62 results on '"Attila Braun"'
Search Results
2. GALE force in platelet production
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Attila Braun and Elmina Mammadova-Bach
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2023
3. Inflammatory Networks in Renal Cell Carcinoma
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Linus Kruk, Medina Mamtimin, Attila Braun, Hans-Joachim Anders, Joachim Andrassy, Thomas Gudermann, and Elmina Mammadova-Bach
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Cancer Research ,Oncology - Abstract
Cancer-associated inflammation has been established as a hallmark feature of almost all solid cancers. Tumor-extrinsic and intrinsic signaling pathways regulate the process of cancer-associated inflammation. Tumor-extrinsic inflammation is triggered by many factors, including infection, obesity, autoimmune disorders, and exposure to toxic and radioactive substances. Intrinsic inflammation can be induced by genomic mutation, genome instability and epigenetic remodeling in cancer cells that promote immunosuppressive traits, inducing the recruitment and activation of inflammatory immune cells. In RCC, many cancer cell-intrinsic alterations are assembled, upregulating inflammatory pathways, which enhance chemokine release and neoantigen expression. Furthermore, immune cells activate the endothelium and induce metabolic shifts, thereby amplifying both the paracrine and autocrine inflammatory loops to promote RCC tumor growth and progression. Together with tumor-extrinsic inflammatory factors, tumor-intrinsic signaling pathways trigger a Janus-faced tumor microenvironment, thereby simultaneously promoting or inhibiting tumor growth. For therapeutic success, it is important to understand the pathomechanisms of cancer-associated inflammation, which promote cancer progression. In this review, we describe the molecular mechanisms of cancer-associated inflammation that influence cancer and immune cell functions, thereby increasing tumor malignancy and anti-cancer resistance. We also discuss the potential of anti-inflammatory treatments, which may provide clinical benefits in RCCs and possible avenues for therapy and future research.
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- 2023
4. Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
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Chao Han, Pengxuan Ren, Medina Mamtimin, Linus Kruk, Edita Sarukhanyan, Chenyu Li, Hans-Joachim Anders, Thomas Dandekar, Irena Krueger, Margitta Elvers, Silvia Goebel, Kristin Adler, Götz Münch, Thomas Gudermann, Attila Braun, and Elmina Mammadova-Bach
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collagen ,blood platelets ,GPVI ,Medicine (miscellaneous) ,thrombosis ,General Biochemistry, Genetics and Molecular Biology ,anti-thrombotic therapies - Abstract
Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.
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- 2023
5. BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation
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Sarah Beck, Markus Sauer, Hannah S. Heil, Attila Braun, Julia Volz, Michael K. Schuhmann, Mara Meub, Thomas Premsler, David Stegner, Guido Stoll, Julia Preu, Inga Scheller, Charly Kusch, Albert Sickmann, Michael Popp, Timo Vögtle, Katrin G. Heinze, Bernhard Nieswandt, and Katherina Hemmen
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Blood Platelets ,0301 basic medicine ,Mice, Transgenic ,Inflammation ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Inositol 1,4,5-Trisphosphate Receptors ,Platelet ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Platelet activation ,Adaptor Proteins, Signal Transducing ,Calcium signaling ,ORAI1 ,Chemistry ,Endoplasmic reticulum ,Thrombosis ,STIM1 ,General Medicine ,Inositol trisphosphate receptor ,Cell biology ,Disease Models, Animal ,030104 developmental biology ,030220 oncology & carcinogenesis ,medicine.symptom ,Research Article - Abstract
Store-operated Ca(2+) entry (SOCE) is the major route of Ca(2+) influx in platelets. The Ca(2+) sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca(2+) channel Orai1 and the inositol trisphosphate receptor (IP(3)R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP(3)R in platelets. Deletion of platelet BIN2 (Bin2(fl/fl,Pf4-Cre) mice) resulted in reduced Ca(2+) store release and Ca(2+) influx in response to all tested platelet agonists. These defects were a consequence of impaired IP(3)R function in combination with defective STIM1-mediated SOC channel activation, while Ca(2+) store content and agonist-induced IP(3) production were unaltered. This severely defective Ca(2+) signaling translated into impaired thrombus formation under flow and a protection of Bin2(fl/fl,Pf4-Cre) mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.
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- 2020
6. Coactosin-like 1 integrates signaling critical for shear-dependent thrombus formation in mouse platelets
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Irina Pleines, Albert Sickmann, Katrin G. Heinze, Oliver Otto, Bernhard Nieswandt, Sarah Beck, Simon Stritt, Markus Bender, Attila Braun, Inga Scheller, Robert Ahrends, and Bing Peng
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Blood Platelets ,Platelet Biology & its Disorders ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Megakaryocyte ,Von Willebrand factor ,von Willebrand Factor ,medicine ,Animals ,Platelet ,Platelet activation ,Thrombus ,Actin ,Mice, Knockout ,biology ,Chemistry ,Microfilament Proteins ,Thrombosis ,Articles ,Hematology ,Platelet Activation ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Platelet Glycoprotein GPIb-IX Complex ,Actin depolymerizing factor ,Hemostasis ,biology.protein ,030215 immunology - Abstract
Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.
- Published
- 2019
7. TRP channel function in platelets and megakaryocytes: basic mechanisms and pathophysiological impact
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Kalina Kazandzhieva, Elmina Mammadova-Bach, Alexander Dietrich, Thomas Gudermann, and Attila Braun
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Pharmacology ,Blood Platelets ,Transient Receptor Potential Channels ,Cations ,Humans ,Pharmacology (medical) ,Calcium ,Calcium Signaling ,Megakaryocytes - Abstract
Transient receptor potential (TRP) proteins form a superfamily of cation channels that are expressed in a wide range of tissues and cell types. During the last years, great progress has been made in understanding the molecular complexity and the functions of TRP channels in diverse cellular processes, including cell proliferation, migration, adhesion and activation. The diversity of functions depends on multiple regulatory mechanisms by which TRP channels regulate Ca
- Published
- 2021
8. FC 003HYPERURICEMIA HAS VASOACTIVE EFFECTS IN CHOLESTEROL CRYSTAL-INDUCED ACUTE KIDNEY INJURY
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Hans-Joachim Anders, Stefanie Steiger, Luying Yang, Elmina Mammadova-Bach, Attila Braun, and Thomas Gudermann
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Transplantation ,Kidney ,medicine.medical_specialty ,Necrosis ,biology ,Cholesterol ,business.industry ,Acute kidney injury ,Renal function ,medicine.disease ,Fibrin ,chemistry.chemical_compound ,medicine.anatomical_structure ,Endocrinology ,chemistry ,Nephrology ,Internal medicine ,medicine ,biology.protein ,Platelet activation ,medicine.symptom ,Interleukin 6 ,business - Abstract
Background and Aims Numerous observational studies have reported an association between hyperuricemia (HU) and cardiovascular disease where atherosclerosis is a leading cause. In advanced atherosclerosis, cholesterol crystal (CC) embolism is a life-threatening complication with an average mortality of 62.8%. Clinical manifestations include skin necrosis, intestinal injury and acute kidney injury (AKI). Autopsies and tissue biopsies reveal CC inside the arterial lumen. In a new model of CC-induced AKI, we recently found that fibrin clots formed around CC obstruct peripheral arteries and cause tissue infarction and organ failure. However, the role of asymptomatic HU in CC-induced AKI is currently unknown. Thus, we hypothesized that asymptomatic HU improves the outcomes after CC-induced AKI. Method In vivo, 6 weeks old Alb-creERT2;Glut9lox/lox and Glut9lox/lox control mice were intraperitoneally injected with tamoxifen to deplete hepatic Glut9 expression. Both groups of mice were placed on a standard chow diet enriched with inosine for two weeks. Afterward, a solution of CCs was injected into the left kidney arteries of mice to induce AKI. Serum and kidneys were collected 24 hours later, and kidney function, infarct size and kidney injury evaluated using GFR measurement, colorimetric assays, ELISA and immunohistochemistry of kidney sections. For in vitro studies, we isolated platelets from healthy mice and cultured them in the presence or absence of soluble uric acid (sUA) prior to CC activation. After stimulation, platelet aggregation assays and flow cytometry were performed. Results Our in vivo data showed that Alb-creERT2;Glut9lox/lox mice developed asymptomatic HU without kidney impairment (serum UA levels 9-15 mg/dL), while Glut9lox/lox control mice remained healthy. HU mice displayed a rapid decline in GFR of approx. 80% as compared to only 30% in healthy mice after CC-induced AKI, which was in line with increased serum BUN and IL-6 levels. The rapid GFR decline was due to vasoconstriction in arteries of the contralateral kidney as determined by αSMA/fibrin staining and a reduced ratio of lumen versus artery area in mice with HU after AKI. HU mice also had significantly less kidney infarct size as well as reduced kidney injury (necrosis and edema) and arterial occlusion. The in vitro data showed that sUA had no impact on platelet aggregation, activation and degranulation as compared to medium control in response to CCs. Conclusion We now show that HU has vasoconstrictive effects in the contralateral kidney by reducing GFR, while at the same time protects mice from CC-induced AKI suggesting a compensatory physiological mechanism of autoregulation to protect nephrons. On the other hand, in patients with chronic kidney disease, HU-related vasoconstriction might lead to increased arterial blood pressure; thus, increasing the risk for cardiovascular complications due to platelet activation and aggregation.
