@phdthesis{Semeniak2018,
  author    = {Semeniak, Daniela},
  title     = {Role of bone marrow extracellular matrix proteins on platelet biogenesis and function},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155857},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis. First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level. Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice. In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation. Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.},
  subject      = {Knochenmark},
  language  = {en}
}
@phdthesis{Heidenreich2018,
  author    = {Heidenreich, Julius Frederik},
  title     = {Characterization of the widely used Rac1-inhibitors NSC23766 and EHT1864 in mouse platelets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic diseases, such as myocardial infarction and stroke. Extracellular agonists induce platelet activation by stimulation of platelet membrane receptors. Signal transduction results in reorganization of the cytoskeleton, shape change, platelet adhesion and aggregation, cumulating in thrombus formation. Several Rho GTPases, including Rac1, Cdc42 and RhoA, are essential mediators of subsequent intracellular transduction of ITAM- and GPCR-signaling. Therefore, inhibition or knockout can result in severely defective platelet signaling. Mice with platelet specific Rac1-deficiency are protected from arterial thrombosis. This benefit highlights further investigation of Rac1-specific functions and its potential as a new pharmacological target for prevention of cardiovascular diseases. Two newly developed synthetic compounds, NSC23766 and EHT1864, were proposed to provide highly specific inhibition of Rac1 activity, but both drugs have never been tested in Rac1-deficient cell systems to rule out potential Rac1-independent effects. This study revealed significant off-target effects of NSC23766 and EHT1864 that occurred in a dose-dependent fashion in both wild-type and Rac1-deficient platelets. Both inhibitors individually affected resting platelets after treatment, either by altering membrane protein expression (NSC23766) or by a marked decrease of platelet viability (EHT1864). Platelet apoptosis could be confirmed by enhanced levels of phosphatidylserine exposure and decreased mitochondrial membrane potential. Phosphorylation studies of the major effector proteins of Rac1 revealed that NSC23766 and EHT1864 abolish PAK1/PAK2 activation independently of Rac1 in wild-type and knockout platelets, which may contribute to the observed off-target effects. Additionally, this study demonstrated the involvement of Rac1 in G protein-coupled receptor-mediated platelet activation and GPIb-induced signaling. Furthermore, the data revealed that Rac1 is dispensable in the process of integrin IIb 3-mediated clot retraction. This study unveiled that new pharmacological approaches in antithrombotic therapy with Rac1 as molecular target have to be designed carefully in order to obtain high specificity and minimize potential off-target effects.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Popp2018,
  author    = {Popp, Michael},
  title     = {Mechanisms of platelet activation and receptor regulation in genetically modified mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135494},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {This work summarizes the results of studies on several major aspects of platelet activation and platelet receptor regulation. Therefore, this thesis is divided into four parts. Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic disease states, such as myocardial infarction and stroke. Agonist-induced elevation in cytosolic Ca2+ concentrations is essential for platelet activation in hemostasis and thrombosis. The principal route of Ca2+ influx in platelets is store-operated calcium entry (SOCE). The calcium sensor molecule stromal interaction molecule 1 (STIM1) regulates SOCE by activating the membrane calcium channel protein Orai1, but the exact mechanisms of this interaction are not fully understood. Using affinity chromatography to screen for STIM1 interacting proteins in platelets, bridging integrator 2 (BIN2), an adapter protein belonging to the family of BAR proteins that is mainly expressed in the hematopoietic system, was identified. Newly generated BIN2 KO mice were viable and fertile but their platelets displayed markedly impaired SOCE in response to thapsigargin (TG) as well as agonists acting on immunoreceptor tyrosine-based activation motif (ITAM) or G protein-coupled receptors. This SOCE defect resulted in impaired (hem)ITAM induced platelet activation, aggregate formation under flow and procoagulant activity. As a consequence, mice lacking BIN2 in platelets were protected from occlusive arterial thrombus formation and thrombo-inflammatory cerebral infarct progression in a model of experimental stroke. These results identify BIN2 as a critical regulator of platelet SOCE in thrombosis and thrombo-inflammatory disease. Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets. 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. It was hypothesized that Hic-5 is a novel regulator of integrin αIIbβ3 activation in mice. As demonstrated in the second part of this thesis, 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. The Rho GTPase family members RhoA and Rac1 play major roles in platelet activation at sites of vascular injury. Little is known about possible redundant functions of these Rho GTPases in regulating platelet function. To investigate functional redundancies of RhoA and Rac1 in platelet production and function, mice with MK- and platelet-specific double- deficiencies in RhoA and Rac1 were generated. RhoA/Rac1 double-deficiency phenocopied the respective single knockouts without any additional effects in the double-knockout animals, demonstrating for the first time a functional non-redundancy of RhoA and Rac1 in platelet function. Antibodies against platelet glycoproteins (GP) trigger platelet destruction in immune thrombocytopenia (ITP) by binding to Fcγ receptors (FcγRs) on immune cells. However, antibodies against the platelet collagen receptor GPVI exert powerful anti-thrombotic action in vivo by inducing ectodomain shedding of the receptor associated with a transient thrombocytopenia. As shown in the final part of this thesis, blockade or deficiency of the inhibitory FcγRIIB abolished sequestration of anti-GPVI opsonized platelets in the hepatic vasculature and GPVI shedding. This process was mediated by liver sinusoidal endothelial cells (LSEC), the major FcγRIIB expressing cell type in the body. Furthermore, LSEC FcγRIIB mediated hepatic platelet sequestration and contributed to thrombocytopenia in mice treated with antibodies against αIIbβ3, the major target antigen in human ITP. These results reveal a novel and unexpected function of hepatic FcγRIIB in the processing of antibody-opsonized platelets.},
  subject      = {H{\"a}mostase},
  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}
}