@phdthesis{ZimmermannneePapp2024,
  author    = {Zimmermann [n{\´e}e Papp], Lena},
  title     = {Platelets as modulators of blood-brain barrier disruption and inflammation in the pathophysiology of ischemic stroke},
  doi       = {10.25972/OPUS-30285},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302850},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Ischemia-reperfusion injury (I/R injury) is a common complication in ischemic stroke (IS) treatment, which is characterized by a paradoxical perpetuation of tissue damage despite the successful re-establishment of vascular perfusion. This phenomenon is known to be facilitated by the detrimental interplay of platelets and inflammatory cells at the vascular interface. However, the spatio-temporal and molecular mechanisms underlying these cellular interactions and their contribution to infarct progression are still incompletely understood. Therefore, this study intended to clarify the temporal mechanisms of infarct growth after cerebral vessel recanalization. The data presented here could show that infarct progression is driven by early blood-brain-barrier perturbation and is independent of secondary thrombus formation. Since previous studies unravelled the secretion of platelet granules as a molecular mechanism of how platelets contribute to I/R injury, special emphasis was placed on the role of platelet granule secretion in the process of barrier dysfunction. By combining an in vitro approach with a murine IS model, it could be shown that platelet α-granules exerted endothelial-damaging properties, whereas their absence (NBEAL2-deficiency) translated into improved microvascular integrity. Hence, targeting platelet α-granules might serve as a novel treatment option to reduce vascular integrity loss and diminish infarct growth despite recanalization. Recent evidence revealed that pathomechanisms underlying I/R injury are already instrumental during large vessel occlusion. This indicates that penumbral tissue loss under occlusion and I/R injury during reperfusion share an intertwined relationship. In accordance with this notion, human observational data disclosed the presence of a neutrophil dominated immune response and local platelet activation and secretion, by the detection of the main components of platelet α-granules, within the secluded vasculature of IS patients. These initial observations of immune cells and platelets could be further expanded within this thesis by flow cytometric analysis of local ischemic blood samples. Phenotyping of immune cells disclosed a yet unknown shift in the lymphocyte population towards CD4+ T cells and additionally corroborated the concept of an immediate intravascular immune response that is dominated by granulocytes. Furthermore, this thesis provides first-time evidence for the increased appearance of platelet-leukocyte-aggregates within the secluded human vasculature. Thus, interfering with immune cells and/or platelets already under occlusion might serve as a potential strategy to diminish infarct expansion and ameliorate clinical outcome after IS.},
  subject      = {Schlaganfall},
  language  = {en}
}
@phdthesis{ZeeshangebMajeed2014,
  author    = {Zeeshan [geb. Majeed], Saman},
  title     = {Implementation of Bioinformatics Methods for miRNA and Metabolic Modelling},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-102900},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Dynamic interactions and their changes are at the forefront of current research in bioinformatics and systems biology. This thesis focusses on two particular dynamic aspects of cellular adaptation: miRNA and metabolites. miRNAs have an established role in hematopoiesis and megakaryocytopoiesis, and platelet miRNAs have potential as tools for understanding basic mechanisms of platelet function. The thesis highlights the