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 - 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 - Kobsar, Anna A1 - Koehnlechner, Karina A1 - Klingler, Philipp A1 - Niklaus, Marius A1 - Zeller-Hahn, Julia A1 - Koessler, Angela A1 - Weber, Katja A1 - Boeck, Markus A1 - Koessler, Juergen T1 - The effect of short-term refrigeration on platelet responsiveness JF - Scientific Reports N2 - Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we could show that cold-induced attenuation of inhibitory signaling is an important mechanism promoting platelet reactivity. For developing strategies in blood banking, it is required to elucidate the time-dependent onset of facilitated platelet activation. Thus, freshly prepared platelet-rich-plasma (PRP) was stored for 1 and 2 h at CT (2–6 °C) or at RT (20–24 °C), followed by subsequent comparative analysis. Compared to RT, basal and induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation levels were decreased under CT within 1 h by approximately 20%, determined by Western blot analysis and flow cytometry. Concomitantly, ADP- and collagen-induced threshold aggregation values were enhanced by up to 30–40%. Furthermore, platelet-covered areas on collagen-coated slides and aggregate formation under flow conditions were increased after storage at CT, in addition to induced activation markers. In conclusion, a time period of 1–2 h for refrigeration is sufficient to induce an attenuation of inhibitory signaling, accompanied with an enhancement of platelet responsiveness. Short-term refrigeration may be considered as a rational approach to obtain PC with higher functional reactivity for the treatment of hemorrhage. KW - short‑term refrigeration KW - platelet responsiveness KW - cold temperature Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301390 VL - 12 IS - 1 ER - TY - JOUR A1 - Becker, Jürgen C. A1 - Andersen, Mads H. A1 - Hofmeister-Müller, Valeska A1 - Wobser, Marion A1 - Frey, Lidia A1 - Sandig, Christiane A1 - Walter, Steffen A1 - Singh-Jasuja, Harpreet A1 - Kämpgen, Eckhart A1 - Opitz, Andreas A1 - Zapatka, Marc A1 - Bröcker, Eva-B. A1 - thor Straten, Per A1 - Schrama, David A1 - Ugurel, Selma T1 - Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma JF - Cancer Immunology, Immunotherapy N2 - Background Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. Patients and methods This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). Results Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. Conclusion Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma. KW - peptide vaccination KW - melanoma KW - survivin KW - T-cell reactivity KW - therapy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124830 VL - 61 IS - 11 ER - TY - JOUR A1 - Koessler, Juergen A1 - Hermann, Stephanie A1 - Weber, Katja A1 - Koessler, Angela A1 - Kuhn, Sabine A1 - Boeck, Markus A1 - Kobsar, Anna T1 - Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets JF - PLoS One N2 - Background Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms—potentially interfering with experimental results—have not been thoroughly studied. Objectives Since ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation. Results In contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets. Conclusion In conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function. KW - platelets KW - flow cytometry KW - adenosine KW - statistical data KW - platelet activation KW - platelet aggregation KW - phosphorylation KW - blood plasma Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146400 VL - 11 IS - 1 ER - TY - JOUR A1 - Schossee, Nadine A1 - Veit, Gabriele A1 - Gittel, Julia A1 - Viebahn, Johannes A1 - Niklaus, Marius A1 - Klingler, Philipp A1 - Üçeyler, Nurcan A1 - Klinker, Erdwine A1 - Kobsar, Anna A1 - Boeck, Markus A1 - Koessler, Juergen T1 - Profile of the single-use, multiple-pass protein A adsorber column in immunoadsorption JF - Vox Sanguinis N2 - Background and Objectives Immunoadsorptions (IA) are used to remove autoantibodies from the plasma in autoimmune disorders. In this study, we evaluated the effects of a single-use, recombinant staphylococcal protein A-based immunoadsorber on blood composition of the patient. Materials and Methods In a cohort of patients with myasthenia gravis or stiff-person syndrome, essential parameters of blood cell count, coagulation, clinical chemistry or plasma proteins and immunoglobulins (Ig) were measured before and after IA (n = 11). Results In average, IA reduced the levels of total IgG, IgG1, IgG2 and IgG4 by approximately 60%, the acetylcholine receptor autoantibody levels by more than 70%. IgG3, IgA or IgM were diminished to a lower extent. In contrast to fibrinogen or other coagulation factors, the column markedly removed vitamin K-dependent coagulation factors II, VII, IX and X by approximately 40%–70%. Accordingly, international normalized ratio and activated partial thromboplastin time were increased after IA by 59.1% and 32.7%, respectively. Coagulation tests