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- 2021
9. Platelet-Cancer Interplay: Molecular Mechanisms and New Therapeutic Avenues
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Attila, Braun, Hans-Joachim, Anders, Thomas, Gudermann, and Elmina, Mammadova-Bach
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Oncology ,anti-platelet therapies ,platelets ,cancer ,metastasis ,Review ,thrombosis - Abstract
Although platelets are critically involved in thrombosis and hemostasis, experimental and clinical evidence indicate that platelets promote tumor progression and metastasis through a wide range of physical and functional interactions between platelets and cancer cells. Thrombotic and thromboembolic events are frequent complications in patients with solid tumors. Hence, cancer modulates platelet function by directly inducing platelet-tumor aggregates and triggering platelet granule release and altering platelet turnover. Also, platelets enhance tumor cell dissemination by activating endothelial cell function and recruiting immune cells to primary and metastatic tumor sites. In this review, we summarize current knowledge on the complex interactions between platelets and tumor cells and the host microenvironment. We also critically discuss the potential of anti-platelet agents for cancer prevention and treatment.
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- 2021
10. Platelet life without TMEM163: no dense granules
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Attila Braun and Elmina Mammadova-Bach
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Blood Platelets ,Pathology ,medicine.medical_specialty ,business.industry ,Chemistry ,Immunology ,Cell Biology ,Hematology ,Cytoplasmic Granules ,Platelets and Thrombopoiesis ,Biochemistry ,eye diseases ,Text mining ,medicine ,Platelet ,business - Abstract
Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1(sdy) and Pldn(pa))–, BLOC-2 (Hps6(ru))–, or AP-3 (Ap3b1(pe))–deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker–positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.
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- 2021
11. Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
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Johannes Balkenhol, Kristin V. Kaltdorf, Elmina Mammadova-Bach, Attila Braun, Bernhard Nieswandt, Marcus Dittrich, and Thomas Dandekar
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Blood Platelets ,Interspecies comparison ,Interactome ,genetic structures ,lcsh:QH426-470 ,Proteome ,Mouse ,lcsh:Biotechnology ,Network ,behavioral disciplines and activities ,Mice ,ddc:570 ,lcsh:TP248.13-248.65 ,Animals ,Guanine Nucleotide Exchange Factors ,Humans ,Platelet ,Thrombosis ,Platelet Activation ,Signaling ,lcsh:Genetics ,nervous system ,Cascade ,Transcriptome ,psychological phenomena and processes ,Signal Transduction ,Research Article ,Human - Abstract
Background: Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results: The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always uniprot notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.Conclusions: Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
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- 2020
12. Profilin 1–mediated cytoskeletal rearrangements regulate integrin function in mouse platelets
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Simon Stritt, Simona Sorrentino, Xiaoping Du, Sarah Beck, John H. Hartwig, Harald Schulze, Julien Viaud, Johannes Heck, Inga Birkholz, Markus Bender, Bernhard Nieswandt, K. Tanuj Sapra, Ohad Medalia, Attila Braun, Frédérique Gaits-Iacovoni, Regenerative Medicine and Skeleton research lab (RMeS), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, University of Zurich, and Nieswandt, Bernhard
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Blood Platelets ,0301 basic medicine ,2720 Hematology ,Integrin ,610 Medicine & health ,[SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,macromolecular substances ,Mice ,Profilins ,03 medical and health sciences ,0302 clinical medicine ,Profilin-1 ,10019 Department of Biochemistry ,Animals ,Platelet ,Cytoskeleton ,ComputingMilieux_MISCELLANEOUS ,biology ,Chemistry ,Integrin beta1 ,Integrin beta3 ,[SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN] ,Hematology ,Stimulus Report ,Cell biology ,030104 developmental biology ,Profilin ,030220 oncology & carcinogenesis ,Hemostasis ,biology.protein ,570 Life sciences ,Function (biology) - Abstract
Key Points Profilin 1–mediated cytoskeletal dynamics regulate platelet β1- and β3-integrin function and turnover. Profilin 1 deficiency in platelets impairs hemostasis and results in a marked protection from arterial thrombosis.
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- 2018
13. TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in Mice
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Wenchun Chen, René P. Zahedi, Vladimir Chubanov, Anne-Laure Perraud, Attila Braun, Karen Wolf, Michael G. Tomlinson, Carsten Schmitz, Simon Stritt, Susanna Zierler, Thomas Gudermann, Bernhard Nieswandt, Isabelle C. Becker, Thomas Dandekar, Deviyani Rao, Heike M. Hermanns, Sanjeev K. Gotru, Masayuki Matsushita, Guido Stoll, Peter Kraft, and Peter J. Noy
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0301 basic medicine ,Phospholipase C ,Chemistry ,Kinase ,Store-operated calcium entry ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,TRPM7 ,Platelet ,Platelet activation ,GPVI ,Kinase activity ,Cardiology and Cardiovascular Medicine ,030217 neurology & neurosurgery - Abstract
Objective— TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg 2+ and Ca 2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. Approach and Results— Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice ( Trpm7 R/R ) causes a marked signaling defect in platelets. Trpm7 R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca 2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca 2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. Conclusions— Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca 2+ entry in platelets. The protection of Trpm7 R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.
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- 2018
14. Loss of Orai2-Mediated Capacitative Ca
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David, Stegner, Sebastian, Hofmann, Michael K, Schuhmann, Peter, Kraft, Alexander M, Herrmann, Sandy, Popp, Marlen, Höhn, Michael, Popp, Vanessa, Klaus, Antonia, Post, Christoph, Kleinschnitz, Attila, Braun, Sven G, Meuth, Klaus-Peter, Lesch, Guido, Stoll, Robert, Kraft, and Bernhard, Nieswandt
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Mice, Knockout ,Neurons ,Stroke ,Mice ,Cell Death ,Acute Disease ,ORAI2 Protein ,Animals ,Calcium ,Calcium Signaling ,Neuroprotection ,Brain Ischemia - Abstract
Background and Purpose- Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca
- Published
- 2019
15. Platelet glycoprotein VI promotes metastasis through interaction with cancer cell-derived galectin-3
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Elmina Mammadova-Bach, Charlotte Schonhart, Katharina A. Remer, Thomas Dandekar, Alan T. Nurden, Philipp Burkard, Paquita Nurden, Bernhard Nieswandt, Sergey Shityakov, Jesus Gil-Pulido, Diego Mezzano, Scott I. Abrams, Magdolna Dank, David Stegner, Attila Braun, Edita Sarukhanyan, and Laszlo Nehez
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0301 basic medicine ,Blood Platelets ,Male ,Lung Neoplasms ,Galectin 3 ,Immunology ,Syk ,Breast Neoplasms ,Platelet Membrane Glycoproteins ,Biochemistry ,Metastasis ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Platelet ,Platelet activation ,Protein Interaction Maps ,Neoplasm Metastasis ,Mice, Inbred BALB C ,Chemistry ,Cell Biology ,Hematology ,medicine.disease ,Platelet Activation ,Extravasation ,Mice, Inbred C57BL ,030104 developmental biology ,Galectin-3 ,Cancer cell ,Colonic Neoplasms ,Cancer research ,Female ,GPVI ,030215 immunology - Abstract
Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM)-signaling and thereby regulates diverse functions including platelet adhesion, aggregation and procoagulant activity. GPVI has been proposed as a safe antithrombotic target as its inhibition is protective in models of arterial thrombosis with only minor effects on hemostasis. Here, we demonstrate that genetic deficiency of platelet GPVI in mice decreases experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses show that mouse, as well as human GPVI, supports platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knock-out approach, we identified Galectin-3 as the major counter-receptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed Galectin-3 utilizes ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab)2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study reveals a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.
- Published
- 2019
16. TRPM7 is the central gatekeeper of intestinal mineral absorption essential for postnatal survival
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Camilla Recordati, Masayuki Matsushita, Attila Braun, David G. Simmons, Eleonora Zakharian, Lorenz Mittermeier, Lusine Demirkhanyan, Vladimir Chubanov, Andreas Breit, Benjamin Stadlbauer, Anne Hilgendorff, and Thomas Gudermann
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inorganic chemicals ,0301 basic medicine ,Physiology ,TRPM7 ,TRPM Cation Channels ,Endogeny ,magnesium ,Kidney ,03 medical and health sciences ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,medicine ,Animals ,Homeostasis ,Mineral absorption ,Kinase activity ,Intestinal Mucosa ,Mice, Knockout ,Minerals ,Multidisciplinary ,calcium ,Chemistry ,TRP channels ,Point mutation ,zinc ,Transporter ,Biological Sciences ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,PNAS Plus ,Signal transduction ,030217 neurology & neurosurgery - Abstract
Significance Zn2+, Mg2+, and Ca2+ are the most abundant divalent metals in mammals. Different categories of cation-selective channels and transporters are thought to control the levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these minerals are poorly understood. Using genetic mouse models together with biophysical and biochemical analysis, we show that the channel-kinase TRPM7 is a master regulator of the organismal balance of divalent cations. TRPM7 activity is primarily required in the intestine, while TRPM7 function in the kidney—commonly thought to be essential—is expendable. Hence, against current thinking, organismal balance of multiple divalent cations predominantly relies on a common gatekeeper, TRPM7, rather than on individual specialized channels/transporters., Zn2+, Mg2+, and Ca2+ are essential minerals required for a plethora of metabolic processes and signaling pathways. Different categories of cation-selective channels and transporters are therefore required to tightly control the cellular levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these essential minerals are poorly understood. Herein, we identify a central and indispensable role of the channel-kinase TRPM7 for organismal mineral homeostasis. The function of TRPM7 was assessed by single-channel analysis of TRPM7, phenotyping of TRPM7-deficient cells in conjunction with metabolic profiling of mice carrying kidney- and intestine-restricted null mutations in Trpm7 and animals with a global “kinase-dead” point mutation in the gene. The TRPM7 channel reconstituted in lipid bilayers displayed a similar permeability to Zn2+ and Mg2+. Consistently, we found that endogenous TRPM7 regulates the total content of Zn2+ and Mg2+ in cultured cells. Unexpectedly, genetic inactivation of intestinal rather than kidney TRPM7 caused profound deficiencies specifically of Zn2+, Mg2+, and Ca2+ at the organismal level, a scenario incompatible with early postnatal growth and survival. In contrast, global ablation of TRPM7 kinase activity did not affect mineral homeostasis, reinforcing the importance of the channel activity of TRPM7. Finally, dietary Zn2+ and Mg2+ fortifications significantly extended the survival of offspring lacking intestinal TRPM7. Hence, the organismal balance of divalent cations critically relies on one common gatekeeper, the intestinal TRPM7 channel.