possible role of miRNAs in regulating protein translation in platelet lifespan with relevance to platelet apoptosis and identifying involved pathways and potential key regulatory molecules. Furthermore, corresponding miRNA/target mRNAs in murine platelets are identified. Moreover, key miRNAs involved in aortic aneurysm are predicted by similar techniques. The clinical relevance of miRNAs as biomarkers, targets, resulting later translational therapeutics, and tissue specific restrictors of genes expression in cardiovascular diseases is also discussed. In a second part of thesis we highlight the importance of scientific software solution development in metabolic modelling and how it can be helpful in bioinformatics tool development along with software feature analysis such as performed on metabolic flux analysis applications. We proposed the "Butterfly" approach to implement efficiently scientific software programming. Using this approach, software applications were developed for quantitative Metabolic Flux Analysis and efficient Mass Isotopomer Distribution Analysis (MIDA) in metabolic modelling as well as for data management. "LS-MIDA" allows easy and efficient MIDA analysis and, with a more powerful algorithm and database, the software "Isotopo" allows efficient analysis of metabolic flows, for instance in pathogenic bacteria (Salmonella, Listeria). All three approaches have been published (see Appendices).},
  subject      = {miRNS},
  language  = {en}
}
@phdthesis{WeigelverhHoffmann2024,
  author    = {Weigel [verh. Hoffmann], Mathis Leonard},
  title     = {Thrombozytenfunktionsanalyse als potenzielles Instrument zur Fr{\"u}herkennung von Sepsis},
  doi       = {10.25972/OPUS-35819},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358193},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Sepsis ist ein h{\"a}ufiges und akut lebensbedrohliches Syndrom, das eine Organfunktionsst{\"o}rung in Folge einer dysregulierten Immunantwort auf eine Infektion beschreibt. Eine fr{\"u}hzeitige Diagnosestellung und Therapieeinleitung sind von zentraler Bedeutung f{\"u}r das {\"U}berleben der Patient:innen. In einer Pilotstudie konnte unsere Forschungsgruppe mittels Durchflusszytometrie eine ausgepr{\"a}gte Hyporeaktivit{\"a}t der Thrombozyten bei Sepsis nachweisen, die einen potenziell neuen Biomarker zur Sepsis-Fr{\"u}herkennung darstellt. Zur Evaluation des Ausmaßes und Entstehungszeitpunktes der detektierten Thrombozytenfunktionsst{\"o}rung wurden im Rahmen der vorliegenden Arbeit zus{\"a}tzlich zu Patient:innen mit Sepsis (SOFA-Score ≥ 2; n=13) auch hospitalisierte Patient:innen mit einer Infektion ohne Sepsis (SOFA-Score < 2; n=12) rekrutiert. Beide Kohorten wurden zu zwei Zeitpunkten (t1: <24h; t2: Tag 5-7) im Krankheitsverlauf mittels Durchflusszytometrie und PFA-200 untersucht und mit einer gesunden Kontrollgruppe (n=28) verglichen. Ph{\"a}notypische Auff{\"a}lligkeiten der Thrombozyten bei Sepsis umfassten: (i) eine ver{\"a}nderte Expression verschiedener Untereinheiten des GPIb-IX-V-Rezeptorkomplexes, die auf ein verst{\"a}rktes Rezeptor-Shedding hindeutet; (ii) ein ausgepr{\"a}gtes Mepacrin-Beladungsdefizit, das auf eine zunehmend reduzierte Anzahl von δ-Granula entlang des Infektion-Sepsis Kontinuums hinweist; (iii) eine Reduktion endst{\"a}ndig gebundener Sialins{\"a}ure im Sinne einer verst{\"a}rkten Desialylierung. Die funktionelle Analyse der Thrombozyten bei Sepsis ergab bei durchflusszytometrischer Messung der Integrin αIIbβ3-Aktivierung (PAC-1-Bindung) eine ausgepr{\"a}gte generalisierte Hyporeaktivit{\"a}t gegen{\"u}ber multiplen Agonisten, die abgeschw{\"a}cht bereits bei Infektion nachweisbar war und gem{\"a}ß ROC-Analysen gut zwischen Infektion und Sepsis diskriminierte (AUC >0.80 f{\"u}r alle Agonisten). Im Gegensatz dazu zeigten Thrombozyten bei Sepsis und Analyse mittels PFA-200 