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.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Bc. Kateřina Nováková Supervisor: RNDr. Gabriela Červená, Ph.D. plk. MUDr. Miloš Bohoněk, Ph.D. Title of diploma thesis: Whole blood - New Blood Component Containing Platelets Background: The aim of this thesis was to evaluate in vitro quality parameters and haemostatic function of leucodepleted WB and their comparison with non-leucodepleted WB. The same way the quality parameters (RBC, PLT, HB and hemolysis) of erythrocyte blood component were measured and compared in day D14, D21, D35 and D42. Methods: WB collected from 30 healthy group A donors was divided into two groups - leucodepleted (LWB), using in-line platelet-sparing filters and collected to a blood bag system IMUFLEX® WB-SP (Terumo BCT, USA) and non-leucodepleted WB (NLWB), colletected to a blood bag system CompoFlex® Single System (Fresenius Kabi, Germany). Both groups were stored at 42řC for 14 days and following parameters were measured in days D0, D1, D3, D5, D10 and D14: WBC, RBC, PLT, HB, hemolysis, pH, TEG, FVIII, TT, PT, aPTT, aggregometry, concentration of PF4 and sCD40L (ELISA). Moreover, in days D0, D7 and D14 was measured the level of expression of platelet activation marker CD62P (P-selectin), CD42b, CD61 by...

Platelet products can be used, thanks to the broad range of bioactive molecules, either as a supplement for cell cultering in vitro alone or for development of cell- or cell-free scaffolds in diverse fields in regenerative medicine. The aim of this study was to prepare several types of platelet products. The concentration of selected molecules were observed. Subsequently, these products were tested with cell cultures in vitro alone or in combination with nanofibres scaffolds prepared by electrospinning or centrifugal spinning. It was found out, that platelets products contains chemokine RANTES and growth factor PDGF in the highest concentrations. It was further discovered the content of pro and antiinflammatory in terleukins and other growth factors. Platelet lysat in concentration 7% is sufficient to replace FBS in keratinocytes and fibroblasts cultures. In the other experiments, platelets in different concentrations were adhered to the scaffolds prepared by electrospinning and centrifugal spinning. Thus prepared scaffolds promote the proliferation and viability of all tested cell types in dose-dependent manner. In the last experiment, the individual components of platelet concentrate were separated and characterized. Their effect to the cell culture were tested. It was examinated the synergic...

Le brain-derived neurotrophic factor (BDNF) est une protéine de la famille des neurotrophines ayant été initialement découverte au système nerveux central, où elle est impliquée dans la mémoire et l‘apprentissage par la régulation de la croissance et de la survie neuronale. Les effets du BDNF sont médiés par le tropomyosin receptor kinase B (TrkB) et le récepteur pan-neurotrophique de 75 kDa (p75NTR). Le BDNF est le résultat du clivage d’une protéine précurseur, le proBDNF, laquelle a plutôt des effets pro-apoptotiques sur les neurones. Malgré sa découverte au cerveau, le BDNF est retrouvé en concentrations beaucoup plus importantes dans la circulation sanguine, où il est majoritairement contenu dans les plaquettes. Il est rapporté que ces cellules peuvent contenir des concentrations de BDNF allant de 100 à 1000 fois celles retrouvées au cerveau et que celles-ci peuvent être altérées par certaines maladies neurologiques. Malgré les importantes concentrations de BDNF qu’elles contiennent, très peu d’études ont investigué la présence du proBDNF ainsi que des récepteurs TrkB et p75NTR dans les plaquettes. Dans ces études, l’identification de ces protéines au niveau plaquettaire ne représentait pas un objectif primaire et les résultats obtenus ne sont souvent pas présentés. Jusqu’à présent, le proBDNF et les récepteurs TrkB et p75NTR n’ont pas été répertoriés dans les plaquettes. L’objectif principal de ce mémoire était d’investiguer la présence du proBDNF ainsi que des récepteurs TrkB et p75NTR dans les plaquettes de volontaires sains humains et de caractériser ces protéines dans le cas où elles seraient présentes. Les résultats obtenus suggèrent que les plaquettes expriment chacune de ces trois protéines, mais que les isoformes retrouvées au niveau plaquettaire diffèrent de celles retrouvées