Attila Braun

Publications of Attila Braun

• Antithrombotic Potential of Blockers of Store-Operated Calcium Channels in Platelets.

  Authors: Roger van Kruchten, Attila Braun, Marion A H Feijge, Marijke J E Kuijpers, Ronmy Rivera-Galdos, Peter Kraft, Guido Stoll, Christoph Kleinschnitz, Edouard M Bevers, Bernhard Nieswandt, Johan W M Heemskerk

  Arteriosclerosis, thrombosis, and vascular biology. 05/2012;

  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 channelsOBJECTIVE: 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. CONCLUSIONS: 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.

• Critical role for stromal interaction molecule 1 in cardiac hypertrophy.

  Authors: Jean-Sébastien Hulot, Jérémy Fauconnier, Deepak Ramanujam, Antoine Chaanine, Fleur Aubart, Yassine Sassi, Sabine Merkle, Olivier Cazorla, Aude Ouillé, Morgan Dupuis [......] Joachim Schmitt, Attila Braun, Ludovic Bénard, Youakim Saliba, Bernhard Laggerbauer, Bernhard Nieswandt, Alain Lacampagne, Roger J Hajjar, Anne-Marie Lompré, Stefan Engelhardt

  Circulation. 08/2011; 124(7):796-805.

  Cardiomyocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling inCardiomyocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in cardiomyocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. A potential route is store-operated Ca2+ entry, a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). Store-operated Ca2+ entry can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+ entry with respect to cardiac hypertrophy in vitro and in vivo. Consistent with earlier reports, we found drug-inducible store-operated Ca2+ entry in neonatal rat cardiomyocytes, which was dependent on STIM1. Although this STIM1-dependent, drug-inducible store-operated Ca2+ 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 (i.e., 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. By controlling a previously unrecognized sarcolemmal current, STIM1 promotes cardiac hypertrophy.

• STIM and Orai in platelet function.

  Authors: David Varga-Szabo, Attila Braun, Bernhard Nieswandt

  Cell calcium. 05/2011; 50(3):270-8.

  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 toPhysiological 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.

• STIM and Orai in hemostasis and thrombosis.

  Authors: Attila Braun, Timo Vogtle, David Varga-Szabo, Bernhard Nieswandt

  Frontiers in bioscience : a journal and virtual library. 01/2011; 17:2144-60.

  At sites of vascular injury, platelets rapidly adhere to the exposed subendothelial extracellular matrix, become activated and, together with the coagulation system, form a plug that seals theAt sites of vascular injury, platelets rapidly adhere to the exposed subendothelial extracellular matrix, become activated and, together with the coagulation system, form a plug that seals the lesion. This process is essential to prevent blood loss, however, under pathological conditions it may lead to vessel occlusion. Agonist-induced elevation of intracellular Ca(2+) levels is essential for platelet activation. It occurs through two different mechanisms: Ca(2+) release from internal stores, involving phospholipase C (PLC)-dependent generation of inositol-1,4,5-trisphosphate (IP3) and activation of IP3 sensitive channels in the store membrane, and Ca(2+) influx across the plasma membrane. Store operated Ca(2+) entry (SOCE), triggered by store depletion, is the main influx pathway for extracellular Ca(2+) in platelets, but the molecular mechanism underlying this pathway has long remained elusive. In the last years, however, the Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the channel protein Orai1 emerged as the key players in platelet SOCE. This review summarizes the current knowledge about the role of these proteins in platelet physiology and thrombus formation and discusses their suitability as antithrombotic targets.

• Differentially regulated GPVI ectodomain shedding by multiple platelet-expressed proteinases.

  Authors: Markus Bender, Sebastian Hofmann, David Stegner, Athena Chalaris, Michael Bösl, Attila Braun, Jürgen Scheller, Stefan Rose-John, Bernhard Nieswandt

  Blood. 10/2010; 116(17):3347-55.

  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 diseasedGlycoprotein 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 Adam17(ex/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.

• Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

  Authors: Karen Gilio, Roger van Kruchten, Attila Braun, Alejandro Berna-Erro, Marion A H Feijge, David Stegner, Paola E J van der Meijden, Marijke J E Kuijpers, David Varga-Szabo, Johan W M Heemskerk, Bernhard Nieswandt

  The Journal of biological chemistry. 07/2010; 285(31):23629-38.

  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 toIn 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.

• Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

  Authors: Margitta Elvers, David Stegner, Ina Hagedorn, Christoph Kleinschnitz, Attila Braun, Marijke E J Kuijpers, Michael Boesl, Qin Chen, Johan W M Heemskerk, Guido Stoll, Michael A Frohman, Bernhard Nieswandt

  Science signaling. 01/2010; 3(103):ra1.

  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 adhesivePlatelet 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.

• Stromal Interaction Molecules 1 and 2 Are Key Regulators of Autoreactive T Cell Activation in Murine Autoimmune Central Nervous System Inflammation.

  Authors: Michael K Schuhmann, David Stegner, Alejandro Berna-Erro, Stefan Bittner, Attila Braun, Christoph Kleinschnitz, Guido Stoll, Heinz Wiendl, Sven G Meuth, Bernhard Nieswandt

  Journal of immunology (Baltimore, Md. : 1950). 12/2009;

  Calcium (Ca(2+)) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca(2+) entryCalcium (Ca(2+)) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca(2+) entry pathway in nonexcitable cells, including T cells, is store-operated Ca(2+) entry (SOCE), wherein depletion of intracellular Ca(2+) stores upon receptor stimulation causes subsequent influx of extracellular Ca(2+) across the plasma membrane. Stromal interaction molecule (STIM) 1 is the Ca(2+) 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 (MOG(35-55))-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-gamma/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.

• STIM1-independent T cell development and effector function in vivo.

  Authors: Niklas Beyersdorf, Attila Braun, Timo Vögtle, David Varga-Szabo, Ronmy Rivera Galdos, Stephan Kissler, Thomas Kerkau, Bernhard Nieswandt

  Journal of immunology (Baltimore, Md. : 1950). 04/2009; 182(6):3390-7.

  Store-operated Ca(2+) 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 Ca(2+) sensorStore-operated Ca(2+) 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 Ca(2+) sensor stromal interaction molecule 1 (STIM1). In vitro analyses showed that SOCE and Ag receptor complex-triggered Ca(2+) 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.

• Profilin 1 is required for abscission during late cytokinesis of chondrocytes.

  Authors: Ralph T Böttcher, Sebastian Wiesner, Attila Braun, Reiner Wimmer, Alejandro Berna, Nadav Elad, Ohad Medalia, Alexander Pfeifer, Attila Aszódi, Mercedes Costell, Reinhard Fässler

  The EMBO journal. 04/2009;

  Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency isProfilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1-mediated actin filament elongation. Neither an actin nor a poly-proline binding-deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.

• STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death.

  Authors: Alejandro Berna-Erro, Attila Braun, Robert Kraft, Christoph Kleinschnitz, Michael K Schuhmann, David Stegner, Thomas Wultsch, Jens Eilers, Sven G Meuth, Guido Stoll, Bernhard Nieswandt

  Science signaling. 01/2009; 2(93):ra67.

  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 aExcessive 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.

• STIM1 is essential for Fc{gamma} receptor activation and autoimmune inflammation.

  Authors: Attila Braun, J Engelbert Gessner, David Varga-Szabo, Shahzad N Syed, Stephanie Konrad, David Stegner, Timo Vogtle, Reinhold E Schmidt, Bernhard Nieswandt

  Blood. 11/2008;

  Fcgamma receptors (FcgammaRs) on mononuclear phagocytes trigger autoantibody and immune complex-induced diseases through coupling the self-reactive IgG response to innate effector pathways, such asFcgamma receptors (FcgammaRs) on mononuclear phagocytes trigger autoantibody and immune complex-induced diseases through coupling the self-reactive IgG response to innate effector pathways, such as phagocytosis, and the recruitment of inflammatory cells. FcRgamma-based activation is critical in the pathogenesis of these diseases, although the contribution of FcgammaR-mediated calcium signaling in autoimmune injury is unclear. Here we show that macrophages lacking the endoplasmic reticulum resident calcium sensor STIM1 cannot activate FcgammaR-induced Ca(2+) entry and phagocytosis. As a direct consequence, STIM1 deficiency results in resistance to experimental immune thrombocytopenia and anaphylaxis, autoimmune hemolytic anemia, and acute pneumonitis. These results establish STIM1 as a novel and essential component of FcgammaR activation and also indicate that inhibition of STIM1-dependent signaling might become a new strategy to prevent or treat IgG-dependent immunological diseases.

• Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation.