- Published
- 2019
17. Platelet Serotonin in Arterial and Deep Vein Thrombosis
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C. Bode, K. Wolf, Maximilian Mauler, Attila Braun, Daniel Duerschmied, K. Naber, P. Kröning, Nadine Gauchel, and C. Schönichen
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medicine.medical_specialty ,medicine.anatomical_structure ,business.industry ,Internal medicine ,Deep vein ,medicine ,Cardiology ,Platelet ,Serotonin ,business ,medicine.disease ,Thrombosis - Published
- 2019
18. Defective Zn
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Sanjeev, Kiran Gotru, Johanna P, van Geffen, Magdolna, Nagy, Elmina, Mammadova-Bach, Julia, Eilenberger, Julia, Volz, Georgi, Manukjan, Harald, Schulze, Leonard, Wagner, Stefan, Eber, Christian, Schambeck, Carsten, Deppermann, Sanne, Brouns, Paquita, Nurden, Andreas, Greinacher, Ulrich, Sachs, Bernhard, Nieswandt, Heike M, Hermanns, Johan W M, Heemskerk, and Attila, Braun
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inorganic chemicals ,Adult ,Blood Platelets ,Male ,Platelet Storage Pool Deficiency ,Adolescent ,Gray Platelet Syndrome ,Article ,Mice ,Cytosol ,Nephelometry and Turbidimetry ,Animals ,Homeostasis ,Humans ,Child ,Blood Coagulation ,Mice, Knockout ,Fibrin ,Microscopy, Confocal ,Membrane Proteins ,Blood Proteins ,Platelet Activation ,Healthy Volunteers ,Coagulation system ,Zinc ,Microscopy, Fluorescence ,Hermanski-Pudlak Syndrome ,Metals ,biological sciences ,health occupations ,bacteria ,Female - Abstract
Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.
- Published
- 2018
19. Micromolar concentrations of citalopram or escitalopram inhibit glycoprotein VI-mediated and integrin αIIbβ3-mediated signaling in human platelets
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Wenchun Chen, Yu-Lun Tseng, Meng-Ling Chiang, Chi-Yu Tseng, Attila Braun, and Jane Pei-Chen Chang
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0301 basic medicine ,Blood Platelets ,Serotonin ,Platelet Aggregation ,Syk ,Platelet Glycoprotein GPIIb-IIIa Complex ,Platelet Membrane Glycoproteins ,Pharmacology ,Citalopram ,Toxicology ,behavioral disciplines and activities ,03 medical and health sciences ,0302 clinical medicine ,Platelet Adhesiveness ,mental disorders ,Crotalid Venoms ,medicine ,Escitalopram ,Humans ,Syk Kinase ,Platelet ,Lectins, C-Type ,Platelet activation ,Pseudopodia ,Phosphorylation ,Adaptor Proteins, Signal Transducing ,Dose-Response Relationship, Drug ,Chemistry ,Receptors, IgG ,Fibrinogen ,Membrane Proteins ,Convulxin ,030104 developmental biology ,p21-Activated Kinases ,030220 oncology & carcinogenesis ,GPVI ,Proto-Oncogene Proteins c-akt ,Platelet Aggregation Inhibitors ,Selective Serotonin Reuptake Inhibitors ,medicine.drug ,Signal Transduction - Abstract
Collagen and convulxin induce platelet aggregation through glycoprotein VI (GPVI)-FcRγ-Syk signaling pathway. In addition, fibrinogen induces platelet activation through integrin αIIbβ3-FcγRIIa-Syk signaling pathway. We previously reported that high concentrations of selective serotonin reuptake inhibitors (SSRI) reduce platelet aggregation induced by collagen. We further investigated the effects of SSRI on GPVI- and αIIbβ3-mediated signaling pathway. Citalopram and escitalopram, two relatively pure SSRI, were used in this study. Both citalopram and escitalopram concentration-dependently inhibited convulxin-induced platelet aggregation, serotonin (5-HT) release and the activation of αIIbβ3. 5-HT concentration in washed platelets was unchanged after short-term treatment with citalopram. The additional 5-HT failed to fully rescue the inhibitory effect of citalopram on convulxin-induced aggregation. Convulxin-induced phosphorylation of Syk, LAT, and Akt was inhibited by citalopram and escitalopram. Citalopram inhibited the interaction between FcRγ and Syk, whereas the phosphorylation of FcRγ in response to convulxin remained unaltered. Further, citalopram inhibited the increase of the interaction between serotonin transporter and Syk induced by convulxin. In the presence of Mn2+, escitalopram inhibited the formation of lamellipodia on immobilized fibrinogen. Escitalopram did not influence the binding of fibrinogen to platelets. It inhibited the phosphorylation of Syk and PAK triggered by the adhesion on fibrinogen. Our data demonstrate that micromolar concentrations of citalopram and escitalopram inhibit GPVI- and αIIbβ3-mediated platelet functions. The mechanism of the inhibitory effect of citalopram or escitalopram is not the influence on the activation of GPVI or the interaction between fibrinogen and αIIbβ3, but the interaction between Syk and its upstream molecules.
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- 2018
20. Autocrine and paracrine regulatory functions of platelet serotonin
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Elmina Mammadova-Bach, Daniel Duerschmied, Attila Braun, and Maximilian Mauler
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0301 basic medicine ,Blood Platelets ,Serotonin ,business.industry ,Inflammation ,Hematology ,General Medicine ,Pharmacology ,Serotonergic ,03 medical and health sciences ,Paracrine signalling ,Autocrine Communication ,030104 developmental biology ,Blood plasma ,Paracrine Communication ,Medicine ,Humans ,Platelet ,medicine.symptom ,business ,Autocrine signalling ,Wound healing - Abstract
Platelets serotonin (5-hydroxytrytamine, 5-HT) uptake and storage in dense granules is tightly regulated by the serotonergic transport system in the blood. Several 5-HT transporters (5-HTTs) have been identified in the vasculature and blood cells, beyond them 5-HTT is the major 5-HT transporter in platelets. Abnormal 5-HT concentrations in the blood plasma or increased platelet 5-HT uptake or abnormal release contribute to the development of various diseases in the vasculature. Consequently, several clinical trials suggested the positive therapeutic effects of 5-HTT blockade in the circulation. Inhibition of 5-HT strongly attenuates autocrine and paracrine functions of platelets, influencing platelet aggregation, vascular contraction, permeability, tissue repair, wound healing, immunity and cancer. Here, we highlight the current state of basic biological research regarding the hemostatic and non-hemostatic functions of platelet-derived 5-HT in normal and disease conditions. We also describe the physiological consequences of targeting platelet 5-HT functions in thrombosis, stroke, inflammation and cancer to overcome common health problems.
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- 2018
21. Cutting Edge: Imbalanced Cation Homeostasis in MAGT1-Deficient B Cells Dysregulates B Cell Development and Signaling in Mice
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Attila Braun, Niklas Beyersdorf, Timo Vögtle, Heike M. Hermanns, Alma Zernecke, Jesus Gil-Pulido, Andreas Diefenbach, Vladimir Chubanov, Thomas Gudermann, Thomas Kerkau, Bernhard Nieswandt, Sanjeev K. Gotru, Katharina A. Remer, and Isabelle C. Becker
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0301 basic medicine ,Male ,T-Lymphocytes ,Immunology ,Antigens, CD19 ,Lymphocyte Activation ,CD19 ,03 medical and health sciences ,Mice ,Antigen ,Cations ,Cation homeostasis ,medicine ,Immunology and Allergy ,Animals ,Homeostasis ,Cation Transport Proteins ,B cell ,Protein Kinase C ,Mice, Knockout ,B-Lymphocytes ,biology ,Chemistry ,breakpoint cluster region ,Marginal zone ,Cell biology ,Hematopoiesis ,Killer Cells, Natural ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,Phosphorylation ,Leukocyte Common Antigens ,Calcium ,Signal Transduction - Abstract
Cation homeostasis, in relation to various immune-suppressive diseases, is a novel field of investigation. Recently, patients with a loss-of-function mutation in magnesium transporter 1 (MAGT1) were reported to present a dysregulated Mg2+ homeostasis in T lymphocytes. Using Magt1-knockout mice (Magt1−/y), we show that Mg2+ homeostasis was impaired in Magt1−/y B cells and Ca2+ influx was increased after BCR stimulation, whereas T and NK cell function was unaffected. Consequently, mutant B cells displayed an increased phosphorylation of BCR-related proteins differentially affecting protein kinase C activation. These in vitro findings translated into increased frequencies of CD19+ B cells and marginal zone B cells and decreased frequencies of plasma cells among CD45+ splenocytes in vivo. Altogether, our study demonstrates for the first time, to our knowledge, that abolished MAGT1 function causes imbalanced cation homeostasis and developmental responses in B cells. Therefore, this study might contribute to a further understanding of B cell–related pathologies.
