@article{DrubeWeberLoschinskietal.2015,
  author    = {Drube, Sebastian and Weber, Franziska and Loschinski, Romy and Beyer, Mandy and Rothe, Mandy and Rabenhorst, Anja and G{\"o}pfert, Christiane and Meininger, Isabel and Diamanti, Michaela A. and Stegner, David and H{\"a}fner, Norman and B{\"o}ttcher, Martin and Reinecke, Kirstin and Herdegen, Thomas and Greten, Florian R. and Nieswandt, Bernhard and Hartmann, Karin and Kr{\"a}mer, Oliver H. and Kamradt, Thomas},
  title     = {Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells},
  series = {Oncotarget},
  volume    = {6},
  journal   = {Oncotarget},
  number    = {7},
  doi       = {10.18632/oncotarget.3022},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143681},
  pages     = {5354-5368},
  year      = {2015},
  abstract  = {Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca2\(^{+}\)-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation". This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33. We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo. Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.},
  language  = {en}
}
@article{PoppThielmanNieswandtetal.2015,
  author    = {Popp, Michael and Thielman, Ina and Nieswandt, Bernhard and Stegner, David},
  title     = {Normal Platelet Integrin Function in Mice Lacking Hydrogen Peroxide-Induced Clone-5 (Hic-5)},
  series = {PLoS One},
  volume    = {10},
  journal   = {PLoS One},
  number    = {7},
  doi       = {10.1371/journal.pone.0133429},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125724},
  pages     = {e0133429},
  year      = {2015},
  abstract  = {Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice.},
  language  = {en}
}
@article{AmmarThahoulyHanaueretal.2015,
  author    = {Ammar, Mohamed Raafet and Thahouly, Tamou and Hanauer, Andr{\´e} and Stegner, David and Nieswandt, Bernhard and Vitale, Nicolas},
  title     = {PLD1 participates in BDNF-induced signalling in cortical neurons},
  series = {Scientific Reports},
  volume    = {5},
  journal   = {Scientific Reports},
  number    = {14778},
  doi       = {10.1038/srep14778},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139962},
  year      = {2015},
  abstract  = {The brain-derived neurotrophic factor BDNF plays a critical role in neuronal development and the induction of L-LTP at glutamatergic synapses in several brain regions. However, the cellular and molecular mechanisms underlying these BDNF effects have not been firmly established. Using in vitro cultures of cortical neurons from knockout mice for Pld1 and Rsk2, BDNF was observed to induce a rapid RSK2-dependent activation of PLD and to stimulate BDNF ERK1/2-CREB and mTor-S6K signalling pathways, but these effects were greatly reduced in Pld1\(^{-/-}\) neurons. Furthermore, phospho-CREB did not accumulate in the nucleus, whereas overexpression of PLD1 amplified the BDNF-dependent nuclear recruitment of phospho-ERK1/2 and phospho-CREB. This BDNF retrograde signalling was prevented in cells silenced for the scaffolding protein PEA15, a protein which complexes with PLD1, ERK1/2, and RSK2 after BDNF treatment. Finally PLD1, ERK1/2, and RSK2 partially colocalized on endosomal structures, suggesting that these proteins are part of the molecular module responsible for BDNF signalling in cortical neurons.},
  language  = {en}
}
@article{VieraElMerahbiNieswandtetal.2016,
  author    = {Viera, Jonathan Trujillo and El-Merahbi, Rabih and Nieswandt, Bernhard and Stegner, David and Sumara, Grzegorz},
  title     = {Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight},
  series = {PLoS ONE},
  volume    = {11},
  journal   = {PLoS ONE},
  number    = {6},
  doi       = {10.1371/journal.pone.0157607},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179729},
  year      = {2016},
  abstract  = {Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.},
  language  = {en}
}
@article{DuettingGaitsIacovoniStegneretal.2017,
  author    = {D{\"u}tting, Sebastian and Gaits-Iacovoni, Frederique and Stegner, David and Popp, Michael and Antkowiak, Adrien and van Eeuwijk, Judith M.M. and Nurden, Paquita and Stritt, Simon and Heib, Tobias and Aurbach, Katja and Angay, Oguzhan and Cherpokova, Deya and Heinz, Niels and Baig, Ayesha A. and Gorelashvili, Maximilian G. and Gerner, Frank and Heinze, Katrin G. and Ware, Jerry and Krohne, Georg and Ruggeri, Zaverio M. and Nurden, Alan T. and Schulze, Harald and Modlich, Ute and Pleines, Irina and Brakebusch, Cord and Nieswandt, Bernhard},
  title     = {A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {15838},
  doi       = {10.1038/ncomms15838},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170797},
  year      = {2017},
  abstract  = {Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.},
  language  = {en}
}
@article{StegnervanEeuwijkAngayetal.2017,
  author    = {Stegner, David and van Eeuwijk, Judith M.M. and Angay, Oğuzhan and Gorelashvili, Maximilian G. and Semeniak, Daniela and Pinnecker, J{\"u}rgen and Schmithausen, Patrick and Meyer, Imke and Friedrich, Mike and D{\"u}tting, Sebastian and Brede, Christian and Beilhack, Andreas and Schulze, Harald and Nieswandt, Bernhard and Heinze, Katrin G.},
  title     = {Thrombopoiesis is spatially regulated by the bone marrow vasculature},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {127},
  doi       = {10.1038/s41467-017-00201-7},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170591},
  year      = {2017},
  abstract  = {In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.},
  language  = {en}
}
@phdthesis{Stegner2018,
  author    = {Stegner, David},
