It has been shown previously in our laboratory that zebrafish produce trypsin from their gills when they are under stress, and this trypsin is involved in thrombocyte activation via PAR2 during gill bleeding. In this study, I investigated another role of the trypsin that is secreted from zebrafish. This investigation has demonstrated a novel role of trypsin in zebrafish. Not only did this investigation demonstrate the role of trypsin in zebrafish behavior, but also it showed that PAR2 might be the receptor that is involved in trypsin-mediated behavioral response. In addition, we have shown that Gq and ERK inhibitors are able to block the trypsin pathway and prevent the escaping behavior. Finally, the results of this investigation suggest that the cells that respond to trypsin are surface cells, which have an appearance similar to that of neuromast cells.

Eukaryotic platelets are small cell fragments that are released into the bloodstream from megakaryocytes, and their production is initiated in the bone marrow. They are mainly involved in blood hemostasis and thrombus formation. The newly synthesized platelets are called reticulated platelets or young platelets. Zebrafish thrombocytes are equivalent to mammalian platelets and have similar characteristics and functions. Likewise, zebrafish has both young and mature thrombocytes. Only young thrombocytes as reticulated platelets are labeled with thiazole orange. Similarly, labeling zebrafish thrombocytes with a specific concentration of DiI-C18 showed two populations of thrombocytes (DiI+ and DiI-). Again, only young thrombocytes showed DiI+ labeling. The mechanism of selective labeling of young thrombocytes by is unknown. Furthermore, there is no zebrafish line where young and mature thrombocytes are differentially labeled with fluorescence proteins. Therefore, in this study, we identified and confirmed that the RFP labeled cells of Glofish were young thrombocytes. In addition, we found that myosin light chain 2 (MLC2) promoter is expressed in young thrombocytes. We also generated a transgenic zebrafish line, GloFli fish, where the young and mature thrombocytes are labeled with red and green fluorescence proteins respectively. Furthermore, this study showed a two-fold increase in glycerol-phospholipids (GP) in mature thrombocytes compared to young thrombocytes suggesting the lipid composition may be important for differential labeling. Therefore, we tested the liposomes prepared with different ratios of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and observed that the lower amounts of PE favor the DiI-C18 labeling whereas higher concentrations of PC are less efficient. Also, in both PE and PC, increased concentrations of both resulted in decreased binding. These results are consistent with our observation that mature thrombocytes have higher concentrations GP and thus DiI-C18 may not bind to them efficiently compared to young thrombocytes.

Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.

Zebrafish thrombocytes exhibit characteristics of human platelets and megakaryocytes, making them valuable for studying megakaryopoiesis and thrombopoiesis. Using single-cell RNA sequencing, we analyzed gene expression in young and mature zebrafish thrombocytes. We identified 394 protein-coding genes unique to young thrombocytes, many corresponding with human orthologs, suggesting shared regulatory mechanisms in zebrafish and humans. We hypothesized knocking down these 394 genes should identify the novel regulatory genes that control thrombocyte maturation. To address this, we used the piggyback knockdown method to knock down these genes to study their biological functions in zebrafish thrombopoiesis. We first found the knockdown of nfe2, nfe2l1a, and nfe2l3 reduced both young and mature thrombocyte counts, confirming their role in thrombopoiesis. A comprehensive knockdown screening of the uniquely expressed genes in young thrombocytes identified 7 candidate genes associated with thrombopoiesis. We selected the spi1b gene for further mutant characterization, which revealed its critical role in young thrombocyte development, with homozygous mutations leading to embryonic lethality. Considering megakaryocyte properties in thrombocytes, we studied the potential for polyploidization in zebrafish thrombocytes. The inhibition of AURKA led to the development of polyploid thrombocytes resembling mammalian megakaryocytes, suggesting the retention of genetic programs for megakaryocyte development in zebrafish thrombocytes and providing insights into the evolutionary basis of thrombopoiesis. Thus, our study reveals critical gene expression patterns and regulatory factors in zebrafish thrombocyte development, offering insights into conserved mechanisms relevant to developmental biology and research in thrombosis and hemostasis disorder.

