bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cooperative Calcium Signaling Mediated by Glycoprotein (GP) Ib-IX-V, GPVI and Integrin α2β1 during Platelet Adhesion to Collagen under Flow.
Authors: De Marco Luigi , Cozzi Maria Rita , Mazzucato Mario , Battiston Monica , Perrus Martine Jandrot , Ruggeri Zaverio M. 
Published: April 2020

Abstract
We have investigated signaling mediated by GPIb-IX-V, GPVI and α2β1 during platelet adhesion to collagen type I either in the presence or absence of von Willebrand factor (VWF) A1A2A3 domain under flow conditions (wall shear rate 600 or 3000 s−1). We used platelets labeled with FLUO3-AM and real-time videomicroscopy with high-speed image acquisition to analyze concurrently adhesion, translocation and calcium transients in single platelets interacting with the surface. All experiments were performed in the presence of integrillin (100 μg/ml) to block the possible involvement of αIIbβ3. Platelet adhesion and activation at 600 s−1 were similar in the absence or presence of VWF A1A2A3 domain. Calcium signaling consisted of both short lasting peaks (release from intracellular stores) and long sustained waves (transmembrane flux). In our experimental condition blockade of α2β1 or GPVI with monoclonal antibodies reduced platelet adhesion by 50% and the proportion of adherent platelets that became activated by 20–30%. Concomitant inhibition of both receptors decreased platelet adhesion by &gt;90%. Thus, both α2β1 and GPVI participate in the initial stage of platelet adhesion to collagen type I. At the higher shear rate of 3000 s−1, platelet adhesion and activation was 40–50% less than at 600 s−1 in the absence of VWF A1A2A3 domain; addition of the latter enhanced adhesion and activation by 12 to 14-fold, and &gt;80% of the platelets exhibited stable adhesion during the 30 s observation period. Intracellular Ca++ peaks in adherent platelets reached 2–3 μM. Blockade of GP Ib-IX-V inhibited both platelet adhesion and activation by at least 90%. Blockade of α2β1 markedly reduced the number of adhering platelets, which were mostly (&gt;90%) translocating on the surface and exhibited only short lasting Ca++ transients. Blockade of GPVI greatly decreased the number of adherent platelets, most of which, however, were firmly attached to the surface (&lt;15% exhibiting translocation). Calcium transients in the activated platelet consisted of short lasting peaks and rare long lasting waves reaching concentrations &lt;1.5–2 μM. These results suggest that, on a surface of collagen type I, α2β1 is required for normal platelet arrest following tethering through the VWF A1 domain-GPIb-IX-V interaction at higher shear rates, and Ca++ signaling results from the concerted action of the two receptors reinforced by GPVI. The adhesion potential of platelets exposed to collagen, therefore, appears to depend on signaling triggered by different ligand-receptor interactions varying as a function of flow conditions.
Regulation of Platelet Functions by Targeting STAT3 with a g-Quartet Oligonucleotide.
Authors: Zhou Zhou , Guan Yongli , Sun Wensheng , Jin Naijie , Dong Jing-fei 
Published: April 2020