almost returned to baseline values within 24 h. Blood cell count, electrolytes, total protein or albumin were not essentially affected. No clinical events occurred. Conclusion The single-use, multiple-pass protein A adsorber column is highly efficient to remove IgG1, IgG2 and IgG4 or specific acetylcholine receptor autoantibodies from the plasma. Coagulation parameters should be monitored, since the column has the capacity to largely reduce vitamin K-dependent factors. KW - plasma KW - apheresis technologies KW - apheresis-therapeutic KW - blood processing KW - haemostasis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259689 VL - 117 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 - JOUR A1 - Lapa, Constantin A1 - Kircher, Malte A1 - Hänscheid, Heribert A1 - Schirbel, Andreas A1 - Grigoleit, Götz Ulrich A1 - Klinker, Erdwine A1 - Böck, Markus A1 - Samnick, Samuel A1 - Pelzer, Theo A1 - Buck, Andreas K T1 - Peptide receptor radionuclide therapy as a new tool in treatment-refractory sarcoidosis - initial experience in two patients JF - Theranostics N2 - Sarcoidosis is a multisystem granulomatous disorder of unknown etiology that can involve virtually all organ systems. Whereas most patients present without symptoms, progressive and disabling organ failure can occur in up to 10% of subjects. Somatostatin receptor (SSTR)-directed peptide receptor radionuclide therapy (PRRT) has recently received market authorization for treatment of SSTR-positive neuroendocrine tumors. Methods: We describe the first case series comprising two patients with refractory multi-organ involvement of sarcoidosis who received 4 cycles of PRRT. Results: PRRT was well-tolerated without any acute adverse effects. No relevant toxicities could be recorded during follow-up. Therapy resulted in partial response accompanied by a pronounced reduction in pain (patient #1) and stable disease regarding morphology as well as disease activity (patient #2), respectively. Conclusion: Peptide receptor radionuclide therapy in sarcoidosis is feasible and might be a new valuable tool in patients with otherwise treatment-refractory disease. Given the long experience with and good tolerability of PRRT, further evaluation of this new treatment option for otherwise treatment-refractory sarcoidosis in larger patient cohorts is warranted. KW - peptide receptor KW - PRRT KW - sarcoidosis KW - somatostatin receptors KW - radionuclide therapy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158983 VL - 8 IS - 3 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 - 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 - TY - THES A1 - Trulley, Valerie-Noelle T1 - Einfluss des NO-Donors DEA/NO auf die Integrität der inhibitorischen Signalwege und der Expression purinerger Rezeptoren bei der \(ex\) \(vivo\)-Lagerung von Thrombozyten T1 - Influence of the NO donor DEA/NO on the integrity of the inhibitory signaling pathways and the expression of purinergic receptors in \(ex\) \(vivo\) stored APC (apheresis-derived platelet concentrates) N2 - Im Rahmen der Präparation und Lagerung von TKs entstehen bei Thrombozyten morphologische, funktionelle und hämostatische Defizite, die unter dem Begriff storage lesion zusammengefasst werden. In dieser Dissertation wurde untersucht, ob durch Zugabe des kurzzeitig und reversibel wirksamen NO-Donors DEA/NO zu Apherese-TKs Zeichen der storage lesion über eine 5-tägige Lagerung vermindert werden können. Dafür wurde den Apherese-TKs direkt nach der Herstellung 5 nM DEA/NO zugesetzt. An den Tagen 0 (nach Abklingen der NO-Donor-Wirkung), 2 und 5 wurden verschiedene funktionelle Systeme der Thrombozyten analysiert. Verglichen mit früheren Untersuchungen von unbehandelten TKs ergab sich unter Einsatz von DEA/NO eine abgeschwächte Aktivierung der inhibitorischen Signalwege mit geringerem Anstieg der VASP-Phosphorylierung und des cGMP-Spiegels sowie mit stabilem PDE5A-Gehalt. Gemessen anhand der P-Selektin-Expression und der Fibrinogenbindung zeigte sich ein unverändert niedriger Präaktivierungsgrad der Thrombozyten bei erhaltener Stimulierbarkeit. Bei der Oberflächenexpression von purinergen Rezeptoren war der Rückgang der stimulierten Mobilisation während der 5-tägigen Lagerung im Vergleich zu unbehandelten TKs vermindert. Damit war unter dem Einfluss von DEA/NO eine Abschwächung von Phänomenen der storage lesion zu beobachten. Für eine mögliche klinische Anwendung des NO-Donors DEA/NO bei der TK-Herstellung sind allerdings weitere Studien bezüglich Wirksamkeit und möglicher unerwünschter Wirkungen in vivo notwendig. Darüber hinaus muss eine technische Lösung für die sterile Zugabe von DEA/NO gefunden werden. N2 - During the preparation and storage of apheresis-derived PC (APC), platelets develop morphological, functional and hemostatic deficits (platelet storage lesion). In this dissertation, it was investigated whether the addition of the NO donor DEA/NO with a short-term, reversible inhibitory effect to APC could reduce the signs of storage lesion