au cerveau. Les résultats proposent également que ces différences ne résident pas dans le profil de N-glycosylation des protéines. L’identité exacte des protéines étudiées n’a pas pu être confirmée par séquençage et leur nature demeure donc à confirmer. La présence plaquettaire du proBDNF et des récepteurs TrkB et p75NTR pourrait s’avérer intéressante au niveau des biomarqueurs périphériques de certaines maladies neuronales et psychiatriques. Leur présence pourrait aussi permettre la progression des connaissances dans le domaine de la biologie plaquettaire. / The brain-derived neurotrophic factor (BDNF) is a protein that was initially identified in the central nervous system, where it is involved in learning and memory by promoting neuronal growth and survival. These effects of BDNF are mediated through its binding to the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). Mature BDNF results from the cleavage of its precursor protein proBDNF, which rather has a proapoptotic effect on neurons. While discovered in the brain, BDNF is found in much higher abundance in the blood circulation, where it is mostly contained within platelets. It has been shown that BDNF concentration in platelets can reach up to 1000 times those of the brain, and that peripheral BDNF levels are altered in certain neurological and psychiatric diseases. Despite these important BDNF concentrations in platelets, very few studies assessed the presence of proBDNF, TrkB and p75NTR in these cells. Furthermore, identification of these proteins in platelets was not a main objective of the studies that did assess that question. Consequently, methodology is not always described, and the results are mostly reported as data not shown. Until now, proBDNF, TrkB and p75NTR have not been reported in platelets. The main objective of this master’s thesis was to investigate the presence of proBDNF as well as receptors TrkB and p75NTR in healthy human platelets, and to characterize them if they were found in these cells. The results suggest that platelets express all three proteins, but that the isoforms found in platelets differ from the ones found in the brain. Also, the results show that these differences are not explained by differential N-glycosylation patterns. The identity of the proteins of interest could not be verified by protein sequencing, and their exact nature is yet to be confirmed. The presence of proBDNF as well as the TrkB and p75NTR receptors in platelets could be of interest in the search of peripheral biomarkers for neurological diseases. In addition, presence of these proteins at the platelet level could pave the way for further studies investigating their functions in platelets, and possibly result in advances in our knowledge of platelet biology.

TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) est un immunorécepteur connu pour être exprimé par les neutrophiles et les monocytes/macrophages. Il joue un rôle fondamental dans l’amplification de la réponse inflammatoire via les TLR (Toll-like receptor). A l’aide de plusieurs outils expérimentaux, nous montrons pour la première fois que TREM-1 est exprimé par deux nouveaux types cellulaires : la cellule endothéliale et les plaquettes. La délétion sélective de TREM-1 au niveau endothélial protège du choc septique en réduisant la dysfonction et l’inflammation vasculaire, en modulant le recrutement et l’activation des cellules inflammatoires, et en améliorant la survie. De plus, la modulation pharmacologique via l’utilisation du peptide LR12 ou l'invalidation génétique de TREM-1 altère l'activation plaquettaire et prévient la formation de thrombus. Ces résultats fournissent un nouvel aperçu de la biologie TREM-1 et peuvent expliquer l'action protectrice de la modulation TREM-1 au cours des maladies inflammatoires aiguës comme le choc septique, au-delà de leurs effets sur les cellules myéloïdes. De plus, les agents modulateurs de TREM-1 tels que LR12 pourraient potentiellement être des ajouts utiles dans les thérapies antiplaquettaires dans le cadre de troubles thrombotiques / TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) is an immunoreceptor known to be expressed by neutrophils and monocytes/macrophages. It plays a fundamental role in the amplification of the inflammatory response mediated by TLR (Toll-like receptor) engagement. Using several experimental tools, we show for the first time that TREM-1 is expressed by two other cell types: endothelial cells and platelets. The selective deletion of TREM-1 at the endothelial level protects against septic shock by reducing dysfunction and vascular inflammation, modulating the recruitment and the activation of inflammatory cells, and improves survival. In addition, pharmacological modulation via the use of the LR12 peptide or genetic invalidation of TREM-1 alters platelet activation and prevents the formation of thrombi. These results provide new insights on the TREM-1 biology and may explain the protective effect of the TREM-1 modulation during acute inflammatory diseases such as septic shock beyond its role on myeloid cells. In addition, TREM-1 modulating agents such as LR12 could potentially be useful additions in antiplatelet therapies for thrombotic disorders

Le traitement antiplaquettaire à l’aspirine (ASA) est moins efficace chez certains patients coronariens, ce qui augmente leur risque de développer une thrombose. Des taux sanguins élevés de médiateurs thrombo-inflammatoires, tels que le sCD40L, peuvent expliquer de telles variabilités. Nous avons émis l’hypothèse que, en présence de taux élevés de sCD40L, l’efficacité de l’ASA peut être réduite. Ainsi, nous avons viser à déterminer les effets de l’ASA sur la signalisation et l’agrégation des plaquettes en présence de sCD40L. Les effets de l'ASA sur les plaquettes humaines traitées par le sCD40L, en réponse à des concentrations sub-optimales de collagène ou de thrombine, ont été évalués sur l'agrégation, la sécrétion de thromboxane A2 (TxA2) et la phosphorylation de la p38- « Mitogen-activated protein kinase » (MAPK), le facteur nucléaire-κB (NF-κB), la kinase activée par le TGF-β 1 (TAK-1) et la chaîne légère de la myosine (MLC). Le sCD40L a significativement augmenté la sécrétion de TxA2 dans les plaquettes, en réponse à des doses sub-optimales de collagène et de thrombine, ce qui a été inversé par l'ASA. L'ASA n'a pas inhibé la phosphorylation de p38-MAPK, NF-KB, TAK-1, que ce soit avec une stimulation par le sCD40L seul ou en présence des agonistes plaquettaires. Cependant, Le sCD40L a potentialisé l'agrégation plaquettaire, un effet complètement inversé et partiellement réduit par l'ASA en réponse au collagène et à la thrombine, respectivement. Les effets de l'ASA sur les plaquettes traitées par le sCD40L et stimulées par le collagène étaient liés à l'inhibition du changement de forme des plaquettes et la phosphorylation de la MLC. L'ASA n'affecte pas la signalisation du sCD40L dans les plaquettes, mais empêche son effet sur la sécrétion de TXA2 et l'agrégation plaquettaire en réponse au collagène, via un mécanisme impliquant l'inhibition de la MLC. Le ciblage de l'axe sCD40L dans les plaquettes peut avoir un potentiel thérapeutique chez les patients, présentant des taux élevés de sCD40L, qui ne répondent pas ou moins à l'ASA. / Antiplatelet therapy with Aspirin (ASA) is less efficient in some coronary patients, which increases their risk of developing thrombosis. Elevated blood levels of thrombo-inflammatory mediators, like sCD40L, may explain such variabilities. We hypothesized that in the presence of elevated levels of sCD40L, the efficacy of ASA may be reduced. Accordingly, this study was designed to determine the effects of ASA on sCD40L signalling and aggregation of platelets. The effects of ASA on sCD40L-treated human platelets, in response to suboptimal concentrations of collagen or thrombin, were assessed on aggregation, thromboxane A2 (TxA2) secretion, and phosphorylation of p38-MAPK, NF-κB, TGF-β-activated kinase 1 (TAK-1), and myosin light chain (MLC). sCD40L significantly elevated TxA2 secretion in platelets, in response to suboptimal doses of collagen and thrombin, which was reversed by ASA. ASA did not inhibit phosphorylation of p38-MAPK, NF-κB, TAK-1, either with sCD40L stimulation alone or with platelet agonists. However, sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to collagen and thrombin, respectively. The effects of ASA in sCD40L-treated platelets with collagen were related to inhibition of platelet shape change and MLC phosphorylation. ASA does not affect platelet sCD40L signalling, but prevents its effect on TXA2 secretion and platelet aggregation in response to collagen, via a mechanism implying inhibition of MLC. Targeting sCD40L axis in platelets may have therapeutic potential in patients with elevated levels of sCD40L that are none or less responding to ASA.