  title     = {Novel Aspects of Platelet Signaling and of the Pathogenesis of Immune Thrombocytopenia},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87980},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {This work summarizes the results of studies on three major aspects of platelet signaling and of the pathogenesis of immune thrombocytopenia. Therefore, this thesis is divided into three parts. i) Platelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also trigger 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 (vWF) immobilized on the exposed subendothelial extracellular matrix (ECM). The central importance of GPIb for platelet adhesion is well established, whereas GPV is generally considered to be of minor relevance for platelet physiology and thrombus formation. This study intended to clarify the relevance of this receptor during thrombus formation using Gp5-/- mice and mice with different double-deficiencies in GPV and in other platelet receptors. It was found that GPV and the collagen receptor integrin a2b1 have partially redundant functions in collagentriggered platelet aggregation. Further, it was revealed that GPV limits thrombus formation and impairs hemostasis in vivo. The data presented here demonstrate that the protective effect of GPVI-deficiency (another platelet collagen receptor) in arterial thrombosis and ischemic stroke depends on the expression of GPV. Moreover, it was demonstrated that lack of GPV restores the hemostatic function of mice lacking both GPVI and a2b1 or mice lacking GPVI and the C-type lectin receptor 2 (CLEC-2). Conclusively, GPV-depletion or blockade might have the potential to treat hemorrhagic disease states. ii) Platelets contain the two phospholipase (PL) D isoforms, PLD1 and PLD2, both of which presumably become activated upon platelet stimulation. However, the function of PLD in the process of platelet activation and aggregation has not been definitively explored. Thus, PLD-deficient mice were analyzed. Mice lacking PLD1 or PLD2 were viable, fertile and had normal platelet counts. PLD1 was found to be responsible for the inducible PLD-activity in platelets and to contribute to efficient integrin activation under static conditions. Moreover, flow adhesion experiments revealed that PLD1 is essential for efficient GPIb-mediated integrin activation. Consequently, Pld1-/- mice were protected from arterial thrombosis and ischemic brain infarction without affecting tail bleeding times. Hence, inhibition of PLD1 might be a novel approach for antithrombotic therapy. iii) Cellular activation of platelets or immune cells results in increased cytosolic calcium (Ca2+) levels. Store-operated calcium entry (SOCE) via the STIM1-Orai1 axis is the main route of Ca2+ entry downstream of immunoreceptor tyrosine-based activating motif (ITAM) receptor stimulation in mast cells and T cells. However, the requirement of Ca2+-mobilization in Fcg receptor (FcgR)-signaling and the relevance of STIM2 for T cell SOCE have been unclear. To address these questions, genetically modified mice lacking central molecules of the SOCE machinery were analyzed. Ca2+-measurements revealed that both STIM isoforms contribute to Ca2+-mobilization downstream of T cell receptor activation. Additionally, it was found that FcgR stimulation results in SOCE and is mediated by STIM1 and probably Orai1. Animal models of immune thrombocytopenia (ITP) revealed that SOCE is essential for platelet clearance and that both STIM isoforms contribute to the pathology of ITP. Moreover, in this work it was also demonstrated that STIM1 and Orai1 are essential in IgG-mediated systemic anaphylaxis. STIM2 contributes to IgG-mediated, but not to IgE-mediated anaphylaxis. The data indicate that interference with SOCE might become a new strategy to prevent or treat IgG-dependent autoimmune diseases.},
  subject      = {Thrombozyt},
  language  = {en}
}
@article{SchuhmannKraftBieberetal.2019,
  author    = {Schuhmann, Michael K. and Kraft, Peter and Bieber, Michael and Kollikowski, Alexander M. and Schulze, Harald and Nieswandt, Bernhard and Pham, Mirko and Stegner, David and Stoll, Guido},
  title     = {Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice},
  series = {International Journal of Molecular Science},
  volume    = {20},
  journal   = {International Journal of Molecular Science},
  number    = {8},
  issn      = {1422-0067},
  doi       = {10.3390/ijms20082019},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201700},
  year      = {2019},
  abstract  = {Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{-/-}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.},
  language  = {en}
}
@article{StegnerKlausNieswandt2019,
  author    = {Stegner, David and Klaus, Vanessa and Nieswandt, Bernhard},
  title     = {Platelets as modulators of cerebral ischemia/reperfusion injury},
  series = {Frontiers in Immunology},
  volume    = {10},
  journal   = {Frontiers in Immunology},
  number    = {2505},
  issn      = {1664-3224},
  doi       = {10.3389/fimmu.2019.02505},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195748},
  year      = {2019},
  abstract  = {Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed "reperfusion injury." Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.},
  language  = {en}
}
@article{NavarroStegnerNieswandtetal.2021,
  author    = {Navarro, Stefano and Stegner, David and Nieswandt, Bernhard and Heemskerk, Johan W. M. and Kuijpers, Marijke J. E.},
  title     = {Temporal roles of platelet and coagulation pathways in collagen- and tissue factor-induced thrombus formation},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {1},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23010358},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284219},
  year      = {2021},
  abstract  = {In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.},
  language  = {en}
}