unter Einfluss physiologischer Scherkr{\"a}fte eine normale bis gar beschleunigte Aggregation. Die Reaktivit{\"a}tsmessung von Thrombozyten mittels Durchflusszytometrie stellt weiterhin einen vielversprechenden Biomarker f{\"u}r die Sepsis-Fr{\"u}herkennung dar. F{\"u}r weitere Schlussfolgerungen ist jedoch eine gr{\"o}ßere Kohorte erforderlich. In nachfolgenden Untersuchungen sollten zudem mechanistische Ursachen der beschriebenen ph{\"a}notypischen und funktionellen Auff{\"a}lligkeiten von Thrombozyten bei Infektion und Sepsis z.B. mittels Koinkubationsexperimenten untersucht werden.},
  subject      = {Sepsis},
  language  = {de}
}
@phdthesis{Wangorsch2013,
  author    = {Wangorsch, Gaby},
  title     = {Mathematical modeling of cellular signal transduction},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87746},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {A subtly regulated and controlled course of cellular processes is essential for the healthy functioning not only of single cells, but also of organs being constituted thereof. In return, this entails the proper functioning of the whole organism. This implies a complex intra- and inter-cellular communication and signal processing that require equally multi-faceted methods to describe and investigate the underlying processes. Within the scope of this thesis, mathematical modeling of cellular signaling finds its application in the analysis of cellular processes and signaling cascades in different organisms. ...},
  subject      = {Mathematische Modellierung},
  language  = {en}
}
@phdthesis{Voegtle2014,
  author    = {V{\"o}gtle, Timo},
  title     = {Studies on receptor signaling and regulation in platelets and T cells from genetically modified mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-97114},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Receptors with tyrosine-based signaling motifs control essential functions of hematopoietic cells, including lymphocytes and platelets. Downstream of the platelet receptor glycoprotein (GP) VI and the T cell receptor (TCR) the immunoreceptor tyrosine-based activation motif (ITAM) initiates a signaling cascade that involves kinases, adapter and effector proteins and finally leads to cellular activation. This thesis summarizes the results of three studies investigating different aspects of receptor signaling and regulation in platelets and T cells. In the first part, the impact of constitutive Ca2+ influx on TCR signaling and T cell physiology was investigated using a transgenic mouse line with a mutation in the Ca2+ sensor stromal interaction molecule 1 (STIM1). The elevated cytoplasmic Ca2+ level resulted in an altered phosphorylation pattern of the key enzyme phospholipase (PL) Cγ1 in response to TCR stimulation, but without affecting its enzymatic activity. Withdrawal of extracellular Ca2+ or inhibition of the phosphatase calcineurin restored the normal phosphorylation pattern. In addition, there was a decrease in the release of Th2-type cytokines interleukin 4, 5 and 13 upon stimulation in vitro. The second part of the thesis deals with the role of the adapter protein growth factor receptor-bound protein 2 (Grb2) in platelets using a megakaryocyte/platelet-specific knockout mouse line. Loss of Grb2 severely impaired signaling of GPVI and C-type lectin-like receptor 2 (CLEC-2), a related hemITAM receptor. This was attributed to defective stabilization of the linker for activation of T cells (LAT) signalosome and resulted in reduced adhesion, aggregation, Ca2+ mobilization and procoagulant activity downstream of (hem)ITAM-coupled receptors in vitro. In contrast, the signaling pathways of G protein-coupled receptors (GPCRs) and the integrin αIIbβ3, which do not utilize the LAT signalosome, were unaffected. In vivo, the defective (hem)ITAM signaling caused prolonged bleeding