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- 2017
22. Twinfilin 2a regulates platelet reactivity and turnover in mice
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Pekka Lappalainen, Xiaoping Du, Sarah Beck, Bernhard Nieswandt, Timo Vögtle, Simon Stritt, Markku Hakala, Attila Braun, Katrin G. Heinze, Markus Bender, and Isabelle C. Becker
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0301 basic medicine ,Blood Platelets ,Integrins ,Platelet disorder ,Immunology ,Arp2/3 complex ,Apoptosis ,macromolecular substances ,Biochemistry ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Animals ,Platelet activation ,Cytoskeleton ,biology ,Chemistry ,Microfilament Proteins ,Actin remodeling ,Thrombosis ,Cell Biology ,Hematology ,Arteries ,Actin cytoskeleton ,Thrombocytopenia ,3. Good health ,Cell biology ,Actin Cytoskeleton ,030104 developmental biology ,Profilin ,030220 oncology & carcinogenesis ,biology.protein ,MDia1 - Abstract
Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.
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- 2017
23. Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis
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Tobias Ruck, Kerstin Göbel, Susann Pankratz, Stefan Bittner, Bernhard Nieswandt, Alexander M. Herrmann, Sven G. Meuth, David Stegner, Heinz Wiendl, Michael K. Schuhmann, Johanna Breuer, Christoph Kleinschnitz, and Attila Braun
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Cell adhesion molecule ,Immunology ,Experimental autoimmune encephalomyelitis ,Intercellular Adhesion Molecule-1 ,Soluble cell adhesion molecules ,Biology ,Intercellular adhesion molecule ,medicine.disease ,Cell biology ,Myelin oligodendrocyte glycoprotein ,medicine ,biology.protein ,Immunology and Allergy ,Lymphocyte homing receptor ,Phospholipase D1 - Abstract
Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune-mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1-deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, a model of human MS. The lack of PLD1 reduced chemokine-mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1-deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1-dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.
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- 2014
24. Profilin 1 is required for peripheral nervous system myelination
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Dies Meijer, Joana Faria, João B. Relvas, Attila Braun, Laura Montani, Ueli Suter, Jorge A. Pereira, Robin J.M. Franklin, Nuno G. Dias, Tina Buerki-Thurnherr, Ralph T. Böttcher, Rui Loja Fernandes, Ana Filipa Gonçalves, Klaus-Armin Nave, Yves Benninger, Mercedes Costell, and Molecular Genetics
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Nervous system ,rac1 GTP-Binding Protein ,Neurogenesis ,Cèl·lules ,Schwann cell ,RAC1 ,CDC42 ,Axonal Transport ,Biotecnologia ,Mice ,Profilins ,Peripheral Nervous System ,medicine ,Animals ,Integrin-linked kinase ,Peripheral Nerves ,Pseudopodia ,Axon ,Molecular Biology ,Cells, Cultured ,Myelin Sheath ,Mice, Knockout ,biology ,Neuropeptides ,Cell biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Profilin ,nervous system ,Immunology ,biology.protein ,Schwann Cells ,Lamellipodium ,Proteïnes ,Developmental Biology - Abstract
Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actin-binding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrin-linked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviral-mediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed, SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin β1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation.
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- 2014
25. Author response: Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival
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Yuriy Shymkiv, Emiel P. C. van der Vorst, Christian Weber, Thomas Gudermann, Hans Zischka, Christin Leitzinger, Annette Schürmann, David G. Simmons, Annika Wisnowsky, Susanna Zierler, Alexey G. Ryazanov, Silvia Ferioli, Onder A. Yildirim, Ludmila Sytik, Vindi Jurinovic, Harald Bartsch, Wenke Jonas, Attila Braun, Banu Akdogan, Lorenz Mittermeier, Claudia Einer, Karl Sotlar, Friedrich Torben, and Vladimir Chubanov
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Magnesium transport ,business.industry ,TRPM6 ,Medicine ,Physiology ,business ,Prenatal development - Published
- 2016
26. p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo
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Hans-Peter Elsässer, Katharina Schlereth, Axel Pagenstecher, Bernhard Nieswandt, Michael Wanzel, Thorsten Stiewe, Andreas Rosenwald, Attila Braun, and Oleg Timofeev
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ddc:616 ,genetic structures ,DNA damage ,Mutant ,Cooperativity ,Plasma protein binding ,DNA-binding domain ,Biology ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,chemistry.chemical_compound ,lcsh:Biology (General) ,chemistry ,Protein quaternary structure ,lcsh:QH301-705.5 ,Gene ,DNA - Abstract
SummaryFour molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.
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- 2013
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27. Combined In Vivo Depletion of Glycoprotein VI and C-Type Lectin-Like Receptor 2 Severely Compromises Hemostasis and Abrogates Arterial Thrombosis in Mice
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Frauke May, Ina Hagedorn, Timo Vögtle, Brenda A. Finney, Michael R. Bösl, Steve P. Watson, Markus Bender, Katharina A. Remer, Attila Braun, Viola Lorenz, Ina Thielmann, and Bernhard Nieswandt
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Male ,Hemostasis ,business.industry ,Thrombosis ,Platelet Membrane Glycoproteins ,Pharmacology ,Mice, Inbred C57BL ,Mice ,In vivo ,C-type lectin ,Antithrombotic ,Immunology ,Animals ,Medicine ,Lectins, C-Type ,Platelet ,Tyrosine ,GPVI ,Cardiology and Cardiovascular Medicine ,Receptor ,business - Abstract
Objective— Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif–bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. Approach and Results— We investigated whether both (hem)immunoreceptor tyrosine activation motif–bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2–depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. Conclusions— These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti–CLEC-2–based agents in the prevention of thrombotic diseases.
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- 2013
28. CLP36 Is a Negative Regulator of Glycoprotein VI Signaling in Platelets
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Markus Bender, Michael R. Bösl, Thomas Premsler, Bernhard Nieswandt, Shuchi Gupta, Attila Braun, Albert Sickmann, Zoltan Nagy, Martina Morowski, and Heike M. Hermanns
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Blood Platelets ,Male ,medicine.medical_specialty ,Platelet Aggregation ,Physiology ,Blotting, Western ,PDZ domain ,Gene Expression ,Immunoreceptor Tyrosine-Based Activation Motif ,Inositol 1,4,5-Trisphosphate ,Platelet Membrane Glycoproteins ,Biology ,Mice ,chemistry.chemical_compound ,Microscopy, Electron, Transmission ,Internal medicine ,Immunoreceptor tyrosine-based activation motif ,medicine ,Animals ,Platelet ,Platelet activation ,Phosphorylation ,Mice, Knockout ,Microscopy, Confocal ,Reverse Transcriptase Polymerase Chain Reaction ,Thrombosis ,Tyrosine phosphorylation ,LIM Domain Proteins ,Cell biology ,Mice, Inbred C57BL ,Endocrinology ,chemistry ,Tyrosine ,Calcium ,Female ,Blood Coagulation Tests ,Collagen ,Signal transduction ,GPVI ,Cardiology and Cardiovascular Medicine ,Protein Binding ,Signal Transduction ,Transcription Factors - Abstract
Rationale: At sites of vascular injury, exposed subendothelial collagens not only trigger sudden platelet adhesion and aggregation, thereby initiating normal hemostasis, but also can lead to acute ischemic diseases, such as myocardial infarction or stroke. The glycoprotein (GP) VI/Fc receptor γ-chain complex is a central regulator of these processes because it mediates platelet activation on collagens through a series of tyrosine phosphorylation events downstream of the Fc receptor γ-chain–associated immunoreceptor tyrosine-based activation motif. GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. Objective: We studied the role of PDZ and LIM domain family member CLP36 in platelet physiology in vitro and in vivo. Methods and Results: We report that CLP36 acts as a major inhibitor of GPVI immunoreceptor tyrosine-based activation motif signaling in platelets. Platelets from mice either expressing a low amount of a truncated form of CLP36 lacking the LIM domain ( Clp36 ΔLIM ) or lacking the whole protein ( Clp36 −/− ) displayed profound hyperactivation in response to GPVI agonists, whereas other signaling pathways were unaffected. This was associated with hyperphosphorylation of signaling proteins and enhanced Ca 2+ mobilization, granule secretion, and integrin activation downstream of GPVI. The lack of functional CLP36 translated into accelerated thrombus formation and enhanced procoagulant activity, assembling a prothrombotic phenotype in vivo. Conclusions: These data reveal an inhibitory function of CLP36 in GPVI immunoreceptor tyrosine-based activation motif signaling and establish it as a key regulator of arterial thrombosis.
