex (NPC) while Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 towards the nuclear side of the NPC by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.