times, however, thrombus formation was only affected under conditions where GPCR signaling was impaired (upon acetylsalicylic acid treatment). These results establish Grb2 as an important adapter protein in the propagation of GPVI- and CLEC-2-induced signals. Finally, the proteolytic regulation of the immunoreceptor tyrosine-based switch motif (ITSM)-bearing receptor CD84 in platelets was investigated. This study demonstrated that in mice CD84 is cleaved by two distinct and independent proteolytic mechanisms upon platelet activation: shedding of the extracellular part, which is exclusively mediated by a disintegrin and metalloproteinase (ADAM) 10 and cleavage of the intracellular C-terminus by the protease calpain. Finally, the analysis of soluble CD84 levels in the plasma of transgenic mice revealed that shedding of CD84 by ADAM10 occurs constitutively in vivo.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Volz2020,
  author    = {Volz, Julia},
  title     = {Studies on the influence of platelets on vascular integrity in primary tumors and the role of BIN2 in platelet calcium signaling},
  doi       = {10.25972/OPUS-21742},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217427},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively renders tumor vessels highly permeable, causing massive intratumoral hemorrhage. While these results establish platelets as potential targets for anti-tumor therapy, depletion is not a treatment option due to the essential role of platelets for hemostasis. This thesis demonstrates for the first time that functional inhibition of glycoprotein (GP) VI on the platelet surface rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion but without inducing systemic bleeding complications. Both, the intratumoral bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, GPVI inhibition increased intra-tumoral accumulation of co-administered chemotherapeutic agents, thereby resulting in a profound anti-tumor effect. In summary, this thesis manifests platelet GPVI as a key regulator of vascular integrity specifically in growing tumors, serving as a potential basis for the development of anti-tumor strategies. In the second part of this thesis, light is shed on the modulating role of bridging integrator 2 (BIN2) in platelet Ca2+ signaling. Stromal interaction molecule 1 (STIM1) mediated store-operated calcium entry (SOCE) is the major route of Ca2+ influx in platelets, triggered by inositol trisphosphate receptor (IP3R)-dependent Ca2+ store release. In this thesis, the BAR domain superfamily member BIN2 was identified as the first Ca2+ signaling modulator, interacting with both, STIM1 and IP3R in platelets. Deletion of BIN2 resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. These results establish BIN2 as a central regulator of platelet Ca2+ signaling. The third part of this thesis focuses on the effect of the soluble neuronal guidance protein Sema7A on platelet function. Rosenberger et al. discovered that Sema7A cleavage from red blood cells increases the formation of platelet-neutrophil complexes, thereby reinforcing thrombo-inflammation in myocardial ischemia-reperfusion injury (MIRI). This thesis establishes soluble Sema7A as a stimulator of platelet thrombus formation via its interaction with platelet GPIbα, thereby reinforcing PNC formation. Thus, interfering with the GPIb-Sema7A interaction during MIRI represents a potential strategy to reduce cardiac damage and improve clinical outcome following MI.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Vielreicher2008,
  author    = {Vielreicher, Martin Christian},