Abstract
Collagen exposed on the subendothelial matrix supports platelet adhesion and aggregation at the site of vessel injury. Its interaction with the platelet membrane receptor GP V/FcRγ chain initiates intracellular signals that recruit the hematopoietic tyrosine kinase Syk to immunoreceptor tyrosine activation motifs (ITAMs) within the FcRγ cytoplasmic tail, leading a cascade of intracellular signal events that eventually activate platelets. In the present study, we demonstrated that collagen and the collagen-related peptide (CRP) stimulation also dose-dependently (2.5–10 mg/ml, 10 min) phosphorylated the signal transducers and activators of transcription 3 (STAT3) at the tyrosine residue 705 (Tyr705) in platelets, indicating that STAT3 participates in collagen-induced platelet activation, likely through a non-transcriptional mechanism. To test this hypothesis, we determined whether a STAT3-blocking G-rich oligonucleotide (T40214, GGGCGGGCGGGCGGGC) blocks platelet activation and aggregation induced by collagen or CRP. T40214 was designed based on G-rich DNA sequences, which were originally identified in the telomeres DNA to have the ability to form the protein-binding rigid G-quartet structures. It was formed through four guanine (G) bases into cyclic Hoogsteen H-bonding. NMR determined the tertiary structure of T40214 as two stacked identical G-quartets in the center and two G-C-G-C loop domains on the top and bottom, respectively. It has previously been shown to inhibit STAT3 phosphorylation in cancer cell lines and to induce tumor apoptosis in vivo. T40214 was delivered into platelets using nano-particle polyethylenimine (PEI) as the carrier. The treatment of washed platelets with T40214 inhibited STAT3 phosphorylation induced by collagen (10 mg/ml, 10 min), while the scramble oligonucleotides did not. Incubation of washed platelets and platelets-rich plasma (PRP) with T40214 inhibited collagen-induced platelets aggregation. Finally, thrombus formation on immobilized collagen was significantly reduced when whole blood treated with T40214 (10ug/ml) under a wall shear stress of 30 dyn/cm2. Results demonstrate a novel role of STAT3 in regulating the collagen-induced intracellular signal and platelet functions. The STAT3-blocking G-quartet offers a new class of potential platelet-regulating reagents derived from screening tertiary structure of the G-rich DNA sequences that form protein-binding G-quartets.
The Calcineurin/NFAT Signaling Pathway Is a Target for the Collagen Type I-Induced Calcium Response in Megakaryocytes and Mediates Expression of Megakaryocytic Genes.
Authors: Kyttaelae Satu , Habermann Ivonne , Bornhaeuser Martin , Ehninger Gerhard , Kiani Alexander 
Published: April 2020

Abstract
Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.
The Cytokine Signal Inhibitor Lnk Promotes αIIbβ3 Integrin Outside-In Signaling through β3 Tyrosine Phosphorylation in Platelets.
Authors: Eto Koji , Takizawa Hitoshi , Takaki Satoshi , Nishikii Hidekazu , Oda Atsushi , Nakauchi Hiromitsu 
Published: April 2020

Abstract
Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk−/− mice exhibit a marked increase in numbers of hematopoietic stem cells, megakaryocytes and platelets, presumably due to the lack of negative regulation in thrombopoietin-mediated signals by Lnk. We previously reported that Lnk might play an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk−/− platelets exhibited defects in full spreading on fibrinogen, clot retraction and formation of thrombi on collagen under flow conditions while they showed normal inside-out signaling (Blood, 106 (11):115a, 2005). However the mechanism(s) in which Lnk participates in αIIbβ3 outside-in signaling had not been elucidated. Here we report that in normal platelets Lnk forms a complex with c-Src, Syk, Fyn and adhesion and degranulation promoting adaptor protein (ADAP) but not SLP-76 in a manner dependent on αIIbβ3 ligation and Src kinase activation. c-Src-, but not Syk-, mediated tyrosine phosphorylation of C-terminus in Lnk appeared to be indispensable for the complex formation and Lnk-mediated function. Furthermore we have shown that Lnk is required for the association of Fyn to αIIbβ3 and for β3 subunit tyrosine phosphorylation while activation of non-receptor tyrosine kinases (c-Src and Syk) in proximity to αIIbβ3 is independent of Lnk. Thus, these results provide new insights into Lnk function and the mechanism by which Lnk contributes to integrin signaling in the adhesion responses of platelets.
Distinct Role for Fyn-Kinase in αIIbß3-Mediated Signaling.
Authors: Reddy Kumar B. , Plow Edward F. 
Published: April 2020

Abstract
Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signaling. αIIbβ3 has been shown to interact with multiple SFK members. We analyzed the role of Fyn in αIIbβ3-signaling and hemostasis. Fyn associated with αIIbβ3 in resting as well as thrombin aggregated platelets. In contrast, the interaction between αIIbβ3 and another SFK member, cSrc, was seen predominantly in resting but not in thrombin aggregated platelets. Based on this difference, we explored if the Fyn−/− mice exhibit any abnormalities in hemostasis and platelet function. Fyn−/− mice had significantly prolonged second bleeding times compared to wild-type (WT) controls (3.61 min for Fyn−/− compared to 1.04 min for WT mice, n=11). Platelets from Fyn−/− mice also exhibited delayed spreading on fibrinogen coated surfaces. Fyn, but not cSrc, localized to focal adhesions in CHO cells adherent to fibrinogen via αIIbβ3, and association of Fyn and cSrc depends on different segments of the cytoplasmic tail of the β3 subunit in this model cell system In the CHO cell system, Fyn mediated αIIbβ3-dependent cell spreading in the absence of Rho-GTPase recruitment. Using mutant forms of Fyn, we found that its kinase activity is required for its localization to focal adhesions and to mediate αIIbβ3-dependent cell spreading. These results show previously unidentified roles for Fyn in αIIbβ3-signaling and hemostasis.
Cyclic AMP-Dependent Protein Kinase Mediates Glycoprotein Ibα Shedding from Platelet.
Authors: Dai Kesheng , Yan Rong , Cheng Hong , Bodnar Richard , Ruan Changgeng 
Published: April 2020

