TY - JOUR A1 - Koessler, Juergen A1 - Klingler, Philipp A1 - Niklaus, Marius A1 - Weber, Katja A1 - Koessler, Angela A1 - Boeck, Markus A1 - Kobsar, Anna T1 - The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness JF - TH Open N2 - Introduction  Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods  Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results  In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion  ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies. KW - platelet physiology KW - cold storage KW - adenosine diphosphate KW - purinergic receptors KW - inhibitory signaling Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229387 VL - 4 IS - 3 ER - TY - JOUR A1 - Niklaus, Marius A1 - Klingler, Philipp A1 - Weber, Katja A1 - Koessler, Angela A1 - Kuhn, Sabine A1 - Boeck, Markus A1 - Kobsar, Anna A1 - Koessler, Juergen T1 - Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands JF - TH Open N2 - Background  Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets. Materials and Methods  Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H2DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet–neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging. Results  Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet–neutrophil contacts. Conclusion  Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils. KW - receptors KW - immunity KW - cell-cell interactions KW - platelet physiology Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301401 VL - 6 IS - 3 SP - e156 EP - e167 ER - TY - THES A1 - Schüpferling, Anne Marie Heidi T1 - Der Einfluss der Proteasomhemmung durch Bortezomib auf die Aktivierbarkeit humaner Thrombozyten T1 - The role of proteasom activity for activating signaling in human platelets N2 - Bortezomib, ein selektiver und potenter Proteasominhibitor, wird experimentell in der Tumorzellforschung sowie therapeutisch in der Therapie des Multiplen Myeloms eingesetzt. Die Wirkung auf die Thrombozytenfunktion war bislang unzureichend untersucht. Daher evaluiert diese Studie die dosisabhängige Wirkung von Bortezomib auf die Viabilität, die Aggregation von gewaschenen Thrombozyten und auf aktivierende Signalwege in gewaschenen Thrombozyten. Die Thrombozytenviabilität war bei hohen Bortezomibkonzentrationen von 100 - 200 µM vermindert. Passend dazu verminderten 100 - 200 µM Bortezomib die Phosphorylierung der ERK1/2 und der Akt/PKB in humanen Thrombozyten. Im Gegensatz dazu hatten diese hohen Bortezomibkonzentrationen keinen Einfluss auf das Niveau der p38 MAP Kinase-Phosphorylierung in aktivierten Thrombozyten. Die Thrombozytenaggregation, induziert durch hohe Konzentrationen von Kollagen oder TRAP-6, blieb unter 0,1 nM - 200 µM Bortezomib unverändert. Zusammenfassend lässt sich sagen, dass Bortezomib weder die essenziellen, aktivierenden Signalwege noch die Initialisierung der Aggregation relevant beeinflusst. Das zeigt, dass diese Prozesse in Thrombozyten nicht abhängig von der Proteasomaktivität sind. Supramaximale Inhibierung des Proteasomsystems mit Bortezomibkonzentrationen von 100 µM oder mehr führen möglicherweise zu veränderter Thrombozytenreaktionsfähigkeit, welche unter Umständen durch unspezifische und potenziell toxische Effekte mit erniedrigter Zellviabilität verursacht werden. N2 - Bortezomib, a selective and potent proteasome inhibitor, is used experimentally in tumor cell research and therapeutically in the treatment of multiple myeloma. Its effect on platelet function has been insufficiently studied. Therefore, this study evaluates the dose-dependent effects of bortezomib on viability, aggregation of washed platelets and activating signaling pathways in washed platelets. Platelet viability was tampered after incubation with high bortezomib concentrations of 100 - 200 µM. Fittingly, 100 - 200 µM bortezomib decreased phosphorylation of ERK1/2 and Akt/PKB in human platelets. In contrast, these high concentrations of bortezomib had no effect on the level of p38 MAP kinase phosphorylation in activated platelets. Furthermore platelet aggregation induced by high concentrations of collagen or TRAP-6 remained unchanged under the influence 0.1 nM - 200 