  title     = {Fluoreszenz-mikroskopische Untersuchung der Inaktivierung der Tyrosinkinase SRC im Integrin alphaIIb-beta3 -Signalweg},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-26743},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Essentiell f{\"u}r die Blutstillung (Haemostase) ist die Thrombozyten- oder Blutplaettchen-Adhaesion und die Thrombus-Bildung. Beide Vorgaenge werden hauptsaechlich durch den Thrombozyten-Rezeptor Integrin alphaIIb-beta3 vermittelt. Nach Bindung des Liganden Fibrinogen aendert sich die Rezeptor-Konformation, Integrine assoziieren und ein intrazellulaeres Signalnetzwerk wird aktiviert, welches die Organisation des Aktin-Zytoskeletts steuert. Diese Zytoskelett-Reorganisationen sind Grundlage f{\"u}r zellulaere Adhaesions- und Aggregations-Prozesse. Die Signalvermittlung vom Integrin zum Zytoskelett wird durch die Protein-Tyrosinkinase Src eingeleitet, deren Aktivitaetszustand den Signalweg reguliert. Bei der Src-Aktivierung wird Tyrosin 418 durch Autokatalyse phosphoryliert. Die Kinase muss jedoch wieder inaktiviert werden. Dies {\"u}bernimmt in Plaettchen ausschliesslich die Tyrosinkinase Csk (C-terminale Src Kinase) durch Phosphorylierung von Tyrosin 529 im C-terminalen Ende des Proteins. Die Csk-vermittelte Inaktivierung von Src stellt den entscheidenden Kontrollschritt des alphaIIb-beta3-vermittelten Signalwegs dar. Obwohl bekannt ist, dass die Src-Aktivierung bei der Zelladhaesion an den Zellraendern der Lamellipodien geschieht und man den Mechanismus und die Kinetik der Src-Csk Interaktion genauer versteht, ist bislang immer noch unbekannt, wo und wie Src inaktiviert wird bzw. welche Rolle der Src-Inaktivierung genau zukommt. FRET (Fluoreszenz-Resonanz-Energie-Transfer) ist ein physikalischer Effekt, mit dem Interaktionen beliebiger fluoreszenzmarkierter Proteine mikroskopisch detektiert werden koennen. Diese Technik wurde genutzt, um die Src-Csk-Interaktion waehrend der alphaIIb-beta3-vermittelten Fibrinogen-Adhaesion in einer etablierten Thrombozyten-Modellzelllinie (A5-CHO) direkt visualisierbar zu machen. Es zeigten sich starke Src-Csk Interaktionen (FRET-Signale) an den Zellraendern aktiver Lamellipodien und zusaetzlich in Fokalkontakten, wo beide Proteine mit Vinculin, einem Fokalkontakte-Marker, co-lokalisierten. Die Proteininteraktionen folgten einem hochdynamischen Ablauf. Nach der Akkumulation der Src-Csk Komplexe an den Zellraendern wanderten sie in Abstaenden von 2-3 Minuten nach innen, fragmentierten und bildeten schliesslich stabile Fokal-Adhaesionen. FRET-Signale an den Zellraendern fanden sich vor allem in ruhenden Lamellipodien bzw., waehrend des Lamellipodien-R{\"u}ckzugs, in wachsenden Lamellipodien traten die FRET-Signale dort dagegen nicht auf. In unabhaengigen biochemischen Tests im Zeitfenster der FRET-Beobachtungen wurde ein spezifischer Anstieg der Src-Tyr529-Phosphorylierung (Inaktivierung) und eine parallele Abnahme der Src-Tyr418-Phosphorylierung (Aktivierung) gemessen. Weiterf{\"u}hrende Ergebnisse lieferten Versuche mit Src- und Csk-Mutanten. Die Co-Expression von Wildtyp-Src mit Kinase-inaktivem CskK222R hatte weder einen Effekt auf die Adhaesion und Ausbreitung der Zellen noch auf die Praesenz von FRET, es aenderte sich jedoch drastisch die zellulaere Verteilung der FRET-Signale sowie das Wachstum und die Form der Lamellipodien. Die Co-Expression von Wildtyp-Csk mit konstitutiv aktivem SrcY529F verursachte dagegen eine stark verringerte Adhaesionsfaehigkeit und Hemmung der Lamellipodien-Bildung. Die Fokal-Adhaesionspunkte in diesen Zellen waren sehr schwach und ueberdimensioniert und lagen ungeordnet verteilt in der Adhaesionsebene. Zusaetzlich verursachte SrcY529F eine starke Ueberaktivierung des Zytoskeletts und das fast vollstaendige Verschwinden der FRET-Signale. Die ermittelten Daten zeigen, dass die enge Kontrolle der Src-Aktivitaet durch Csk eine bedeutende Rolle f{\"u}r die funktionelle Zell-Adhaesion and -Ausbreitung spielt. Co-Immunpraezipitations-Resultate und Messungen der Menge an markiertem Protein in Zellen, in welchen FRET detektierbar war, untermauern zusaetzlich unsere These, zum ersten Mal die Src-Regulation durch Csk in lebenden Zellen direkt beobachtbar gemacht zu haben. Dieser neue FRET-Ansatz kann auch als Reporter-System f{\"u}r Prozesse der Src-Inaktivierung in anderen Signalwegen und Zellen angewendet werden. Das Messprinzip kann weiterhin auf das Studium der Inaktivierung weiterer Mitglieder der Familie der Src-Kinasen (in verschiedensten Signalwegen) erweitert werden.},