1981 entwickelten Gerd Binnig und Heinrich Rohrer bei IBM in Zürich das "Scanning Tunneling Microscope". Damit wurde erstmalig das lokal hochaufgelöste Erfassen (bis in den atomaren Auflösungsbereich) von Objekteigenschaften im Nahfeld inerter Oberflächen möglich. Dies und insbesondere die Weiterentwicklung der Technologie und die spätere (1986) Etablierung der Rasterkraftmikroskopie (Atomic Force Microscopy - AFM), die diese Auflösungsmöglichkeiten der Rastersondenmikroskope auch an Non-Konduktoren (nicht leitende Untersuchungsoberflächen) realisieren konnte, stellte die Geburtsstunde einer neuen mikroskopischen Ära auf dem Gebiet der biomedizinischen Grundlagenforschung dar (Kapitel 1.3). Das Studium der umfangreichen Literaturquellen zu diesem Thema und der direkte wissenschaftliche Kontakt und Erfahrungsaustausch mit anderen AFM- Arbeitsgruppen ließen im Initialstadium dieser vorliegenden Arbeit bereits erkennen, dass in der kardiovaskulären Grundlagenforschung zunehmend rasterkraftmikroskopische Versuchsansätze bearbeitet und kardiologisch interessante Fragestellungen mittels dieser Methode begleitend untersucht wurden (Kapitel 1.4). Das Ziel dieser vorliegenden Arbeit bestand darin, kardiovaskulär relevante Zellen und Einzelproteine in vivo und interventionelle Materialien (Stents) rasterkraftmikroskopisch zu untersuchen, wobei die Etablierung und technisch aufwendige Optimierung dieser neuen mikroskopischen (Kapitel 3.1) und der zellspezifisch präparatorischen Methoden (Kapitel 3.2) an diesen Untersuchungsobjekten im Mittelpunkt stehen sollte. Die im Rahmen dieser Arbeit untersuchten endothelialen Zellen und H9C2-Myozyten stammten aus, in unserem Forschungslabor etablierten, immortalen Kulturzelllinien. Die adulten und Kardiomyozyten neonataler Ratten, die kardial- fibrozytären Zellen sowie die Thrombozyten wurden primär isoliert und als Primärkulturzellen kultiviert (Kapitel 3.2.3 und 3.2.4). Außerdem wurden vitale aortale Endothelzellen unterschiedlicher Tiere (Ratte, Meerschwein, Kaninchen) im Gewebsverband der thorakalen Aorta untersucht (Kapitel 4.2). Die Zellen wurden initial, im Rahmen der Etablierungsphase mittels unterschiedlicher Methoden fixiert und nachfolgend rasterkraftmikroskopisch untersucht und dargestellt. Der Etablierungsprozess der Methodik begann mit der Abbildung luftgetrockneter Zellen (Kapitel 4.1.1) unter Raumbedingungen und setzte sich über verschiedene Modifikationen der Zellpräparation (z.B. Glutardialdehydfixation, Cryofixation), des Abbildungsmodus (Contact-, Non-Contact-, Tapping-Mode) und der Abbildungsbedingungen (Raumbedingungen, zellphysiologische Umgebung) fort, so dass schließlich die Abbildung vitaler Zellen (Kapitel 4.1.2 und Kapitel 4.2 - 4.5) in ihrer strukturellen und funktionellen Umgebung (z.B. aortale Endothelzellen im Gewebsverband) etabliert werden konnte und routinemäßig reproduzierbar war. An stabilen oder künstlich stabilisierten Strukturen der o.g. vitalen Zellen wurden erste orientierende Messungen der bioelastischen Eigenschaften (Kraft-Abstands-Kurven, Kapitel 4.1.2.1) durchgeführt. Außerdem haben wir im Einzelfall, wenn technisch und apparativ möglich, andere hochauflösende strukturanalytische Verfahren (z.B. TEM) als mikroskopische Referenzuntersuchungen herangezogen (Kapitel 4.1.2; 4.4.1; 4.6), wobei z.T. erstaunliche Übereinstimmung zwischen den AFM- Daten und den strukturanalytischen Daten der Referenzmethoden nachweisbar waren. Ein strukturell durch Elektronenmikroskopie und Röntgendiffraktionsanalyse sehr gut beschriebenes komplexes Funktionsprotein, das 20-S-Proteasom, wurde mittels der Rasterkraftmikroskopie abgebildet und vermessen und die so gewonnenen strukturanalytischen Daten mit den bekannten strukturellen