@phdthesis{Schurr2023,
  author    = {Schurr, Yvonne},
  title     = {Studies on the role of cytoskeletal-regulatory and -crosslinking proteins in platelet function},
  doi       = {10.25972/OPUS-21892},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-218924},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Cytoskeletal reorganization in platelets is highly regulated and important for proper platelet function during activation and aggregation at sites of vascular injury. In this thesis, the role of three different cytoskeletal-regulatory and -crosslinking proteins was studied in platelet physiology using megakaryocyte- and platelet-specific knockout mice. The generation of branched actin filaments is regulated by nucleation promoting factors (NPF) and the Arp2/3 complex. (1.) The WAVE complex is a NPF, which upregulates the Arp2/3 complex activity at the plasma membrane. As shown in this thesis, the loss of the WAVE complex subunit Cyfip1 in mice did not alter platelet production and had only a minor impact on platelet activation. However, Cyfip1 played an essential role for branching of actin filaments and consequently for lamellipodia formation in vitro. The importance of lamellipodia for thrombus formation and stability has been controversially discussed. Cyfip1-deficient platelets were able to form a stable thrombus ex vivo and in vivo and a hemostatic plug comparable to controls. Moreover, Cyfip1-deficient mice maintained vascular integrity at the site of inflammation. These data show that platelet lamellipodia formation is not required for hemostatic function and pathophysiological thrombus formation. (2.) The WASH complex is another NPF, which mediates actin filament polymerization on endosomal vesicles via the Arp2/3 complex. Loss of the WASH complex subunit Strumpellin led to a decreased protein abundance of the WASH protein and to a 20\% reduction in integrin αIIbβ3 surface expression on platelets and megakaryocytes, whereas the expression of other surface receptors as well as the platelet count, size, ex vivo thrombus formation and bleeding time remained unaltered. These data point to a distinct role of Strumpellin in maintaining integrin αIIbβ3 expression and provide new insights into regulatory mechanisms of platelet integrins. (3.) MACF1 has been described as a cytoskeletal crosslinker of microtubules and F-actin. However, MACF1-deficient mice displayed no alterations in platelet production, activation, thrombus formation and hemostatic function. Further, no compensatory up- or downregulation of other proteins could be found that contain an F-actin- and a microtubule-binding domain. These data indicate that MACF1 is dispensable for platelet biogenesis, activation and thrombus formation. Nevertheless, functional redundancy among different proteins mediating the cytoskeletal crosstalk may exist.},
  subject      = {Cytoskeleton},
  language  = {en}
}
@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{Spindler2020,
  author    = {Spindler, Markus},
  title     = {The role of the adhesion and degranulation promoting adapter protein (ADAP) in platelet production},
  doi       = {10.25972/OPUS-20097},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200977},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Although this process is fundamental to maintain normal platelet counts in circulation only little is known about the regulation of directed proplatelet formation. As revealed in this thesis, ADAP (adhesion and degranulation promoting adapter protein) deficiency (constitutive as well as MK and platelet-specific) resulted in a microthrombocytopenia in mice, recapitulating the clinical hallmark of patients with mutations in the ADAP gene. The thrombocytopenia was caused by a combination of an enhanced removal of platelets from the circulation by macrophages and a platelet production defect. This defect led to an ectopic release of (pro)platelet-like particles into the bone marrow compartment, with a massive accumulation of such fragments around sinusoids. In vitro studies of cultured BM cell-derived MKs revealed a polarization defect of the demarcation membrane system, which is dependent on F-actin dynamics. ADAP-deficient MKs spread on collagen and fibronectin displayed a reduced F-actin content and podosome density in the lowest confocal plane. In addition, ADAP-deficient MKs exhibited a reduced capacity to adhere on Horm collagen and in line with that the activation of beta1-integrins in the lowest confocal plane of spread MKs was diminished. These results point to ADAP as a novel regulator of terminal platelet formation. Beside ADAP-deficient mice, three other knockout mouse models (deficiency for profilin1 (PFN1), Wiskott-Aldrich-syndrome protein (WASP) and Actin-related protein 2/3 complex subunit 2 (ARPC2)) exist, which display ectopic release of (pro)platelet-like particles. As shown in the final part of the thesis, the pattern of the ectopic release of (pro)platelet-like particles in these genetically modified mice (PFN1 and WASP) was comparable to ADAP-deficient mice. Furthermore, all tested mutant MKs displayed an adhesion defect as well as a reduced podosome density on Horm collagen. These results indicate that similar mechanisms might apply for ectopic release.},
  language  = {en}
}
@phdthesis{Stetter2021,
  author    = {Stetter, Maurice},
  title     = {LC3-associated phagocytosis seals the fate of the second polar body in \(Caenorhabditis\) \(elegans\)},
  doi       = {10.25972/OPUS-23198},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231981},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {This work investigates the death and degradation of the second polar body of the nematode C. elegans in order to improve our understanding how pluripotent undifferentiated cells deal with dying cells. With the use of fluorescence microscopy this work demonstrates that both polar bodies loose membrane integrity early. The second polar body has contact to embryonic cells and gets internalized, dependent on the Rac1-ortholog CED-10. The polar body gets degraded via LC3-associated phagocytosis. While lysosome recruitment depends on RAB-7, LC3 does not improve lysosome recruitment but still accelerates polar body degradation. This work establishes the second polar body as a genetic model to study cell death and LC3-associated phagocytosis and has revealed further aspects of phagosome maturation and degradation.},
  subject      = {Polk{\"o}rper},
  language  = {en}
}