@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{SchellergebBirkholz2020, author = {Scheller [geb. Birkholz], Inga}, title = {Studies on the role of actin-binding proteins in platelet production and function in mice}, doi = {10.25972/OPUS-16858}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168582}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Platelet activation and aggregation at sites of vascular injury involves massive cytoskeletal re-organization, which is required for proper platelet function. Moreover, the cytoskeleton plays central roles in megakaryo- and thrombopoiesis. Thus, cytoskeletal protein aberrations can be the underlying reason for many pathological phenotypes. Although intensive research is carried out to identify the key players involved in cytoskeletal reorganization, the signaling cascades orchestrating these complex processes are still poorly understood. This thesis investigates the role of three actin-binding proteins, Coactosin-like (Cotl) 1, Profilin (Pfn) 1 and Thymosin (T) β4, in platelet formation and function using genetically modified mice. ADF-H-containing proteins such as Twinfilin or Cofilin are well characterized as regulators of thrombopoesis and cytoskeletal reorganization. Although Cotl1 belongs to the ADF-H protein family, lack of Cotl1 did not affect platelet count or cytoskeletal dynamics. However, Cotl1-deficiency resulted in significant protection from arterial thrombus formation and ischemic stroke in vivo. Defective GPIb-vWF interactions and altered second wave mediator release present potential reasons for the beneficial effect of Cotl1-deficiency. These results reveal an unexpected function of Cotl1 as a regulator of thrombosis and hemostasis, establishing it as a potential target for a safe therapeutic therapy to prevent arterial thrombosis or ischemic stroke. Recent studies showed that the organization of the circumferential actin cytoskeleton modulates calpain-mediated αIIbβ3 integrin closure, thereby also controlling αIIbβ3 integrin localization. The second part of this thesis identified the actin-sequestering protein Pfn1 as a central regulator of platelet integrin function as Pfn1-deficient platelets displayed almost abolished αIIbβ3 integrin signaling. This translated into a profound protection from arterial thrombus formation and prolonged tail bleeding times in vivo which was caused by enhanced calpain-dependent integrin closure. These findings further emphasize the importance of a functional actin cytoskeleton for intact platelet function in vitro and in vivo. Tβ4 is a moonlighting protein, acting as one of the major actin-sequestering proteins in cells of higher eukaryotes and exerting various paracrine functions including anti-inflammatory, immunomodulatory and pro-angiogenic effects. Although excessively studied, its role for cytoskeletal dynamics, the distinction between endo- and exogenous protein function and its uptake and release mechanisms are still poorly understood. Constitutive Tβ4-deficiency resulted in thrombocytopenia accompanied by a largely diminished G-actin pool in platelets and divergent effects on platelet reactivity. Pre-incubation of platelets with recombinant Tβ4 will help to understand the function of endo- and exogenous protein, which is under current investigation.}, subject = {Thrombozyt}, language = {en} } @article{BalkenholKaltdorfMammadovaBachetal.2020, author = {Balkenhol, Johannes and Kaltdorf, Kristin V. and Mammadova-Bach, Elmina and Braun, Attila and Nieswandt, Bernhard and Dittrich, Marcus and Dandekar, Thomas}, title = {Comparison of the central human and mouse platelet signaling cascade by systems biological analysis}, series = {BMC Genomics}, volume = {21}, journal = {BMC Genomics}, doi = {10.1186/s12864-020-07215-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230377}, year = {2020}, abstract = {Background Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81\%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. Conclusions Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.}, language = {en} } @article{RommelMildeEberleetal.2020, author = {Rommel, Marcel G. E. and Milde, Christian and Eberle, Regina and Schulze, Harald and Modlich, Ute}, title = {Endothelial-platelet interactions in influenza-induced pneumonia: A potential therapeutic target}, series = {Anatomia, Histologia, Embryologia}, volume = {49}, journal = {Anatomia, Histologia, Embryologia}, number = {5}, doi = {10.1111/ahe.12521}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213610}, pages = {606 -- 619}, year = {2020}, abstract = {Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro-thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza-mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza-induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.}, language = {en} }