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- 2012
29. Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival
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Attila Braun, Christian Weber, Ludmila Sytik, Susanna Zierler, Vindi Jurinovic, Annette Schürmann, Lorenz Mittermeier, Banu Akdogan, Hans Zischka, David G. Simmons, Christin Leitzinger, Yuriy Shymkiv, Thomas Gudermann, Emiel P. C. van der Vorst, Annika Wisnowsky, Claudia Einer, Friedrich Torben, Vladimir Chubanov, Karl Sotlar, Alexey G. Ryazanov, Onder A. Yildirim, Harald Bartsch, Silvia Ferioli, Wenke Jonas, RS: CARIM - R1.01 - Blood proteins & engineering, RS: CARIM - R3.07 - Structure-function analysis of the chemokine interactome for therapeutic targeting and imaging in atherosclerosis, Promovendi CD, Pathologie, and Biochemie
- Subjects
0301 basic medicine ,medicine.medical_specialty ,kidney ,placenta ,Mouse ,QH301-705.5 ,Science ,Embryonic Development ,TRPM Cation Channels ,Biology ,magnesium ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Gene Knockout Techniques ,Mice ,longevity ,Pregnancy ,ddc:570 ,Internal medicine ,TRPM6 ,Placenta ,medicine ,Animals ,Biology (General) ,Yolk sac ,Intestinal Mucosa ,Human Biology and Medicine ,intestine ,Yolk Sac ,Kidney ,General Immunology and Microbiology ,General Neuroscience ,Embryogenesis ,Wild type ,General Medicine ,Biophysics and Structural Biology ,Phenotype ,Survival Analysis ,Prenatal development ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Medicine ,Female ,trophoblast stem cells ,Research Article - Abstract
Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood. DOI: http://dx.doi.org/10.7554/eLife.20914.001, eLife digest A balanced diet contains a variety of minerals such as magnesium ions, which are required for many chemical reactions in our body. A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth. Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people. Despite its prominent role in human health, our understanding of how the body maintains the right balance of magnesium ions remains extremely vague. Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell. There are thought to be around ten types of these magnesium ion channels in human cells, but we do not know what roles any of them perform in the body. One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants. However, the loss of TRPM6 in mice disrupts how mouse embryos develop, suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic. To address this question, Chubanov et al. studied mice with mutations that disrupted the production of TRPM6 in specific tissues only. The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body. Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan, growth defects and poor health by disrupting important biochemical reactions. Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals. The findings of Chubanov et al. demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood. The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets. DOI: http://dx.doi.org/10.7554/eLife.20914.002
- Published
- 2016
30. Defective diacylglycerol‐induced Ca2+entry but normal agonist‐induced activation responses in TRPC6‐deficient mouse platelets
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Irina Pleines, Alexander Dietrich, Bernhard Nieswandt, Ina Thielmann, Attila Braun, G. Ramanathan, Shuchi Gupta, Frauke May, Christine Mannhalter, and David Varga-Szabo
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Blood Platelets ,medicine.medical_specialty ,Time Factors ,ORAI1 Protein ,Platelet Glycoprotein GPIIb-IIIa Complex ,Ferric Compounds ,TRPC6 ,Diglycerides ,Mice ,Transient receptor potential channel ,Chlorides ,Internal medicine ,TRPC6 Cation Channel ,medicine ,Animals ,Platelet ,Protease-activated receptor ,Calcium Signaling ,RNA, Messenger ,Stromal Interaction Molecule 1 ,Platelet activation ,TRPC ,TRPC Cation Channels ,Diacylglycerol kinase ,Mice, Knockout ,Membrane Glycoproteins ,ORAI1 ,Chemistry ,Secretory Vesicles ,Thrombin ,Thrombosis ,Hematology ,Platelet Activation ,Cell biology ,Adenosine Diphosphate ,Mice, Inbred C57BL ,Disease Models, Animal ,C-Reactive Protein ,Endocrinology ,Gene Expression Regulation ,Calcium Channels - Abstract
Summary. Background: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca2+ concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store-operated calcium entry (SOCE) through Orai1 was shown to be the main Ca2+ influx pathway in murine platelets, but there are additional non-store-operated Ca2+ (non-SOC) and receptor operated Ca2+ (ROC) channels expressed in the platelet plasma membrane.Objective: Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested.Methods: To investigate the function of TRPC6 in platelet Ca2+ signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays.Results: In the mutant platelets, DAG activated Ca2+ influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca2+ responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6.Conclusion: Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca2+ influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.
- Published
- 2012
31. Critical Role for Stromal Interaction Molecule 1 in Cardiac Hypertrophy
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Jérémy Fauconnier, Stefan Engelhardt, Ludovic Benard, Yassine Sassi, Fleur Cohen Aubart, Lahouaria Hadri, Joachim P. Schmitt, Aude Ouillé, Antoine H. Chaanine, Youakim Saliba, Roger J. Hajjar, Alain Lacampagne, Sabine Merkle, Morgan Dupuis, Deepak Ramanujam, Silke Mühlstedt, Bernhard Nieswandt, Bernhard Laggerbauer, Anne-Marie Lompré, Attila Braun, Jean-Sébastien Hulot, Dongtak Jeong, Olivier Cazorla, Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Régulation de l'expression génétique chez les microorganismes (REGCM), Centre National de la Recherche Scientifique (CNRS), Systèmes interfaciaux à l'echelle nanometrique (SIEN), Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)
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0303 health sciences ,medicine.medical_specialty ,Voltage-dependent calcium channel ,STIM1 ,030204 cardiovascular system & hematology ,Biology ,In vitro ,Cell biology ,Muscle hypertrophy ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,[SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,In vivo ,Physiology (medical) ,Internal medicine ,medicine ,Gene silencing ,Myocyte ,[SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie ,Patch clamp ,Cardiology and Cardiovascular Medicine ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology - Abstract
Background— Cardiomyocytes use Ca 2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca 2+ triggers signaling in cardiomyocytes in the presence of the rapid and large Ca 2+ fluctuations that occur during excitation-contraction coupling. A potential route is store-operated Ca 2+ entry, a drug-inducible mechanism for Ca 2+ signaling that requires stromal interaction molecule 1 (STIM1). Store-operated Ca 2+ entry can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca 2+ entry with respect to cardiac hypertrophy in vitro and in vivo. Methods and Results— Consistent with earlier reports, we found drug-inducible store-operated Ca 2+ entry in neonatal rat cardiomyocytes, which was dependent on STIM1. Although this STIM1-dependent, drug-inducible store-operated Ca 2+ entry was only marginal in adult cardiomyocytes isolated from control hearts, it increased significantly in cardiomyocytes isolated from adult rats that had developed compensated cardiac hypertrophy after abdominal aortic banding. Moreover, we detected an inwardly rectifying current in hypertrophic cardiomyocytes that occurs under native conditions (ie, in the absence of drug-induced store depletion) and is dependent on STIM1. By manipulating its expression, we found STIM1 to be both sufficient and necessary for cardiomyocyte hypertrophy in vitro and in the adult heart in vivo. Stim1 silencing by adeno-associated viruses of serotype 9–mediated gene transfer protected rats from pressure overload–induced cardiac hypertrophy. Conclusion— By controlling a previously unrecognized sarcolemmal current, STIM1 promotes cardiac hypertrophy.
- Published
- 2011
32. Differentially regulated GPVI ectodomain shedding by multiple platelet–expressed proteinases
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Athena Chalaris, Markus Bender, Bernhard Nieswandt, Jürgen Scheller, Stefan Rose-John, Attila Braun, David Stegner, Michael R. Bösl, and Sebastian Hofmann
- Subjects
Blood Platelets ,ADAM10 ,Immunology ,Platelet Membrane Glycoproteins ,ADAM17 Protein ,Biochemistry ,ADAM10 Protein ,Mice ,Calmodulin ,Disintegrin ,Animals ,Platelet ,Platelet activation ,Thrombopoiesis ,biology ,Proteolytic enzymes ,Membrane Proteins ,Cell Biology ,Hematology ,Molecular biology ,Mitochondria ,Cell biology ,Mice, Inbred C57BL ,ADAM Proteins ,Ectodomain ,Mutation ,biology.protein ,Amyloid Precursor Protein Secretases ,GPVI ,Megakaryocytes ,Gene Deletion ,Peptide Hydrolases - Abstract
Glycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseased vessels in the setting of myocardial infarction or stroke. GPVI is an attractive target for antithrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and/or ectodomain shedding. Metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family have been proposed to mediate this ectodomain shedding, but direct evidence for this is lacking. Here, we studied GPVI shedding in vitro and in vivo in newly generated mice with a megakaryocyte–specific ADAM10 deficiency and in Adam17ex/ex mice, which lack functional ADAM17. We demonstrate that GPVI cleavage in vitro can occur independently through either ADAM10 or ADAM17 in response to distinct stimuli. In contrast, antibody (JAQ1)–induced GPVI shedding in vivo occurred in mice lacking both ADAM10/ADAM17 in their platelets, suggesting the existence of a third GPVI cleaving platelet enzyme. This was supported by in vitro studies on ADAM10/ADAM17 double–deficient platelets. These results reveal that ectodomain shedding of GPVI can be mediated through multiple differentially regulated platelet–expressed proteinases with obvious therapeutic implications.
- Published
- 2010
33. Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation
- Author
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Marijke J.E. Kuijpers, Roger van Kruchten, Marion A.H. Feijge, Karen Gilio, Alejandro Berna-Erro, Johan W. M. Heemskerk, Bernhard Nieswandt, David Stegner, Attila Braun, Paola E. J. van der Meijden, David Varga-Szabo, Promovendi CD, Ondersteunend personeel CD, Biochemie, and RS: CARIM School for Cardiovascular Diseases
- Subjects
Blood Platelets ,inorganic chemicals ,ORAI1 Protein ,Mice, Transgenic ,Platelet Membrane Glycoproteins ,Platelet membrane glycoprotein ,Models, Biological ,Biochemistry ,Mice ,chemistry.chemical_compound ,Thrombin ,Prothrombinase ,medicine ,Animals ,Platelet ,Stromal Interaction Molecule 1 ,Thrombus ,Molecular Biology ,Membrane Glycoproteins ,Coagulants ,Chemistry ,Thrombosis ,Cell Biology ,Phosphatidylserine ,medicine.disease ,Mice, Inbred C57BL ,Coagulation ,Biophysics ,Calcium ,Female ,Calcium Channels ,GPVI ,Signal Transduction ,medicine.drug - Abstract
In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.