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.

GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by flow cytometry. We used various thrombocyte agonists to study differences in aggregation between the control and knockdown blood samples. The study showed that knockdown of GPR17 resulted in no significant differences in percent thrombocyte aggregation between control and agonist treated samples except for a slight increase in collagen-treated samples. Thus, it appears that GPR17 has no significant role in hemostasis.

Obstructive sleep apnea (OSA) is a common medical condition that occurs in a considerable percentage of the population. Substantial evidence shows that patients with OSA have an increased incidence of hypertension compared with individuals without OSA, and that OSA is a risk factor for the development of hypertension. It is established that OSA may be implicated in stroke and transient ischemic attacks. OSA is associated with coronary heart disease, heart failure, and cardiac arrhythmias. Pulmonary hypertension may be associated with OSA, especially in patients with pre-existing pulmonary disease. Although the exact cause that links OSA with cardiovascular disease is unknown, there is evidence that OSA is associated with a group of proinflammatory and prothrombotic factors that have been identified as important in the development of atherosclerosis. OSA is associated with increased daytime and nocturnal sympathetic activity. Autonomic abnormalities seen in patients with OSA include increased resting heart rate, decreased R-R interval variability, and increased blood pressure variability. Both atherosclerosis and OSA are associated with endothelial dysfunction, increased C-reactive protein, interleukin 6, fibrinogen, plasminogen activator inhibitor, and reduced fibrinolytic activity. OSA has been associated with enhanced platelet activity and aggregation. Leukocyte adhesion and accumulation on endothelial cells are common in both OSA and atherosclerosis. Clinicians should be aware that OSA may be a risk factor for the development of cardiovascular disease.

Titre : Nano-plaquettes à mobilité dirigée et adhésion amplifiée pour l’administration locale: vers un nouveau traitement des maladies vésicalesAbstract : L’administration locale des médicaments, définie comme une voie d’administration où la substance active est directement administrée sur ou proche de la cible ou tissus souhaités, permet d’apporter des grandes quantités des médicaments avec moins d’effets secondaires, et permet une simplification du système nanoparticulaire du fait de la non-extravasation des médicaments. Dans ce contexte, le projet de recherche de cette thèse s’est focalisé sur la voie intra-vésicale comme voie d’administration locale car il existe un besoin clinique de la part des patients, n’étant pas encore résolu. Malgré les hypothétiques avantages fournis par l’administration locale des médicaments, la voie intra-vésicale présente certaines limitations qui diminuent l’efficacité des traitements et l’observance des patients. La plupart des médicaments pour le traitement des maladies vésicales, notamment pour le cancer de la vessie et les cystites interstitielles, sont sous forme de solutions ou suspensions administrées de manière intra-vésicale via un cathéter qui passe à travers l’urètre. Dès leur arrivée à la vessie, les substances actives sont fortement diluées par les urines et éliminées rapidement lors de la miction. Cela conduit à une diminution des concentrations des substances actives au plus proche de l’épithélium, nécessitant plusieurs instillations intra-vésicales, réalisées par des praticiens hospitaliers, pour atteindre des concentrations thérapeutiques. Il y a donc un réel besoin de développer des nouvelles formulations permettant de contrecarrer les phénomènes décrits au préalable.L’objectif de cette thèse de doctorat est de créer un nouveau système nanoparticulaire de morphologie non-sphérique qui serait susceptible d’avoir un mouvement diffèrent et dirigé ainsi qu’une adhésion amplifiée. En conséquence, nous attendons de ces systèmes qu’ils apportent des concentrations en substances actives plus importantes que les systèmes nanoparticulaires sphériques et formulations galéniques traditionnelles.Aux cours de nos travaux expérimentaux, nous avons réussi à développer un système nanoparticulaire de morphologie hexagonale et aplatie. Ces nanoparticules, appellées nano-plaquettes, sont conçues à partir de l’auto-assemblage des molécules d’α-CD et des chaines alkyles greffées sur les squelettes de polysaccharides tels que l’acide hyaluronique, la chondroïtine sulfate ou l’héparine. Ces systèmes présentent l’originalité de ne pas avoir de substance active encapsulé parce que les molécules