Abstract
Extracellular domain of platelet glycoprotein (GP) Ibα contains ligand binding sites for von Willebrand factor (VWF) and α-thrombin. GPIbα binding to VWF exposed at the injured vessel initiates thrombus formation, thus it plays key roles in thrombosis and hemostasis. While a lot of research has been performed to elucidate the critical roles for GPIbα in platelet activation, little is known about the negative regulatory mechanisms of this adhesion receptor. Here we show that inhibition of cAMP-dependent protein kinase (PKA) resulted in the shedding of GPIbα from platelet. GPIbα was shed after platelets were incubated with PKA inhibitors (H89, PKI) in a dose-dependent manner. PKA mediated GPIbα shedding was inhibited by calpain inhibitors (MDL 28170, E64d, calpain inhibitor-I, calpain inhibitor-II) in a dose-dependent manor, suggesting that shedding of GPIbα is a result of calpain cleavage. Time course experiment revealed that PKA mediated GPIbα shedding occurred as a late event, 10 minutes after platelet activation. Flow cytometry and western-blot data suggested that the cleavage site was at N-terminal of residues 484 and 485 on GPIbα. These residues are responsible for disulfide bond linkage to GPIbβ. Though the size of GPIbα shed fragments from platelet treated with H89 was the same as platelet treated with calcium ionophore A23187 or thrombin, yet the intensity of platelet activation, the amount of GPIbα shedding, and redistribution of GPIbα were different, suggesting that the mechanism of PKA inhibition-initiated GPIbα shedding is different from the shedding caused by A23187 or thrombin. Platelets treated with the PKA inhibitor, H89, presented significant decrease in ristocetin induced platelet aggregation and platelet adhesion on VWF under shear stress. In conclusion, these data provide new evidence that inhibition of PKA results in calpain mediated GPIbα shedding which may play a role in limiting thrombus infinite formation after platelet activation.
DYRK3 Encodes an Inhibitor of Erythroid Differentiation and Is Expressed at High Levels in Patients with Anemia.
Authors: Steensma David P. , Porcher Julie C. , Chadderton Antony , Laubscher Kevin , Jaworski Deb , Lager Joanne J. , Bell William , Hoffman Bryan E. , Erickson-Miller Connie L. 
Published: April 2020

Abstract
Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis.
Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor.
Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied.
Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.
Detection of Hypoxia-Inducible Gene Manipulation.
Authors: Imagawa Shigehiko , Horie Masaki , Matsumoto Ken , Hirano Ikuo , Suzuki Norio , Yamamoto Masayuki 
Published: April 2020