µM bortezomib. In conclusion, bortezomib does not relevantly affect essential activating signaling pathways or the initialization of aggregation. This indicates that these processes in platelets are not dependent on proteasome activity. Supramaximal inhibition of the proteasome system with bortezomib concentrations of 100 µM or above may lead to altered platelet responsiveness, which may be accompanied by nonspecific and potentially toxic effects associated with decreasing cell viability. KW - Thrombozyt KW - Proteasom KW - Bortezomib KW - Thrombozytenaggregation KW - Thrombozyten KW - Proteasomsystem KW - Thrombozytenaktivierung KW - Viabilität KW - Proteasomhemmung Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-327551 ER - TY - THES A1 - Klingler, Philipp T1 - Exploration of proteasome interactions with human platelet function T1 - Untersuchung von Proteasom-Wechselwirkungen mit der Funktion humaner Thrombozyten N2 - Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research. Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition. Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments. Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation. As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets. N2 - Thrombozyten sind kernlose Zellfragmente, welche aus Megakaryozyten gebildet werden. Sie spielen eine fundamentale Rolle in der Hämostase, aber es gibt immer mehr Hinweise, dass Thrombozyten auch in immunologischen Prozessen involviert sind. Trotz Fehlen eines Zellkerns haben humane Thrombozyten die Fähigkeit zur de novo Proteinsynthese und besitzen außerdem ein voll funktionstüchtiges Proteasomsystem, welches in kernhaltigen Zellen über den Proteinabbau an Prozessen wie dem Fortschreiten des Zellzyklus oder der Apoptose beteiligt ist. Die physiologische Bedeutung des Proteasomsystems in humanen Thrombozyten ist nicht vollständig geklärt und ist aus diesem Grund Gegenstand aktueller Forschung. Daher war es Ziel dieser Studie, die Rolle des Proteasomsystems für die funktionellen Eigenschaften humaner Thrombozyten zu erforschen. Für die Experimente wurde Citrat-Vollblut von gesunden Spendern gewonnen und mit Proteasom-Hemmstoffen vorinkubiert. Es wurde neben dem gängigen Bortezomib der potente und selektive Proteasom-Inhibitor Carfilzomib als zweiter Inhibitor eingesetzt, um substanzspezifische Effekte auszuschließen und zu bestätigen, dass die beobachteten Veränderungen auf der Proteasom-Inhibition beruhen. Die irreversibel induzierte Thrombozytenaktivierung und -aggregation wurde durch die Hemmung des Proteasoms mit Bortezomib bis zu 24 Stunden nicht beeinflusst. Allerdings führte die Proteasom-Hemmung zu einer verstärkten Schwellenwertaggregation und -agglutination um bis zu 25 % sowie zu einer partiellen Abschwächung der induzierten VASP-Phosphorylierung um etwa 10-15 %. Die Expression verschiedener Rezeptoren blieb nahezu unbeeinflusst. Stattdessen konnte unter Proteasom-Inhibition eine erhöhte Enzymaktivität der PP2A beobachtet werden, begleitet von einer erleichterten Thrombozytenadhäsion an beschichteten Oberflächen bei statischen Versuchen ober bei Flusskammerversuchen. Carfilzomib, welches erstmals für funktionelle Experimente mit menschlichen Thrombozyten in vitro eingesetzt wurde, führte zu einer dosisabhängigen Abnahme der Proteasom-Aktivität mit einer Akkumulation von poly ubiquitylierten Proteinen. Wie Bortezomib mündete die Behandlung mit Carfilzomib in einer verstärkten Schwellenwertaggregation und abgeschwächter VASP-Phosphorylierung. Die wichtigste Schlussfolgerung dieser Arbeit ist, dass die Inhibition des Proteasoms die Reaktivität humaner Thrombozyten erhöht, gekennzeichnet durch eine Abschwächung der hemmenden Signalwege der Thrombozyten und durch eine zusätzliche Erhöhung der PP2A-Enzymaktivität, was zu einer erleichterten Thrombozytenadhäsion unter statischen Verhältnissen und unter Flussbedingungen führt. Das Proteasomsystem scheint an der Förderung der hemmenden Gegenregulation in Thrombozyten beteiligt zu sein. Das Potenzial von Proteasom Inhibitoren, thromboembolische Nebenwirkungen bei Patienten auszulösen, muss in weiteren Studien geklärt werden, ebenso wie