  Authors: Attila Braun, David Varga-Szabo, Christoph Kleinschnitz, Irina Pleines, Markus Bender, Madeleine Austinat, Michael Bosl, Guido Stoll, Bernhard Nieswandt

  Blood. 11/2008;

  Platelet activation and aggregation at sites of vascular injury is essential for primary hemostasis, but is also a major pathomechanism underlying myocardial infarction and stroke. Changes inPlatelet activation and aggregation at sites of vascular injury is essential for primary hemostasis, but is also a major pathomechanism underlying myocardial infarction and stroke. Changes in [Ca(2+)]i are a central step in platelet activation. In non-excitable cells receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated calcium (SOC) channels. STIM1 has been identified as an endoplasmic reticulum (ER)-resident Ca(2+) 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 Ca(2+) 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 a mild bleeding time prolongation. These results establish Orai1 as the long-sought platelet SOC channel and a crucial mediator of ischemic cardio- and cerebrovascular events.

• The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction.

  Authors: David Varga-Szabo, Attila Braun, Christoph Kleinschnitz, Markus Bender, Irina Pleines, Mirko Pham, Thomas Renné, Guido Stoll, Bernhard Nieswandt

  The Journal of experimental medicine. 08/2008; 205(7):1583-91.

  Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction andPlatelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction and stroke. Agonist-induced elevation of [Ca(2+)](i) is a central step in platelet activation, but the underlying mechanisms are not fully understood. A major pathway for Ca(2+) entry in nonexcitable cells involves receptor-mediated release of intracellular Ca(2+) stores, followed by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) has been identified as the Ca(2+) sensor in the endoplasmic reticulum (ER) that activates Ca(2+) release-activated channels in T cells, but its role in mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact function has been elusive, because these cells lack a normal ER and Ca(2+) is stored in a tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking STIM1 display early postnatal lethality and growth retardation. STIM1-deficient platelets have a marked defect in agonist-induced Ca(2+) responses, and impaired activation and thrombus formation under flow in vitro. Importantly, mice with STIM1-deficient platelets are significantly protected from arterial thrombosis and ischemic brain infarction but have only a mild bleeding time prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.

• Store-operated Ca(2+) entry in platelets occurs independently of transient receptor potential (TRP) C1.

  Authors: David Varga-Szabo, Kalwant Authi, Attila Braun, Markus Bender, Archana Ambily, Sheila Hassock, Thomas Gudermann, Alexander Dietrich, Bernhard Nieswandt

  Pflugers Archiv : European journal of physiology. 07/2008;

  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-operatedChanges 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.

• A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy.

  Authors: Anja Fritsch, Stefan Loeckermann, Johannes S Kern, Attila Braun, Michael R Bösl, Thorsten A Bley, Hauke Schumann, Dominik von Elverfeldt, Dominik Paul, Miriam Erlacher, Dirk Berens von Rautenfeld, Ingrid Hausser, Reinhard Fässler, Leena Bruckner-Tuderman

  The Journal of clinical investigation. 06/2008; 118(5):1669-79.

  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 isDystrophic 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.

• Trps1 regulates proliferation and apoptosis of chondrocytes through Stat3 signaling.

  Authors: Hiroki Suemoto, Yasuteru Muragaki, Katsuhiro Nishioka, Misako Sato, Akira Ooshima, Shunji Itoh, Ikuji Hatamura, Michitaka Ozaki, Attila Braun, Erika Gustafsson, Reinhard Fässler

  Developmental biology. 01/2008; 312(2):572-81.

  Mutations in the TRPS1 gene lead to the tricho-rhino-phalangeal syndrome, which is characterized by skeletal defects and abnormal hair development. The TRPS1 gene encodes an atypical member of theMutations in the TRPS1 gene lead to the tricho-rhino-phalangeal syndrome, which is characterized by skeletal defects and abnormal hair development. The TRPS1 gene encodes an atypical member of the GATA-type family of transcription factors. Here we show that mice with a disrupted Trps1 gene develop a chondrodysplasia characterized by diminished chondrocyte proliferation and decreased apoptosis in growth plates. Our analyses revealed that Trps1 is a repressor of Stat3 expression, which in turn controls chondrocyte proliferation and survival by regulating the expression of cyclin D1 and Bcl2. Our conclusion is supported (i) by siRNA-mediated depletion of Stat3 in Trps1-deficient chondrocytes, which normalized the expression of cyclin D1 and Bcl2, (ii) by overexpression of Trps1 in ATDC5 chondrocytes, which diminished Stat3 levels and increased proliferation and apoptosis, and (iii) by mutational analysis of the GATA-binding sites in the Stat3 gene, which revealed that their integrity is critical for the direct association with Trps1 and for Trps1-mediated repression of Stat3. Altogether our findings identify Trps1 as a novel regulator of chondrocytes proliferation and survival through the control of Stat3 expression.