  subject      = {Antigen CD41},
  language  = {de}
}
@phdthesis{Thielmann2014,
  author    = {Thielmann, Ina},
  title     = {Function and regulation of phospholipase D in blood platelets: in vitro and in vivo studies in mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-99179},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Summary Platelet activation and aggregation are crucial for primary hemostasis but can also result in occlusive thrombus formation. Agonist induced platelet activation involves different signaling pathways leading to the activation of phospholipases (PL) which produce second messengers. While the role of PLCs in platelet activation is well established, less is known about the relevance of PLDs. In the current study, the function and regulation of PLD in platelets was investigated using genetic and pharmacological approaches. In the first part of this thesis, adhesion, activation and aggregation of platelets from mice lacking PLD2 or both PLD1 and PLD2 were analyzed in vitro and in vivo. While the absence of PLD2 resulted in slightly reduced PLD activity in platelets, it had no detectable effect on the platelet function in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective alpha-granule release and protection in a model of ferric chloride induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. These results revealed, for the first time, redundant roles of PLD1 and PLD2 in platelet alpha-granule secretion and indicate that this may be relevant for pathological thrombus formation. Thus, PLD might represent a promising target for antithrombotic therapy. Thus, this hypothesis was tested more directly in the second part of this thesis. The effects of pharmacological inhibition of PLD activity on hemostasis, thrombosis and thrombo-inflammatory brain infarction in mice were assessed. Treatment of platelets with the reversible, small molecule PLD inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI) led to a specific blockade of PLD activity that was associated with reduced -granule release and integrin activation. Mice that received FIPI at a dose of 3 mg/kg displayed reduced occlusive thrombus formation upon chemical injury of carotid arteries or mesenterial arterioles. Similarly, FIPI-treated mice had smaller infarct sizes and significantly better motor and neurological function 24 hours after transient middle cerebral artery occlusion. This protective effect was not associated with major intracerebral hemorrhage or prolonged tail bleeding times. Thus, pharmacological PLD inhibition might represent a safe therapeutic strategy to prevent arterial thrombosis or ischemic stroke. After revealing a central role for PLD in thrombo-inflammation, the regulation of PLD activity in platelets was analyzed in the last part of the thesis. Up to date, most studies made use of inhibitors potentially exerting off-target effects and consequently PLD regulation is discussed controversially. Therefore, PLD activity in mice genetically lacking potential modulators of PLD activity was determined to address these controversies. These studies revealed that PLD is tightly regulated during initial platelet activation. While integrin outside-in signaling and Gi signaling was dispensable for PLD activation, it was found that PLC dependent pathways were relevant for the regulation of PLD enzyme activity.},
  subject      = {Phospholipase D},