de polymère elles mêmes agissent à la fois en tant que substance active et de véhicule. Ces nano-plaquettes ont montré un mouvement en milieu isotrope et statique très diffèrent des nano-sphères utilisées comme contrôle. En effet, la majorité d’entre elles diffuse de manière plus importante et dirigée, avec des trajectoires rectilignes. Grâce à leur mouvement et aux propriétés inhérentes liées à leur forme, ces systèmes se sont montrés particulièrement intéressants vis-à-vis des interactions avec des cellules. Ils adhèrent mieux et plus longtemps à la muqueuse vésicale, elles sont mieux internalisées par des cellules et sont éliminées plus lentement une fois adhérées à la surface de l’urothélium.Un modelé in vivo de Syndrome de la Vessie Douloureuse / Cystite Interstitielle développé chez le rat nous a permis de montrer l’efficacité thérapeutique des nano-plaquettes, notamment celle constituées d’acide hyaluronique. En effet, elles présentent une meilleure bioaccumulation dans la vessie et une meilleure activité anti-inflammatoire et de régénération de la muqueuse urothéliale.Ces systèmes nanoparticulaires, conçues lors de nos travaux de thèse, constituent une approche innovante, rationnelle et efficace pouvant ouvrir de nouvelles voies de recherche pour le traitement des maladies vésicales. / Title: Directed-mobility and enhanced-adhesion nano-platelets for local drug delivery: towards a new treatment of bladder diseases.Abstract: Local drug delivery, defined as the administration route where the drug is delivered directly or very close to its target or tissue, allows to bring large amounts of drugs with reduced side effects, in comparison with systemic administration. In this context, our research project has been focused on the intravesical drug delivery as local administration route, because there is a real need to develop new pharmaceutical formulations to thwart several limitations. Despite the advantages provided by the local drug delivery, intravesical drug delivery exhibited some issues which are decreasing the therapeutic efficacy and the patient compliance to the treatment. Most of therapies for the treatment of bladder diseases are simple drug solutions or suspensions administered intravesically by using a catheter through the urethra in order to reach easily the bladder and, consequently, the urothelium. Since the drug is administered into the bladder, drug dilution is occurring because the continuous production of urine. Furthermore, active substances are being eliminated during washout when bladder urine voiding is happening. These two processes lead to the decrease of local drug concentration close to the urothelium. Patients need repeated catheterization, performed by health care practitioners, to reach therapeutic dose of the drug. Therefor, there is a need of new drug formulations to avoid these main limitations.The main goal of this PhD thesis was to create and design a new nanoparticulate system with non-spherical shape susceptible to move in a different manner compared to spherical nanoparticles. These systems may exhibit an amplified mucoadhesion allowing to bring more important amounts of drug than classical and nanoparticle administration.During this thesis, we developed a new nanoparticulate system presenting non-spherical, hexagonal and flattened shape. The driven force for the design of these nanoparticles was the self-assembling of α-cyclodextrin molecules with alkyl chains grafted on the polymer skeleton. Polymers used belong to a polysaccharide family called glycosaminoglycans including hyaluronic acid, chondroitin sulfate or heparin. This original and innovative nanoparticulate system does not encapsulate an active drug. Our polysaccharide will act, at the same time, as the active drug and the carrier. These nanoparticles, called now nano-platelets have shown different movement behavior than the spherical ones. Indeed, they diffuse more rapidly in a straight-line way. Thanks to their oriented and directed motion and to their intrinsic properties, due to the shape, these systems have shown a better mucoadhesion on the bladder tissue, a better uptake in different cell lines and they were far less rapidly eliminated from the urothelium mucosa.An in vivo model of Bladder Painful Syndrome / Interstitial Cystitis in rats demonstrated the therapeutic efficacy of nano-platelets, especially for hyaluronic acid nanoparticles. Indeed, they demonstrated a better bioaccumulation into the bladder and a better therapeutic efficacy as anti-inflammatory and urothelium regenerating agents.These nanoparticulate systems, designed during this work, represent a new innovative, rational and effectiveness approach allowing to open new research pathways for the treatment of bladder diseases.