• An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice.

  Authors: Johannes Grosse, Attila Braun, David Varga-Szabo, Niklas Beyersdorf, Boris Schneider, Lutz Zeitlmann, Petra Hanke, Patricia Schropp, Silke Mühlstedt, Carolin Zorn, Michael Huber, Carolin Schmittwolf, Wolfgang Jagla, Philipp Yu, Thomas Kerkau, Harald Schulze, Michael Nehls, Bernhard Nieswandt

  The Journal of clinical investigation. 12/2007; 117(11):3540-50.

  Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediatedChanges in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

• gamma-Parvin is dispensable for hematopoiesis, leukocyte trafficking, and T-cell-dependent antibody response.

  Authors: Haiyan Chu, Ingo Thievessen, Michael Sixt, Tim Lämmermann, Ari Waisman, Attila Braun, Angelika A Noegel, Reinhard Fässler

  Molecular and cellular biology. 04/2006; 26(5):1817-25.

  Integrins regulate cell behavior through the assembly of multiprotein complexes at the site of cell adhesion. Parvins are components of such a multiprotein complex. They consist of three membersIntegrins regulate cell behavior through the assembly of multiprotein complexes at the site of cell adhesion. Parvins are components of such a multiprotein complex. They consist of three members (alpha-, beta-, and gamma-parvin), form a functional complex with integrin-linked kinase (ILK) and PINCH, and link integrins to the actin cytoskeleton. Whereas alpha- and beta-parvins are widely expressed, gamma-parvin has been reported to be expressed in hematopoietic organs. In the present study, we report the expression pattern of the parvins in hematopoietic cells and the phenotypic analysis of gamma-parvin-deficient mice. Whereas alpha-parvin is not expressed in hematopoietic cells, beta-parvin is only found in myeloid cells and gamma-parvin is present in both cells of the myeloid and lymphoid lineages, where it binds ILK. Surprisingly, loss of gamma-parvin expression had no effect on blood cell differentiation, proliferation, and survival and no consequence for the T-cell-dependent antibody response and lymphocyte and dendritic cell migration. These data indicate that despite the high expression of gamma-parvin in hematopoietic cells it must play a more subtle role for blood cell homeostasis.

• Consequences of loss of PINCH2 expression in mice.

  Authors: Fabio Stanchi, Randi Bordoy, Oliver Kudlacek, Attila Braun, Alexander Pfeifer, Markus Moser, Reinhard Fässler

  Journal of cell science. 01/2006; 118(Pt 24):5899-910.

  PINCH2 belongs, together with PINCH1, to a new family of focal adhesion proteins, the members of which are composed of five LIM domains. PINCH1 and PINCH2 interact, through their first LIM domain,PINCH2 belongs, together with PINCH1, to a new family of focal adhesion proteins, the members of which are composed of five LIM domains. PINCH1 and PINCH2 interact, through their first LIM domain, with the integrin-linked kinase and thereby link integrins with several signal transduction pathways. Despite their high similarity, it has been shown that PINCH1 and PINCH2 could exert distinct functions during cell spreading and cell survival. To investigate the function of PINCH2 in vivo, we deleted PINCH2 in mouse using the loxP/Cre system. In contrast to the PINCH1-deficient mice, which die at the peri-implantation stage, PINCH2-null mice are viable, fertile and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder and kidney revealed no apparent abnormalities, but showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, overlapping function in vivo. To further test this possibility, we established PINCH1-null mouse embryonic fibroblasts, which express neither PINCH1 nor PINCH2. We found that in fibroblasts with a PINCH1/2-null background, PINCH2 is able to rescue the spreading and adhesion defects of mutant fibroblasts to the same extent as PINCH1. Furthermore, we show that the LIM1 domain only of either PINCH1 or PINCH2 can prevent ILK degradation despite their failure to localize to focal adhesions. Altogether these results suggest that PINCH1 and PINCH2 share overlapping functions and operate dependently and independently of their subcellular localization.