TY - JOUR A1 - Regn, Michael A1 - Laggerbauer, Bernhard A1 - Jentzsch, Claudia A1 - Ramanujam, Deepak A1 - Ahles, Andrea A1 - Sichler, Sonja A1 - Calzada-Wack, Julia A1 - Koenen, Rory R. A1 - Braun, Attila A1 - Nieswandt, Bernhard A1 - Engelhardt, Stefan T1 - Peptidase inhibitor 16 is a membrane-tethered regulator of chemerin processing in the myocardium JF - Journal of Molecular and Cellular Cardiology N2 - A key response of the myocardium to stress is the secretion of factors with paracrine or endocrine function. Intriguing in this respect is peptidase inhibitor 16 (PI16), a member of the CAP family of proteins which we found to be highly upregulated in cardiac disease. Up to this point, the mechanism of action and physiological function of PI16 remained elusive. Here, we show that PI16 is predominantly expressed by cardiac fibroblasts, which expose PI16 to the interstitium via a glycophosphatidylinositol (-GPI) membrane anchor. Based on a reported genetic association of PI16 and plasma levels of the chemokine chemerin, we investigated whether PI16 regulates post-translational processing of its precursor pro-chemerin. PI16-deficient mice were engineered and found to generate higher levels of processed chemerin than wildtype mice. Purified recombinant PI16 efficiently inhibited cathepsin K, a chemerin-activating protease, in vitro. Moreover, we show that conditioned medium from PI16-overexpressing cells impaired the activation of pro-chemerin. Together, our data indicate that PI16 suppresses chemerin activation in the myocardium and suggest that this circuit may be part of the cardiac stress response. KW - Cells KW - Activation KW - Purification KW - Protein KW - Peptidase inhibitor 16 (PI16) KW - Identification KW - Inflammation KW - Adipokine KW - Metabolism KW - Heart KW - Mice KW - Chemerin KW - RARRES2 KW - TIG2 KW - Protease inhibition KW - Chemerin processing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187039 VL - 99 ER - TY - JOUR A1 - Wolf, Karen A1 - Braun, Attila A1 - Haining, Elizabeth J. A1 - Tseng, Yu-Lun A1 - Kraft, Peter A1 - Schuhmann, Michael K. A1 - Gotru, Sanjeev K. A1 - Chen, Wenchun A1 - Hermanns, Heike M. A1 - Stoll, Guido A1 - Lesch, Klaus-Peter A1 - Nieswandt, Bernhard T1 - Partially Defective Store Operated Calcium Entry and Hem(ITAM) Signaling in Platelets of Serotonin Transporter Deficient Mice JF - PLoS One N2 - Background Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation. Objective To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt\(^{-/-}\)) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke. Results In 5Htt\(^{-/-}\) platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca\(^{2+}\) entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt\(^{-/-}\) platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt\(^{-/-}\) mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt\(^{-/-}\) mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice. Conclusion Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization. KW - platelets KW - serotonin KW - integrins KW - blood flow KW - collagens KW - platelet activation KW - platelet aggregation KW - ischemic stroke Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146399 VL - 11 IS - 1 ER - TY - JOUR A1 - Hofmann, Sebastian A1 - Braun, Attila A1 - Pozgaj, Rastislav A1 - Morowski, Martina A1 - Vögtle, Timo A1 - Nieswandt, Bernhard T1 - Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis JF - PLoS One N2 - Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. \(Cd84^{−/−}\) platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of \(Cd84^{−/−}\) mice. In vivo, \(Cd84^{−/−}\) mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions. KW - flow cytometry KW - CD coreceptors KW - integrins KW - blood KW - platelet aggregation KW - platelet activation KW - cytotoxic T cells KW - platelets Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126477 VL - 9 IS - 12 ER - TY - JOUR A1 - Timofeev, Oleg A1 - Schlereth, Katharina A1 - Wanzel, Michael A1 - Braun, Attila A1 - Nieswandt, Bernhard A1 - Pagenstecher, Axel A1 - Rosenwald, Andreas A1 - Elsässer, Hans-Peter A1 - Stiewe, Thorsten T1 - p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo JF - Cell Reports N2 - Four 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 p53(E177R) 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. KW - mutant p53 KW - senescence KW - mice KW - tumorigenesis KW - restoration KW - damage responses KW - antioxidant function KW - p53-inducible regulator KW - p53-dependent apoptosis KW - cell-cycle arrest Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122168 VL - 3 ER - TY - JOUR A1 - Pfeiffer, Verena A1 - Götz, Rudolf A1 - Xiang, Chaomei A1 - Camarero, Guadelupe A1 - Braun, Attila A1 - Zhang, Yina A1 - Blum, Robert A1 - Heinsen, Helmut A1 - Nieswandt, Bernhard A1 - Rapp, Ulf R. T1 - Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum JF - PLoS ONE N2 - This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures. KW - granule cells KW - hippocampus KW - neurons KW - neuronal dendrites KW - embryos KW - dentate gyrus KW - neuronal differentiation KW - cerebellum Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130304 VL - 8 IS - 3 ER - TY - JOUR A1 - Balkenhol, Johannes A1 - Kaltdorf, Kristin V. A1 - Mammadova-Bach, Elmina A1 - Braun, Attila A1 - Nieswandt, Bernhard A1 - Dittrich, Marcus A1 - Dandekar, Thomas T1 - Comparison of the central human and mouse platelet signaling cascade by systems biological analysis JF - BMC Genomics N2 - 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 Entrez 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. KW - interspecies comparison KW - transcriptome KW - proteome KW - platelet KW - network KW - signaling KW - mouse KW - human KW - interactome KW - cascade Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230377 VL - 21 ER - TY - JOUR A1 - Stritt, Simon A1 - Nurden, Paquita A1 - Favier, Remi A1 - Favier, Marie A1 - Ferioli, Silvia A1 - Gotru, Sanjeev K. A1 - van Eeuwijk, Judith M.M. A1 - Schulze, Harald A1 - Nurden, Alan T. A1 - Lambert, Michele P. A1 - Turro, Ernest A1 - Burger-Stritt, Stephanie A1 - Matsushita, Masayuki A1 - Mittermeier, Lorenz A1 - Ballerini, Paola A1 - Zierler, Susanna A1 - Laffan, Michael A. A1 - Chubanov, Vladimir A1 - Gudermann, Thomas A1 - Nieswandt, Bernhard A1 - Braun, Attila T1 - Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg\(^{2+}\) homeostasis and cytoskeletal architecture JF - Nature Communications N2 - Mg\(^{2+}\) plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg\(^{2+}\)]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7\(^{fl/fl-Pf4Cre}\)) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7\(^{fl/fl-Pf4Cre}\) MKs, which is rescued by Mg\(^{2+}\) supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice. KW - Cytoskeleton KW - homeostasisIon channels KW - thrombopoiesis Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173843 VL - 7 ER -