David Stegner

Publications of David Stegner

• Platelet receptor signaling in thrombus formation.

  Authors: David Stegner, Bernhard Nieswandt

  Journal of molecular medicine (Berlin, Germany). 11/2010; 89(2):109-21.

  Platelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also cause myocardial infarction and stroke. The initial capture ofPlatelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also cause myocardial infarction and stroke. The initial capture of flowing platelets to the injured vessel wall is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor immobilized on the exposed subendothelial extracellular matrix. Tethered platelets are then able to bind to collagens through the immunoglobulin-like receptor GPVI and to initiate cellular activation, a process that is reinforced by G protein-coupled receptors stimulated by locally produced thrombin and soluble mediators released from activated platelets. These signaling events lead to a rise in the cytosolic Ca(2+) concentration, rearrangement of the cytoskeleton, release of granule content, and functional upregulation of integrin adhesion receptors allowing firm adhesion and thrombus growth. Fully activated platelets also undergo a procoagulant conversion thereby facilitating coagulation and thrombus stabilization. This review summarizes the most important receptor systems and signaling mechanisms involved in platelet activation and thrombus formation with special focus on recent discoveries.

• 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.

• 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.