Abmessungen des Proteins verglichen (Kapitel 4.6). Die hierbei detektierten dimensionalen Abweichungen zwischen den AFM- assoziierten Daten und den bekannten strukturanalytischen Daten der Elektronenmikroskopie wurden im Kontext der funktionellen Integrität des Proteins und hinsichtlich möglicher methodischer Fehlereinflüsse (Kapitel 3.1.4.3) diskutiert. Interventionelle Materialien (Stents), die in der täglichen kardiologischen Praxis Anwendung finden, sind hinsichtlich ihrer Ultrastruktur mittels dieser hochsensitiven Abbildungsmethode im Nahfeld von Objektoberflächen untersucht worden. Bezüglich ihrer nativen Oberflächenbeschaffenheit und ihrer mechanischen Alteration durch den Ballon- Dilatationsprozess wurden die Stents sehr detailliert qualitativ und quantitativ (Kapitel 4.7) beschrieben, wobei Prädilektionsstellen der prozedural- assoziierten mechanischen Beanspruchung der Stents durch die hier beschriebene, oberflächensensitive AFM- Methode sehr genau diskriminiert werden konnten. Die präparierten Stents wurden weiterführend mit humanen Thrombozytenkonzentraten inkubiert und die Zell- Stentoberflächenkontakte sowie mögliche Stentoberflächen- induzierte Veränderungen der Thrombozyten sind morphologisch ausführlich beschrieben worden. Letztendlich wurde im Rahmen der vorliegenden Arbeit die spezifische Aktivierung der vitalen Thrombozyten durch pharmakologische Stimulantien (z.B. ADP) mit der, durch den AFM-Abbildungsprozess induzierten Thrombozytenaktivierung (Kapitel 4.5) unter AFM-Bedingungen verglichen und diskutiert. Die Ergebnisse dieser Arbeit weisen, dass mit der AFM-Technologie und objektorientiert optimierten Mess- und Präparationsmethoden ein neues mikroskopisches Analyseverfahren vorliegt, dass zum einen real-dreidimensionale morphologische Bildgebung bis in den submolekularen Auflösungsbereich an vitalen Zellen und präparierten Proteinkomplexen, zum anderen aber gleichermaßen Funktionsanalytik in Form von Messungen zelldynamischer Prozesse wie Migrationsbewegungen und Kontraktionen sowie visko- elastische Quantifizierung von Zellmembranen erlaubt. Der Vorteil gegenüber den meisten gegenwärtig verfügbaren mikroskopischen Methoden liegt in der neu eröffneten Möglichkeit der seriellen, wiederholten und stabil reproduzierbaren Messung an vitalen Zellen und zellulären Substrukturen. Insofern könnte in Zukunft diese neue Technologie eine methodische Bereicherung der mikroskopisch-morphologisch und funktionell orientierten Analysetechnik darstellen. / In 1981 Binnig and Rohrer invented the "Scanning Tunneling Microscope". Thereby it became feasible to high-resolution record the surface-properties of specimens (up to atomic resolution) at the nearfield of inert surfaces. This and in detail the further development of this technology and the establishment of "Atomic Force Microscopy" (1986), that allows implementation of this resolution capabilities in non-conductors or insulating materials represent the birth of a new microscopic era in the field of biomedical basic research (chapter 1.3). The promise of atomic (scanning) force microscopy (AFM) for cardiovascular research is enormous. The perusal of the extensive literature concerning this topic and scientific contact with other researchers reveals initial the capabilities of this method in cardiovascular basic research. Intriguing questions of cardiology may investigate concomitantly with help of scanning-force-micoscopic approaches (chapter 1.4). The aim of this study was to investigate relevant cardiovascular cells and single proteins in-vivo and specific materials (coronary artery stents) with scanning-force-micoscopic setup. The establishment and expensive optimization of this new microscopic method (chapter 3.1) and of the