ihr möglicher Einsatz für die gezielte Beeinflussung der Thrombozytenfunktion zur Verbesserung der hämostatischen Reaktivität der Thrombozyten. KW - Thrombozyt KW - Proteasom KW - Platelet KW - Proteasome KW - Bortezomib Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-321089 ER - TY - JOUR A1 - Koessler, Juergen A1 - Schwarz, Michaela A1 - Weber, Katja A1 - Etzel, Julia A1 - Koessler, Angela A1 - Boeck, Markus A1 - Kobsar, Anna T1 - The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions JF - PLoS ONE N2 - Background: Platelets are important for effective hemostasis and considered to be involved in pathophysiological processes, e.g. in cardiovascular diseases. Platelets provided for research or for therapeutic use are frequently separated from citrated whole blood (WB) stored for different periods of time. Although functionally intact platelets are required, the stability of platelet integrity, e.g. adenosine diphosphate (ADP) mediated responsiveness, has never been thoroughly investigated in citrated WB under ex vivo conditions. Objectives: Platelet integrity was evaluated at different time points in citrated WB units, collected from healthy donors and stored for 5 days at ambient temperature. The analysis included the measurement of activation markers, of induced light transmission aggregometry and of purinergic receptor expression or function. Inhibitory pathways were explored by determination of basal vasodilator-stimulated phosphoprotein (VASP)-phosphorylation, intracellular cyclic nucleotide levels and the content of phosphodiesterase 5A. Fresh peripheral blood (PB) samples served as controls. Results: On day 5 of storage, thrombin receptor activating peptide-6 (TRAP-6) stimulated CD62P expression and fibrinogen binding were comparable to PB samples. ADP induced aggregation continuously decreased during storage. Purinergic receptor expression remained unchanged, whereas the P2Y1 activity progressively declined in contrast to preserved P2Y12 and P2X1 function. Inhibitory pathways were unaffected except for a slight elevation of VASP phosphorylation at Ser\(^{239}\) on day 5. Conclusion: After 5 days of storage in citrated WB, platelet responsiveness to TRAP-6 is sufficiently maintained. However, ADP-mediated platelet integrity is more sensitive to deterioration, especially after storage for more than 2 days. Decreasing ADP-induced aggregation is particularly caused by the impairment of the purinergic receptor P2Y1 activity. These characteristics should be considered in the use of platelets from stored citrated WB for experimental or therapeutic issues. KW - fibrinogen KW - phosphorylation KW - platelets KW - blood KW - specimen storage KW - flow cytometry Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159879 VL - 12 IS - 11 ER - TY - THES A1 - Wiebecke, Sophie Karolina T1 - Einfluss mikrobieller Substanzen von Parodontitis-assoziierten Bakterien auf die Funktion humaner Thrombozyten T1 - The influence of bacterial substances from periodontitis-associated bacteria on the function of human platelets N2 - Diese Arbeit untersuchte die Wirkung von bakteriellen Substanzen auf essenzielle thrombozytäre Funktionen. Bei den Substanzen handelte es sich um Toxine von Bakterien, die mutmaßlich zur Pathogenese der Parodontitis beitragen. Während LTX von Bakterium A. actinomycetemcomitans und C14 von F. nucleatum zur Hemmung der Aggregation und zur Stimulation inhibitorischer Systeme beiträgt, induziert LPS von P. gingivalis eine leichte Aktivierung der Thrombozyten, gekennzeichnet durch eine gering verstärkte P-Selektin-Expression. N2 - In this doctoral thesis the effect of bacterial substances on essential platelet functions was investigated. The substances were toxins from bacteria that contribute to the pathogenesis of the periodontitis. LTX from A.actinomycetem comitans and C14 fom F.nucleatum cause the inhibition of the aggregation and the stimulation of inhibitory systems. LPS from P.gingivalis induces a slight activation of platelets, characterized by an increased P-selectin expression KW - Parodontitis KW - Thrombozytenfunktion Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-329038 ER -