@phdthesis{vanEeuwijk2018,
  author    = {van Eeuwijk, Judith Martina Maria},
  title     = {Studies on thrombopoiesis and spleen tyrosine kinase-mediated signaling in platelets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142933},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis. Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor  chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.},
  subject      = {Thrombose},
  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{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}
}