cell specific preparatory methods (chapter 3.2) represented the center of interest of our inevestigations. The endothelial cells and H9C2-myocytes stem from established imortal cell culture lines. The adult cardiomyocytes and cardiomyocytes of neonatal rats, the fibrocytes and the thrombocytes were primarily cultivated (chapter 3.2.3 and 3.2.4). In addition we investigated aortic endothelial cells of intact aortic tissue of different animals (rat, guinea pig, rabbit - chapter 4.2). During the establish experiments cells underlied different methods of cell-fixation. The primary investigations was performed using air-dried cells (chapter 4.1.1) analyzed in room ambient conditions and were continued by different modifications of cell-preparation. (e.g. glutardialdehyde-fixation, cryo-fixation), of microscopic mode (contact-, non-contact-, tapping-mode) and of cell-specific environmental conditions (from room ambient to cellphysiological medium and temperature). As result we became enabled to investigate (reproducible and routinely) vital cells (chapter 4.1.2 and chapter 4.2 - 4.5) embedded in physiological normal structural und functional ambient conditions (e.g. endothelial cells of intact aortic tisue in-vivo). Additionally, we performed measurements of bio-elastic properties of stable or artificial stabilized structures of named cells (force-distances-curves - chapter 4.1.2.1). If posibble, depending of available technical equipment, we compared our microscopic results with other high-resolution analytical procedures of reference (e.g. TEM - Kapitel 4.1.2; 4.4.1; 4.6) and detected astonishing congruence between the data. Furthermore we analyzed the well-described (electron-microscopy and x-ray-diffraction data) complex 20-S-proteasome using a specific atomic force microscopic setup. Analytical and structural data of these AFM-scans and abovementioned methods were likened (chapter 4.6). The deviations concerning the detected proportions were discussed regarding functional integrity of the protein and with respect to potential methodically determined artifacts. (chapter 3.1.4.3). Assaying (qualitative and quantitative) the surface roughness properties of coronary artery stents, we found significant alterations of stent material induced by balloondilatation. We suppose, that changes in roughness of inner surface of coronary artery stents might induce clinical problems like acute stent-thrombosis and in-stent-restenosis. Finally these stents were coated with human thromboytes to investigate cell-stent-surface interactions. Surface-roughness correllated triggering of thrombocyte adhesion was evaluated by morphological analysis of AFM-scans. Finishing, we have investigated and concluding discussed the specific activation of vital thrombocytes by pharmacological substances (e.g. ADP) and by mechanical stimulation (due to AFM-associated tip-surface-interaction). The results of this work demonstrate, AFM-technology using optimized microscopic setup and object-specific adjusted measurement- and preparation- methods, is an new, powerful, microscopic technique, that allow real-3-dimensional morphological mapping up to submolecular range of resolution in vital cells and protein complexes. Moreover, this technology opens new dimensions in functional analytic of cell migration processes or cellular contractions and in evaluation of visco-elastic quantification of cell membranes. The advantage owed to the most currently available microscopic methods is the option of serial and reproducible measurement of vital cells and subcellular structures. In this respect, this new method might represent a methodical enrichment of the microscopic-morphological and functional oriented analysis-technique in future.