- Published
- 2010
34. STIM1-Independent T Cell Development and Effector Function In Vivo
- Author
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Timo Vögtle, David Varga-Szabo, Thomas Kerkau, Ronmy Rivera Galdos, Bernhard Nieswandt, Niklas Beyersdorf, Stephan Kissler, and Attila Braun
- Subjects
inorganic chemicals ,Adoptive cell transfer ,T cell ,Immunology ,Graft vs Host Disease ,Thymus Gland ,Biology ,Mice ,Interleukin 21 ,Immune system ,T-Lymphocyte Subsets ,medicine ,Animals ,Homeostasis ,Immunology and Allergy ,Cytotoxic T cell ,Stromal Interaction Molecule 1 ,IL-2 receptor ,Antigen-presenting cell ,Cells, Cultured ,Cell Proliferation ,Mice, Knockout ,Mice, Inbred BALB C ,Membrane Glycoproteins ,FOXP3 ,Biological Transport ,Cell Differentiation ,Coculture Techniques ,Mice, Mutant Strains ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Female ,Calcium Channels - Abstract
Store-operated Ca2+ entry (SOCE) is believed to be of pivotal importance in T cell physiology. To test this hypothesis, we generated mice constitutively lacking the SOCE-regulating Ca2+ sensor stromal interaction molecule 1 (STIM1). In vitro analyses showed that SOCE and Ag receptor complex-triggered Ca2+ flux into STIM1-deficient T cells is virtually abolished. In vivo, STIM1-deficient mice developed a lymphoproliferative disease despite normal thymic T cell maturation and normal frequencies of CD4+Foxp3+ regulatory T cells. Unexpectedly, STIM1-deficient bone marrow chimeric mice mounted humoral immune responses after vaccination and STIM1-deficient T cells were capable of inducing acute graft-versus-host disease following adoptive transfer into allogeneic hosts. These results demonstrate that STIM1-dependent SOCE is crucial for homeostatic T cell proliferation, but of much lesser importance for thymic T cell differentiation or T cell effector functions.
- Published
- 2009
35. Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation
- Author
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Attila Braun, David Varga-Szabo, Christoph Kleinschnitz, Irina Pleines, Markus Bender, Madeleine Austinat, Michael Bösl, Guido Stoll, and Bernhard Nieswandt
- Subjects
Blood Platelets ,Pathology ,medicine.medical_specialty ,ORAI1 Protein ,Immunology ,Myocardial Ischemia ,Ischemia ,Excipient ,Pharmacology ,Biochemistry ,Brain Ischemia ,Pathogenesis ,Mice ,medicine ,Animals ,Humans ,Platelet ,Stromal Interaction Molecule 1 ,Thrombus ,Mice, Knockout ,Membrane Glycoproteins ,business.industry ,ORAI1 ,Activator (genetics) ,Body Weight ,Thrombosis ,Cell Biology ,Hematology ,medicine.disease ,Mice, Inbred C57BL ,Calcium ,Female ,Calcium Channels ,business ,medicine.drug - Abstract
Platelet activation and aggregation at sites of vascular injury are essential for primary hemostasis, but are also major pathomechanisms underlying myocardial infarction and stroke. Changes in [Ca2+]i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca2+ stores triggers Ca2+ entry through store-operated calcium (SOC) channels. STIM1 has been identified as an endoplasmic reticulum (ER)–resident Ca2+ sensor that regulates store-operated calcium entry (SOCE) in immune cells and platelets, but the identity of the platelet SOC channel has remained elusive. Orai1 (CRACM1) is the recently discovered SOC (CRAC) channel in T cells and mast cells but its role in mammalian physiology is unknown. Here we report that Orai1 is strongly expressed in human and mouse platelets. To test its role in blood clotting, we generated Orai1-deficient mice and found that their platelets display severely defective SOCE, agonist-induced Ca2+ responses, and impaired activation and thrombus formation under flow in vitro. As a direct consequence, Orai1 deficiency in mice results in resistance to pulmonary thromboembolism, arterial thrombosis, and ischemic brain infarction, but only mild bleeding time prolongation. These results establish Orai1 as the long-sought platelet SOC channel and a crucial mediator of ischemic cardiovascular and cerebrovascular events.
- Published
- 2009
36. Characterization of a novel mutation in exon 10 of the adrenoleukodystrophy gene
- Author
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Esther M. Maier, Andreas Holzinger, Adelbert A. Roscher, S Stöckler-Ipsiroglu, and Attila Braun
- Subjects
Adult ,Male ,congenital, hereditary, and neonatal diseases and abnormalities ,endocrine system diseases ,Nonsense mutation ,Biology ,ATP Binding Cassette Transporter, Subfamily D, Member 1 ,Exon ,Genetics ,medicine ,Humans ,Adrenoleukodystrophy ,Gene ,Cells, Cultured ,Genetics (clinical) ,Messenger RNA ,Membrane Proteins ,nutritional and metabolic diseases ,Translation (biology) ,Exons ,Fibroblasts ,medicine.disease ,Cyclic nucleotide-binding domain ,Mutation ,Mutation (genetic algorithm) ,ATP-Binding Cassette Transporters - Abstract
We have detected a novel mutation in the adrenoleukodystrophy (ALD) gene in skin fibroblasts in primary culture derived from a patient suffering from the adrenocortical insufficiency-only-phenotype of ALD. This nonsense mutation (C2400T/Q672X) is the only mutation reported to date affecting exon 10. It leads to a translation product lacking the 74 C-terminal amino acids. As a consequence of the loss of this region, which immediately follows the putative nucleotide binding domain, the ALD protein (ALDP) was not detectable at all by ALDP-specific monoclonal antibodies. Since ALDP-specific mRNA was readily detected in these fibroblasts, the loss of protein is probably not attributable to RNA instability but may be explained by protein instability. If the Q672X mutation leads in fact to an unstable translation product this would be consistent with the hypothesis that the C-terminus is crucial for ALDP stability.
- Published
- 2008
37. Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1
- Author
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Archana Ambily, Alexander Dietrich, Bernhard Nieswandt, Thomas Gudermann, Kalwant S. Authi, Markus Bender, Sheila R. Hassock, Attila Braun, and David Varga-Szabo
- Subjects
Blood Platelets ,medicine.medical_specialty ,Time Factors ,Physiology ,Clinical Biochemistry ,chemistry.chemical_element ,Calcium ,Ferric Compounds ,TRPC1 ,Mice ,Transient receptor potential channel ,Chlorides ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Humans ,Platelet ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Platelet activation ,Blood Coagulation ,TRPC Cation Channels ,Calcium signaling ,Mice, Knockout ,Chemistry ,Endoplasmic reticulum ,Membrane Proteins ,Thrombosis ,STIM1 ,Platelet Activation ,Neoplasm Proteins ,Cell biology ,Disease Models, Animal ,Endocrinology - Abstract
Changes in [Ca(2+)](i) are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca(2+) sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1.
- Published
- 2008
38. A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy
- Author
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Anja Fritsch, Hauke Schumann, Thorsten A. Bley, Dominik von Elverfeldt, Johannes S. Kern, Miriam Erlacher, S. Loeckermann, Leena Bruckner-Tuderman, Ingrid Hausser, Michael R. Bösl, Dominik Paul, Dirk Berens von Rautenfeld, Reinhard Fässler, and Attila Braun
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Collagen Type VII ,Foot Deformities, Congenital ,RNA Splicing ,Mucocutaneous zone ,Mice, Transgenic ,Biology ,Mice ,Fibrosis ,Forelimb ,medicine ,Animals ,Humans ,Intradermal injection ,Fibroblast ,Mitten deformity ,Cells, Cultured ,Skin ,integumentary system ,Malnutrition ,Epidermolysis bullosa dystrophica ,Soft tissue ,General Medicine ,Fibroblasts ,medicine.disease ,Dermatology ,Epidermolysis Bullosa Dystrophica ,Mice, Inbred C57BL ,Disease Models, Animal ,Phenotype ,medicine.anatomical_structure ,Female ,Hand Deformities, Congenital ,Myofibroblast ,Research Article - Abstract
Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-beta1-mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.
- Published
- 2008
39. Genomic organization of profilin-III and evidence for a transcript expressed exclusively in testis
- Author
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Reinhard Fässler, Attila Aszódi, Alejandro Berna, Attila Braun, Oliver Brandau, and Heide Hellebrand
- Subjects
Male ,DNA, Complementary ,Transcription, Genetic ,Molecular Sequence Data ,Gene Expression ,Mice, Inbred Strains ,macromolecular substances ,In situ hybridization ,Biology ,Kidney ,Mice ,Profilins ,Exon ,Contractile Proteins ,Testis ,Gene expression ,Genetics ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,RNA, Messenger ,Actin-binding protein ,Gene ,In Situ Hybridization ,Messenger RNA ,Base Sequence ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,Microfilament Proteins ,Kidney metabolism ,DNA ,Sequence Analysis, DNA ,General Medicine ,Blotting, Northern ,Molecular biology ,Rats ,Profilin ,biology.protein ,Sequence Alignment - Abstract
Profilins are small, widely expressed actin binding proteins, thought to be key regulators of actin dynamics in living cells. So far, three profilin-genes have been described: profilin-I (PFN1), profilin-II (PFN2) with two splice variants and the recently identified profilin-III (PFN3). Here we describe the genomic organization of the genes encoding human and mouse profilin-III. Both are single exon genes and lie in close vicinity to the renal sodium-phosphate transport gene 2 (SLC34A1, NPT2) which is highly expressed in kidney. Northern hybridization to rat tissues has previously demonstrated expression of an approximately 4.5 kb long profilin-III mRNA transcript in kidney and a mRNA transcript of approximately 1 kb in length in testis. Here we show that mouse profilin-III expression is restricted to testis and that the 4.2 kb profilin-III mRNA in kidney is the result of a slc34a1 transcript which includes the antisense profilin-III open reading frame in its 3'-untranslated region. Finally, we demonstrate by in situ hybridization that profilin-III mRNA is localized to cells in the late stage of spermatogenesis.