  language  = {en}
}
@phdthesis{Teichmann2007,
  author    = {Teichmann, Lino Lars},
  title     = {Stromabw{\"a}rts der cGMP-abh{\"a}ngigen Proteinkinase in Thrombozyten - Physiologische und diagnostische Relevanz von VASP und Identifikation neuer Substrate},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-25843},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2007},
  abstract  = {Clopidogrel hat sich als potentes Medikament zur Verhinderung kardiovaskul{\"a}rer Ereignisse sowohl bei Patienten mit akutem Koronarsyndrom ohne ST-Hebung (non-STE-ACS) als auch bei Patienten mit akutem Myokardinfarkt mit ST-Hebung (STEMI) erwiesen (CURE- und COMMIT-Studie). Insbesondere der Nutzen einer Vorbehandlung mit Clopidogrel bei perkutaner Koronarintervention ist bei Patienten mit non-STE-ACS und STEMI gut belegt (PCI-CURE- und PCI-CLARITY-Studie). Ein betr{\"a}chtlicher Anteil der Patienten zeigt allerdings kein ad{\"a}quates Ansprechverhalten auf Clopidogrel. Wir etablierten deswegen zus{\"a}tzlich zu einem bereits bestehenden FACS-Assay, der den Effekt von Clopidogrel anhand der Phosphorylierung des Proteins VASP quantitativ bestimmt, einen neuartigen auf dem gleichen Prinzip beruhenden enzyme-immuno assay (EIA). In einer Doppelblindstudie mit gesunden Probanden spiegelten im systematischen Vergleich von VASP-EIA, VASP-FACS und anderer verbreiteter Verfahren (Aggregation, p-Selektin Expresssion, PFA100) sowohl die VASP-Assays als auch die Aggregation die Pl{\"a}ttchen-Inhibition deutlich wider. Demgegen{\"u}ber waren weder die p-Selektin Expression noch der PFA100 ein Indikator f{\"u}r den Clopidogrel-Effekt. Die VASP-Assays zeichneten sich im Vergleich zur Aggregation durch bessere Quantifizierbarkeit und eine enge Abh{\"a}ngigkeit von der Zielstruktur des Clopidogrels, dem P2Y12-Rezeptor, aus. So hatte eine Medikation mit Acetylsalicyls{\"a}ure keinen Einfluss auf die VASP-Assays, verminderte allerdings die Aggregation. Weiterhin konnten wir im Rahmen der Probandenstudie durch Verwendung PAR- (protease activated receptor) spezifischer Peptide (SFFLRN, AYPGKF) und dem stabilen Thromboxan A2 Analog U46619 in ex-vivo Versuchen nachweisen, dass die antithrombozyt{\"a}ren Eigenschaften des Clopidogrels zum Teil auf der indirekten Hemmung der Thrombin- und Thromboxan-induzierten Pl{\"a}ttchenaktivierung beruhen. Diese Ergebnisse betonen die zentrale Bedeutung Galpha(i)-vermittelter Signalwege in Thrombozyten. Im zweiten Teil dieser Arbeit strebten wir an, neue Substrate der cGMP-abh{\"a}ngigen Proteinkinase zu identifizieren und das bekannte Substrat VASP weiter zu charakterisieren. Die Pl{\"a}ttchenadh{\"a}sion und -aktivierung an der Zellwand sind initiale Ereignisse der arteriellen Thrombose. Prostacyclin und NO erh{\"o}hen die intrazellul{\"a}re Konzentration zyklischer Nukleotide (cAMP, cGMP). Dies f{\"u}hrt zu einer umfassenden Inhibition der Thrombozyten, haupts{\"a}chlich durch Aktivierung der cAMP- bzw. cGMP-abh{\"a}ngige Proteinkinase (cAK, cGK). Es liegt bisher kein schl{\"u}ssiges Bild davon vor, wie die Substrate der cAK und cGK die Pl{\"a}ttcheninhibition regulieren. Wir identifizierten zun{\"a}chst das adenylylcyclase-associated protein (CAP1) anhand der Analyse des humanen Pl{\"a}ttchen-Phosphoproteoms als neues Substrat der cGK, das innerhalb von Sekunden nach SNP-Stimulation phosphoryliert wird. Die Separation des Phosphoproteoms erfolgte durch 2D-Gelelektrophorese. Wildtyp-CAP1 und Mutanten, bei denen an putativen Phosphorylierungsstellen Serin bzw. Threonin durch Alanin ausgetauscht wurde, wurden anschließend in PtK2-Zellen exprimiert. Bisher konnte die Ko-Transfektion von PtK2-Zellen mit CAP1 und cGK eine Phosphorylierung von CAP1 durch die cGK nicht best{\"a}tigen. Weitere Anstrengungen werden n{\"o}tig sein, CAP1 als cGK-Substrat zu etablieren. Flusskammerversuche zeigten, dass