David Varga-Szabo

Publications of David Varga-Szabo

• 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.

• 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.

• 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.

• 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.

• Rac1 is essential for phospholipase C-gamma2 activation in platelets.

  Authors: Irina Pleines, Margitta Elvers, Amrei Strehl, Miroslava Pozgajova, David Varga-Szabo, Frauke May, Anna Chrostek-Grashoff, Cord Brakebusch, Bernhard Nieswandt

  Pflugers Archiv : European journal of physiology. 09/2008;

  Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+)Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.

• 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.

• Cell adhesion mechanisms in platelets.

  Authors: David Varga-Szabo, Irina Pleines, Bernhard Nieswandt

  Arteriosclerosis, thrombosis, and vascular biology. 04/2008; 28(3):403-12.

  At sites of vascular injury, platelets come into contact with the subendothelial extracellular matrix which triggers their activation and the formation of a hemostatic plug. This process is crucialAt sites of vascular injury, platelets come into contact with the subendothelial extracellular matrix which triggers their activation and the formation of a hemostatic plug. This process is crucial for normal hemostasis, but may also lead to pathological thrombus formation causing diseases such as myocardial infarction or stroke. The initial capture of flowing platelets is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor (vWF) immobilized on exposed collagens. This interaction allows the binding of the collagen receptor GPVI to its ligand and to initiate cellular activation, a process that is reinforced by locally produced thrombin and soluble mediators released from platelets. These events lead to the shift of beta1 and beta3 integrins on the platelet surface from a low to a high affinity state, thereby enabling them to bind their ligands and to mediate firm adhesion, spreading, coagulant activity, and aggregation. This review summarizes the most important structural and functional properties of these adhesion receptors and briefly discusses their potential as targets for antithrombotic therapy.

• Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo.

  Authors: Bernhard Nieswandt, Markus Moser, Irina Pleines, David Varga-Szabo, Sue Monkley, David Critchley, Reinhard Fässler

  The Journal of experimental medicine. 01/2008; 204(13):3113-8.

  Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarctionPlatelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin beta subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.

• 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.

• Diverging signaling events control the pathway of GPVI down-regulation in vivo.

  Authors: Tamer Rabie, David Varga-Szabo, Markus Bender, Rastislav Pozgaj, Francois Lanza, Takashi Saito, Stephen P Watson, Bernhard Nieswandt

  Blood. 08/2007; 110(2):529-35.

  Coronary artery thrombosis is often initiated by platelet activation on collagen-rich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptorCoronary artery thrombosis is often initiated by platelet activation on collagen-rich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptor glycoprotein VI (GPVI) noncovalently associates with the Fc receptor gamma-chain (FcRgamma), which signals through its immunoreceptor-tyrosine-based activation motif (ITAM) via the adaptor LAT leading to the activation of phospholipase Cgamma2 (PLCgamma2). GPVI is a promising antithrombotic target as anti-GPVI antibodies induce the irreversible loss of the receptor from circulating platelets by yet undefined mechanisms in humans and mice and long-term antithrombotic protection in the latter. However, the treatment is associated with transient but severe thrombocytopenia and reduced platelet reactivity to thrombin questioning its clinical usefulness. Here we show that GPVI down-regulation occurs through 2 distinct pathways, namely ectodomain shedding or internalization/intracellular clearing, and that both processes are abrogated in mice carrying a point mutation in the FcRgamma-associated ITAM. In mice lacking LAT or PLCgamma2, GPVI shedding is abolished, but the receptor is irreversibly down-regulated through internalization/intracellular clearing. This route of GPVI loss is not associated with thrombocytopenia or altered thrombin responses. These results reveal the existence of 2 distinct signaling pathways downstream of the FcRgamma-ITAM and show that it is possible to uncouple GPVI down-regulation from undesired side effects with obvious therapeutic implications.