- Published
- 2002
40. Profilin II Is Alternatively Spliced, Resulting in Profilin Isoforms That Are Differentially Expressed and Have Distinct Biochemical Properties
- Author
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Joël Vandekerckhove, Reinhard Fässler, Inge Van Colen, Attila Braun, Anja Lambrechts, Attila Aszódi, Christophe Ampe, Lorene M. Lanier, Johan Robbens, and Veronique Jonckheere
- Subjects
Phosphatidylinositol 4,5-Diphosphate ,Gene isoform ,Time Factors ,Transcription, Genetic ,Blotting, Western ,Molecular Sequence Data ,macromolecular substances ,Biology ,Ligands ,Mice ,Profilins ,Exon ,Contractile Proteins ,Animals ,Humans ,Protein Isoforms ,Tissue Distribution ,Amino Acid Sequence ,Cell Growth and Development ,Molecular Biology ,Gene ,In Situ Hybridization ,Actin ,Expressed Sequence Tags ,Base Sequence ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,Microfilament Proteins ,Alternative splicing ,Intron ,Brain ,Exons ,Cell Biology ,Actin cytoskeleton ,Introns ,Rats ,Alternative Splicing ,Biochemistry ,Profilin ,biology.protein ,Protein Binding - Abstract
We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.
- Published
- 2000
41. Replication and virulence of early protein 0 and long latency transcript deficient mutants of the Aujeszky′s disease (pseudorabies) virus
- Author
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Istvan Fodor, Zsolt Boldogkoi, and Attila Braun
- Subjects
Transcription, Genetic ,Swine ,Immunology ,DNA, Recombinant ,Pseudorabies ,Virulence ,Genome, Viral ,Virus Replication ,medicine.disease_cause ,Microbiology ,Immediate early protein ,Herpesviridae ,Virus ,Cell Line ,Lethal Dose 50 ,Mice ,Viral Proteins ,Latent Virus ,Alphaherpesvirinae ,medicine ,Animals ,Mice, Inbred BALB C ,biology ,biology.organism_classification ,Herpesvirus 1, Suid ,Virology ,Molecular biology ,Virus Latency ,Infectious Diseases ,Trigeminal Ganglion ,Viral replication ,DNA, Viral ,Mutation ,Female ,Virus Activation ,Plasmids - Abstract
Early protein 0 (EP0)-deficient recombinant Aujeszky's disease viruses, Ka-ep0lac and Ba-ep0lac derived from strains Kaplan and Bartha, respectively, were constructed to explore the impact of the mutation on replication, virulence and latency of the virus. Inactivation of the EP0 gene resulted in a mutation of long latency transcript (Cheung et al., 1991) that is located on the complementary DNA strand of EP0 and immediate early protein (IE)175 genes. In infection of immortalized porcine kidney cells, the growth rate and yield of both EP0(-) mutant strains were significantly smaller than that of wild-type virus. Ka-ep0lac was found to be highly virulent, while Ba-ep0lac showed an attenuated phenotype in mice. PCR assay and immunohistochemistry showed that the Ba-ep0lac virus was able to establish latency in the mouse trigeminal ganglia. However, latent virus was not able to reactivate in explant reactivation assays. Accordingly, latent Ba-ep0lac has the potential to be exploited as vectors for the delivery of foreign genes to the nervous system.
- Published
- 2000
42. Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis
- Author
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Kerstin, Göbel, Michael K, Schuhmann, Susann, Pankratz, David, Stegner, Alexander M, Herrmann, Attila, Braun, Johanna, Breuer, Stefan, Bittner, Tobias, Ruck, Heinz, Wiendl, Christoph, Kleinschnitz, Bernhard, Nieswandt, and Sven G, Meuth
- Subjects
Inflammation ,Encephalomyelitis, Autoimmune, Experimental ,Endothelial Cells ,Vascular Cell Adhesion Molecule-1 ,Intercellular Adhesion Molecule-1 ,Mice, Inbred C57BL ,Mice ,Blood-Brain Barrier ,Cell Movement ,Cell Adhesion ,Phospholipase D ,Animals ,Female ,Lymphocytes - Abstract
Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune-mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1-deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, a model of human MS. The lack of PLD1 reduced chemokine-mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1-deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1-dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.
- Published
- 2013
43. Orai1-induced store-operated Ca(2+) entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets
- Author
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David Stegner, Wenchun Chen, Johan W. M. Heemskerk, Attila Braun, Alexander Dietrich, Shuchi Gupta, Stepan Gambaryan, R. Van Kruchten, Ina Thielmann, Heike M. Hermanns, Hariharan Subramanian, Bernhard Nieswandt, Promovendi CD, Biochemie, and RS: CARIM - R1 - Thrombosis and haemostasis
- Subjects
Blood Platelets ,Thapsigargin ,Time Factors ,ORAI1 Protein ,Platelet Aggregation ,Mice, Transgenic ,Phospholipase ,calcium signaling ,TRPC6 ,Diglycerides ,chemistry.chemical_compound ,Mice ,Thromboxane A2 ,platelet activation ,TRPC6 Cation Channel ,Animals ,Platelet activation ,TRPC ,Diacylglycerol kinase ,Calcium signaling ,Glycoproteins ,TRPC Cation Channels ,Mice, Knockout ,Chemistry ,ORAI1 ,Hematology ,Cell biology ,Mice, Inbred C57BL ,Liver ,Phospholipases ,Calcium ,Calcium Channels ,Signal Transduction - Abstract
Summary Background Orai1, the major store-operated Ca2+ entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca2+ entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. Objective We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. Methods Trpc6−/−, Orai1−/− and Orai1−/−/Trpc6−/− mice were generated, and their platelets were analyzed. Results Thapsigargin (TG)-induced SOCE was further reduced in Orai1−/−/Trpc6−/− platelets as compared with Orai1−/− platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipase C and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1−/− platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoprotein VI-mediated thromboxane A2 release was strongly dependent on Orai1-mediated SOCE. Conclusion The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca2+ signaling in platelets.
- Published
- 2013
44. Antithrombotic potential of blockers of store-operated calcium channels in platelets
- Author
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Johan W. M. Heemskerk, Marijke J.E. Kuijpers, Guido Stoll, Roger van Kruchten, Christoph Kleinschnitz, Attila Braun, Peter Kraft, Bernhard Nieswandt, Edouard M. Bevers, Ronmy Rivera-Galdos, Marion A.H. Feijge, Promovendi CD, Biochemie, Farmacologie en Toxicologie, and RS: CARIM School for Cardiovascular Diseases
- Subjects
Blood Platelets ,Boron Compounds ,ORAI1 Protein ,Pharmacology ,Inhibitory postsynaptic potential ,Mice ,Fibrinolytic Agents ,Antithrombotic ,Medicine ,Animals ,Humans ,Platelet ,Thrombus ,thrombosis ,Whole blood ,Calcium metabolism ,Voltage-dependent calcium channel ,business.industry ,ORAI1 ,medicine.disease ,Calcium Channel Blockers ,Platelet Activation ,stroke ,Mice, Inbred C57BL ,platelets ,Calcium ,Calcium Channels ,pharmacology ,Cardiology and Cardiovascular Medicine ,business - Abstract
Objective— Platelet Orai1 channels mediate store-operated Ca 2+ entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channels may provide a novel way of antithrombotic therapy. Therefore, the thromboprotective effect of SOCE blockers directed against platelet Orai1 is determined. Methods and Results— Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Tested antagonists included the known compounds, SKF96365, 2-aminoethyl diphenylborate, and MRS1845 and the novel compounds, Synta66 and GSK-7975A. The potency of SOCE inhibition was in the order of Synta66>2-aminoethyl diphenylborate>GSK-7975A>SKF96365>MRS1845. The specificity of the first 3 compounds was verified with platelets from Orai1-deficient mice. Inhibitory activity on procoagulant activity and high-shear thrombus formation was assessed in plasma and whole blood. In the presence of plasma, all 3 compounds suppressed platelet responses and restrained thrombus formation under flow. Using a murine stroke model, arterial thrombus formation was provoked in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylborate markedly diminished brain infarct size. Conclusion— Plasma-soluble SOCE blockers such as 2-aminoethyl diphenylborate suppress platelet-dependent coagulation and thrombus formation. The platelet Orai1 channel is a novel target for preventing thrombotic events causing brain infarction.
- Published
- 2012
45. Sequence of a putative human housekeeping gene (HK33) localized on chromosome 1
- Author
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Stefan Kammerer, Hartwig Cleve, Winfried Weissenhorn, Attila Braun, and Elisabeth H. Weiss
- Subjects
Untranslated region ,DNA, Complementary ,Transcription, Genetic ,Polyadenylation ,Sequence analysis ,Molecular Sequence Data ,Biology ,Polymerase Chain Reaction ,Complementary DNA ,Genetics ,Humans ,Coding region ,Amino Acid Sequence ,Northern blot ,Gene ,Alternative polyadenylation ,four transcripts ,ubiquitous expression ,chromosomal location ,Genomic Library ,Base Sequence ,Chromosome Mapping ,Sequence Analysis, DNA ,General Medicine ,Blotting, Northern ,Molecular biology ,Housekeeping gene ,Genes ,Chromosomes, Human, Pair 1 - Abstract
A gene ( HK33 ) localized on human chromosome 1 has been detected by crossreaction of its fusion protein with a monospecific antiserum directed against human vitamin-D-binding protein (hDBP; group-specific component). Its cDNA sequence analysis showed no evident homologies neither to the sequence encoding hDBP nor to any other sequence. The largest cDNA clone of 3.2 kb includes a 897-bp coding region and a large 3' untranslated region with at least four polyadenylation sites. Further cDNA amplification using PCR demonstrated a total cDNA length of approx. 3.7 kb. Northern blot analysis revealed signals at about 2.2–2.5 kb and 4.0 kb, the shorter transcripts representing mRNAs using one of the two polyadenylation sites at about 2.0 kb. Synthesis of the 299-amino-acid polypeptide (33 kDa) in the bacterial host, with subsequent Western blot analysis, verified the sequence-specific recognition by the hDBP-specific antiserum. The search of protein databanks revealed no homology of HK33 to any known sequence. Since the gene is transcribed in all cells and tissues tested so far, it is a strong candidate for another housekeeping gene.