die Hemmung der Thrombusformation durch SNP bei VASP-defizienten M{\"a}usen im Vergleich zu Wildtyp-M{\"a}usen (WT) ineffektiv ist und belegen, dass die Inhibition der Agonisten-induzierten Thrombusformation durch den NO/cGMP-Signalweg in VASP-defizienten Pl{\"a}ttchen gest{\"o}rt ist. VASP hatte in unseren Experimenten keinen signifikanten Einfluss auf die GPIIbIIIa-Aktivierung (gemessen mit dem monoklonalen Antik{\"o}rper JON/A), die Serotonin-Sekretion (delta-Granula) und die p-Selektin-Expression (alpha-Granula). Die Ergebnisse legen nahe, dass die VASP-Deletion zu subtilen St{\"o}rungen von Thrombozytenfunktionen f{\"u}hrt, die erst in Experimenten deutlich zu Tage treten, die ihr Zusammenspiel abbilden.},
  subject      = {Blutstillung},
  language  = {de}
}
@phdthesis{Stritt2017,
  author    = {Stritt, Simon},
  title     = {The role of the cytoskeleton in platelet production and the pathogenesis of platelet disorders in humans and mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122662},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Platelets are continuously produced from megakaryocytes (MK) in the bone marrow by a cytoskeleton-driven process of which the molecular regulation is not fully understood. As revealed in this thesis, MK/ platelet-specific Profilin1 (Pfn1) deficiency results in micro- thrombocytopenia, a hallmark of the Wiskott-Aldrich syndrome (WAS) in humans, due to accelerated platelet turnover and premature platelet release into the bone marrow. Both Pfn1-deficient mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyper-stable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. In contrast, Twinfilin2a (Twf2a) was established as a central regulator of platelet reactivity and turnover. Twf2a-deficient mice revealed an age-dependent macrothrombocytopenia that could be explained by a markedly decreased platelet half-life, likely due to the pronounced hyper-reactivity of \(Twf2a^{-/-}\) platelets. The latter was characterized by sustained integrin acti- vation and thrombin generation in vitro that translated into accelerated thrombus formation in vivo. To further elucidate mechanisms of integrin activation, Rap1-GTP-interacting adaptor molecule (RIAM)-null mice were generated. Despite the proposed critical role of RIAM for platelet integrin activation, no alterations in this process could be found and it was concluded that RIAM is dispensable for the activation of β1 and β3 integrins, at least in platelets. These findings change the current mechanistic understanding of platelet integrin activation. Outside-in signaling by integrins and other surface receptors was supposed to regulate MK migration, but also the temporal and spatial formation of proplatelet protrusions. In this the- sis, phospholipase D (PLD) was revealed as critical regulator of actin dynamics and podo- some formation in MKs. Hence, the unaltered platelet counts and production in \(Pld1/2^{-/-}\) mice and the absence of a premature platelet release in the bone marrow of \(Itga2^{-/-}\) mice question the role of podosomes in platelet production and raise the need to reconsider the proposed inhibitory signaling by α2β1 integrins on proplatelet formation. Non-muscle myosin IIA (NMMIIA) has been implicated as a downstream effector of the in- hibitory signals transmitted via α2β1 integrins. Besides Rho-GTPase signaling, also \(Mg^{2+}\) and transient receptor potential melastatin-like 7 (TRPM7) channel α-kinase are known regulators of NMMIIA activity. In this thesis, TRPM7 was identified as major regulator of \(Mg^{2+}\) homeostasis in MKs and platelets. Furthermore, decreased \([Mg^{2+}]_i\) led to deregulated NMMIIA activity and altered cytoskeletal dynamics that impaired thrombopoiesis and resulted in macrothrombocytopenia in humans and mice.},
  subject      = {Thrombozytopoese},
  language  = {en}
}