Abstract
Erythropoietin (Epo) gene expression is controlled by hypoxia-inducible factor-1 (HIF-1); it is positively regulated through the HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. Drugs that inhibit GATA or activate HIF-1 might increase the production of Epo and restore hemoglobin concentration. Epo gene doping and the illicit use of HIF-PHD inhibitor (FG-2216) and GATA inhibitor (K-11706) or HIF-1 activators may be used as new doping practices to increase the number of red blood cells. The broad scope of this research is to develop a system for detecting illegal hypoxia-inducible gene manipulation. We performed hematologic analyses, a treadmill exercise test, microarray and real-time RT-PCR (reverse transcriptase-polymerase chain reaction) to examine the effects of K-11706 and FG-2216 on mice, and compared these effects with those induced by recombinant human Epo (rhEpo) as a positive control. Oral administration of K-11706 for 5 and 8 days, FG-2216 for 5 days, and 14-day intra-peritoneal injection of rhEpo on alternate days significantly increased Epo and hemoglobin concentrations, hematocrit, and endurance performance, respectively, compared with the control. Transgenic lines of mice were generated to evaluate erythropoiesis in living mice. Firefly luciferase reporter line generated with the human β-globin locus control region (Hbb-LCR) is referred as Hbb-LCR-Luc; this mouse model provides a sensitive, noninvasive, and real-time method to monitor erythropoiesis in vivo in response to the administration of K-11706 and FG-2216. The oral administration of K-11706 and FG-2216 for 5 days significantly accumulated Hbb-LCR-Luc activity in the spleen. DNA chip technology was used to investigate the molecular mechanisms responsible for hypoxia-inducible gene manipulation. Total RNA was prepared from the bone marrow cells of mice treated with K-11706 for 28 days, FG-2216 for 5 days, and rhEpo for 14 days, and in untreated mice. The genes that appeared to exhibit a difference in the expression level (increase more or decrease less than five times for K-11706 and rhEpo, two times for FG-2216) were 251 genes for K-11706, 343 genes for FG-2216, and 142 genes for rhEpo. Twenty-eight genes showed a significant difference between the 3 groups, and 4 genes showed it with K-11706 and FG-2216. Among them, real-time RT-PCR revealed that FG-2216 significantly decreased the expression levels of FOS, OSM, IL8rb, Ms4a8a, and Arl5c genes to 0.58, 0.54, 0.56, 0.46, and 0.53-fold compared with the control, respectively. A panel of several candidate genes to detect hypoxia-inducible gene manipulation by real-time RT-PCR will provide a new detection system.
Four Novel Mutations in the Ferrochelatase (FECH) Gene in Patients with Erythropoietic Protoporphyria (EPP).
Authors: Di Pierro Elena , Brancaleoni Valentina , Besana Valeria , Ausenda Sabrina , Care Melanie , Cappellini Maria Domenica 
Published: April 2020

Abstract
Erythropoietic protoporphyria (EPP) is an autosomal dominant disease with incomplete penetrance, due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the insertion of iron in the protoporphyrin molecule in the final step of the heme biosynthetic pathway. The disease is biochemically characterized by increased protoporphyrins in erythrocytes, plasma and faeces and normal levels in urine. The clinical manifestations have a childhood onset, characterized by mild to moderate cutaneous photosensitivity, mild anemia and, in 5–10% of the cases, by progressive hepatic failure. Diagnosis of EPP can be supported by the simple measurement with flow citometry of fluorescent erythrocytes in peripheral blood. The phenotype of EPP results from coinheritance of a mutated allele and a wild-type low expressed allele of the FECH gene that spans about 45 kb on the long arm of chromosome 18. The FECH gene encodes for a precursor of 423 amino acid residues that is targeted to the matrix side of the inner mitochondrial membrane via a 62 amino acid cleavable leader sequence. A single promoter directs both housekeeping and erythroid expression, but two polyadenylation sites produce two mRNAs of different length. The molecular analysis of FECH gene has allowed the identification of more than 100 different mutations responsible for EPP, showing a high genetic heterogeneity. Aim of this study was to search for molecular abnormalities in four patients with clinical and biochemical signs of EPP. Molecular analysis was performed by PCR and direct sequencing of the promoter, the entire coding region and the splicing junctions of the gene. In patients who remained negative for mutations, DNA analysis was completed by long-PCR and chromosome walking. In this study four different molecular defects have been identified: two missense mutations and two large deletions in the first region of gene. One A&gt;G substitution at nucleotide 362 in exon 4 resulting in a Glu to Gly amino acid substitution at position 121; one change C&gt;T in position 782 of exon 7 causing a Ser 261Phe substitution; the c.1-7887_67+2422del10376bp and the c.1-9628_67+2871del12566bp. Even though with different breakpoints, both deletions include a portion of upstream intergenic region, the promoter, the exon 1 and a portion of intron 1 of the FECH gene preventing the expression of the mutated allele. This deletions are probably caused by unequal intragenic recombination between ALU sequences which have been found close to the breakpoints. Recently others deletions involving ALU sequences were described suggesting that large deletions can affect the FECH gene and cause of EPP.