- Published
- 1994
46. STIM and Orai in platelet function
- Author
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Attila Braun, David Varga-Szabo, and Bernhard Nieswandt
- Subjects
Blood Platelets ,ORAI1 Protein ,Physiology ,Apoptosis ,Endoplasmic Reticulum ,Extracellular ,Animals ,Homeostasis ,Humans ,Platelet ,Platelet activation ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Molecular Biology ,Calcium signaling ,ORAI1 ,Chemistry ,Endoplasmic reticulum ,Cell Membrane ,Membrane Proteins ,STIM1 ,Thrombosis ,Cell Biology ,Cell biology ,Neoplasm Proteins ,Immunology ,Calcium ,Calcium Channels ,Intracellular - Abstract
Physiological platelet activation and thrombus formation are essential to stop bleeding in case of vascular injury, whereas inadequate triggering of the same process in diseased vessels can lead to fatal thromboembolism and tissue ischemia of vital organs. A central step in platelet activation is agonist-induced elevation of the intracellular Ca(2+) concentration. This happens on the one hand through the release of Ca(2+) from intracellular stores and on the other hand through Ca(2+) influx from the extracellular space. In platelets, the major Ca(2+) influx pathway is the so-called store operated Ca(2+) entry (SOCE), induced by store depletion. Studies in the last five years discovered the molecular background of platelet SOCE. Stromal interaction molecule 1 (STIM1) and Orai1, two so far unknown molecules, got in the focus of research. STIM1 was found to be the Ca(2+) sensor in the endoplasmic reticulum (ER) membrane, whereas Orai1 was identified as the major store operated Ca(2+) (SOC) channel in the plasma membrane. These two molecules and their role in platelet function and thrombus formation are the topic of the present review with a special focus on apoptosis and apoptosis-like processes in platelet physiology.
- Published
- 2011
47. Impaired α IIb β 3 Integrin Activation and Shear-Dependent Thrombus Formation in Mice Lacking Phospholipase D1
- Author
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Michael A. Frohman, Guido Stoll, Margitta Elvers, Bernhard Nieswandt, Johan W. M. Heemskerk, David Stegner, Christoph Kleinschnitz, Qin Chen, Michael Boesl, Ina Hagedorn, Marijke E. J. Kuijpers, and Attila Braun
- Subjects
Blood Platelets ,Brain Infarction ,Bleeding Time ,Platelet Aggregation ,Integrin ,Platelet Glycoprotein GPIIb-IIIa Complex ,Phospholipase ,Biochemistry ,Article ,Mice ,Phospholipase D ,Animals ,Platelet ,Platelet activation ,Molecular Biology ,Mice, Knockout ,biology ,Phospholipase C ,Chemistry ,Thrombosis ,Cell Biology ,Cell biology ,Type C Phospholipases ,biology.protein ,Phospholipase D1 - Abstract
Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, alpha(IIb)beta(3) integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate-binding protein or immunoreceptor tyrosine-based activation motif-coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1(-/-) mice displayed impaired alpha(IIb)beta(3) integrin activation in response to major agonists and defective glycoprotein Ib-dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.
- Published
- 2010
48. Stromal interaction molecules 1 and 2 are key regulators of autoreactive T cell activation in murine autoimmune central nervous system inflammation
- Author
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Michael K. Schuhmann, Stefan Bittner, Christoph Kleinschnitz, Alejandro Berna-Erro, Heinz Wiendl, Sven G. Meuth, Attila Braun, Guido Stoll, Bernhard Nieswandt, and David Stegner
- Subjects
Stromal cell ,Encephalomyelitis, Autoimmune, Experimental ,T cell ,Immunology ,Biology ,Lymphocyte Activation ,Mice ,Immune system ,Drug Delivery Systems ,T-Lymphocyte Subsets ,medicine ,Immunology and Allergy ,Animals ,Protein Isoforms ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Stromal Interaction Molecule 2 ,Glycoproteins ,Mice, Knockout ,Membrane Glycoproteins ,Experimental autoimmune encephalomyelitis ,STIM1 ,STIM2 ,T lymphocyte ,medicine.disease ,Immunity, Innate ,Mice, Mutant Strains ,Peptide Fragments ,Cell biology ,medicine.anatomical_structure ,Female ,Myelin-Oligodendrocyte Glycoprotein ,Calcium Channels ,Inflammation Mediators ,Intracellular - Abstract
Calcium (Ca2+) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca2+ entry pathway in nonexcitable cells, including T cells, is store-operated Ca2+ entry (SOCE), wherein depletion of intracellular Ca2+ stores upon receptor stimulation causes subsequent influx of extracellular Ca2+ across the plasma membrane. Stromal interaction molecule (STIM) 1 is the Ca2+ sensor in the endoplasmic reticulum, which controls this process, whereas the other STIM isoform, STIM2, coregulates SOCE. Although the contribution of STIM molecules and SOCE to T lymphocyte function is well studied in vitro, their significance for immune processes in vivo has remained largely elusive. In this study, we studied T cell function in mice lacking STIM1 or STIM2 in a model of myelin-oligodendrocyte glycoprotein (MOG35–55)-induced experimental autoimmune encephalomyelitis (EAE). We found that STIM1 deficiency significantly impaired the generation of neuroantigen-specific T cell responses in vivo with reduced Th1/Th17 responses, resulting in complete protection from EAE. Mice lacking STIM2 developed EAE, but the disease course was ameliorated. This was associated with a reduced clinical peak of disease. Deficiency of STIM2 was associated with an overall reduced proliferative capacity of lymphocytes and a reduction of IFN-γ/IL-17 production by neuroantigen-specific T cells. Neither STIM1 nor STIM2 deficiency altered the phenotype or function of APCs. These findings reveal a crucial role of STIM-dependent pathways for T cell function and activation under autoimmune inflammatory conditions, establishing them as attractive new molecular therapeutic targets for the treatment of inflammatory and autoimmune disorders.
- Published
- 2009
49. STIM2 Regulates Capacitive Ca 2+ Entry in Neurons and Plays a Key Role in Hypoxic Neuronal Cell Death
- Author
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Alejandro Berna-Erro, Bernhard Nieswandt, Christoph Kleinschnitz, Guido Stoll, David Stegner, Sven G. Meuth, Thomas Wultsch, Robert Kraft, Jens Eilers, Attila Braun, and Michael K. Schuhmann
- Subjects
Programmed cell death ,Ischemia ,Biology ,Biochemistry ,Tissue Culture Techniques ,Mice ,Mediator ,medicine ,Animals ,Protein Isoforms ,Stromal Interaction Molecule 2 ,Molecular Biology ,Cells, Cultured ,Mice, Knockout ,Neurons ,Membrane Glycoproteins ,Cell Death ,Endoplasmic reticulum ,STIM1 ,Cell Biology ,STIM2 ,medicine.disease ,Cell Hypoxia ,Cell biology ,Mice, Inbred C57BL ,Cytosol ,Organ Specificity ,Hypoxia-Ischemia, Brain ,Calcium ,Female - Abstract
Excessive cytosolic calcium ion (Ca 2+ ) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca 2+ entry (CCE) is a process whereby depletion of intracellular Ca 2+ stores causes the activation of plasma membrane Ca 2+ channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)–resident Ca 2+ sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca 2+ concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca 2+ accumulation in neurons. Neurons from Stim2 −/− mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2 −/− mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.
- Published
- 2009
50. Calcium signaling in platelets
- Author
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David Varga-Szabo, Attila Braun, and Bernhard Nieswandt
- Subjects
Blood Platelets ,ORAI1 ,Chemistry ,STIM1 ,Hematology ,Inositol trisphosphate receptor ,Store-operated calcium entry ,Cell biology ,Transient receptor potential channel ,Homeostasis ,Humans ,Calcium ,Platelet activation ,Calcium Signaling ,TRPC ,Calcium signaling - Abstract
Summary. Agonist-induced elevation in cytosolic Ca2+ concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca2+ release from intracellular stores and Ca2+ entry through the plasma membrane (PM). Ca2+ store release is a well-established process involving phospholipase (PL)C-mediated production of inositol-1,4,5-trisphosphate (IP3), which in turn releases Ca2+ from the intracellular stores through IP3 receptor channels. In contrast, the mechanisms controlling Ca2+ entry and the significance of this process for platelet activation have been elucidated only very recently. In platelets, as in other non-excitable cells, the major way of Ca2+ entry involves the agonist-induced release of cytosolic sequestered Ca2+ followed by Ca2+ influx through the PM, a process referred to as store-operated calcium entry (SOCE). It is now clear that stromal interaction molecule 1 (STIM1), a Ca2+ sensor molecule in intracellular stores, and the four transmembrane channel protein Orai1 are the key players in platelet SOCE. The other major Ca2+ entry mechanism is mediated by the direct receptor-operated calcium (ROC) channel, P2X1. Besides these, canonical transient receptor potential channel (TRPC) 6 mediates Ca2+ entry through the PM. This review summarizes the current knowledge of platelet Ca2+ homeostasis with a focus on the newly identified Ca2+ entry mechanisms.
- Published
- 2009
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