over a 5-day storage period. Therefore 5 nM DEA/NO were added to APC directly after apheresis. On days 0 (after termination of the NO donor effect), 2 and 5, different phenomena of the platelet storage lesion were analyzed. Compared to previous studies of untreated APC, the use of DEA/NO resulted in an attenuated activation of the inhibitory signaling pathways with a lower increase in VASP phosphorylation and cGMP levels and with a stable PDE5A content. Measured by P-selectin expression and fibrinogen binding, the level of platelet preactivation remained unchanged at a low level while the activation capacity was maintained. In the surface expression of purinergic receptors, the decrease of stimulated mobilization was reduced during the 5-day storage period compared to untreated APC. Thus, under the influence of DEA/NO, an attenuation of storage lesion phenomena was observed. Regarding the clinical application of the NO donor DEA/NO in manufacturing of APC, further studies are required in respect to the efficacy and possible adverse effects in vivo. Furthermore, a technical solution for the sterile addition of DEA/NO to APC needs to be established. KW - Thrombozytenkonzentrat KW - Apherese KW - Purinerger Rezeptor KW - Vasodilatator-stimuliertes Phosphoprotein KW - Phosphorylierung KW - NO-Donor KW - DEA/NO KW - Rezeptorexpression KW - Präaktivierung KW - platelet storage lesion KW - Zyklische Nukleotide Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175096 ER - TY - THES A1 - Schossee, Nadine T1 - Einfluss der Immunadsorption mit einer rekombinanten Protein A-basierten Adsorbersäule auf die Zusammensetzung des peripheren Blutes T1 - Effect of immunoadsorption using a recombinant protein A adsorber column on peripheral blood composition N2 - In dieser Arbeit wurden die Effektivität und mögliche Einflüsse der Immunadsorption mit der auf rekombinantem Protein A-basierten Einmalsäule Ligasorb® auf bestimmte Blutparameter bei einer Kohorte von neurologischen Patienten mit Myasthenia gravis oder Stiff-Person-Syndrom evaluiert. Die IA mit der Ligasorb®-Säule senkte effizient die Immunglobulinkonzentrationen IgG1, IgG2 und IgG4 im Patientenblut. Die durchschnittliche Reduktion dieser Plasmaproteine betrug 60%. Die Reduktionsrate der Acetylcholinrezeptor-Autoantikörper der MG Patienten lag bei 70% nach IA. Die Blutkonzentrationen der Serumproteine wie Albumin oder der Komplementfaktoren C3 und C4 wurden durch die IA nicht wesentlich beeinflusst. Die IA zeigten auch keine klinisch relevanten Auswirkungen auf die zelluläre Blutzusammensetzung, insbesondere wurden keine Leukozytosen beobachtet. Die IA hatten außerdem keinen relevanten Einfluss auf die untersuchten klinisch-chemischen Parameter. Nach den IA konnte aber eine relevante Störung der Hämostaseparameter festgestellt werden. Die Globaltests Quick, INR und PTT zeigten nach Behandlung deutlich pathologische Werte. Die nachfolgende Einzelfaktorbestimmung ergab Verminderungen der Vitamin K-abhängigen Gerinnungsfaktoren II, VII, IX und X um 42% bis 67%, die z.B. auf einer direkten Interaktion dieser Faktoren mit der Adsorbermatrix und/oder dem Liganden aus rekombinanten Protein A beruhen könnten. Nach 16 bis 20 Stunden zeigten sich die abweichenden Gerinnungsparameter wieder weitgehend normalisiert. Das Einhalten eines entsprechenden Zeitabstands bis zur Durchführung anderer medizinischer Eingriffe, wie z.B. einer Lumbalpunktion, sowie eine vorherige Kontrolle der Globaltests TPZ und PTT sind daher anzuraten. N2 - Efficacy and potential influence of immunoadsorption (IA) with the single-use, multiple-pass recombinant protein A-adsorber column Ligasorb® on selected blood parameters were evaluated in a cohort of neurological patients with myasthenia gravis or stiff person syndrome. IA with the Ligasorb® is highly efficient to remove IgG1, IgG2, IgG 4 and specific acetylcholine receptor autoantibodies from the plasma. In average, IgG, IgG1, IgG2 and IgG4 were reduced by approximately 60%, the acetylcholine receptor autoantibody levels in patients with myasthenia gravis by more than 70%. IgG3, IgA or IgM were diminished to a lower extent. The slightly reduced levels of albumin or other plasma proteins by 10%-20% may be interpreted as a dilutive effect, since a large volume of citrate solution (with more than 1 L) was administered during the procedures. Regarding the influence on blood composition, the blood cell count or the electrolyte levels were not essentially tampered by IA. IA with the Ligasorb® column seems a safe procedure since no technical problems or clinical events occurred within 24 hours after IA. The adsorber is technically able to process a large amount of plasma. However, the intermittent regeneration cycles of the single column contribute to time-consuming procedures. The long duration of IA with almost 6 h should be considered as a disadvantage. In contrast to fibrinogen or other coagulation factors, the column markedly removed vitamin K-dependent coagulation factors II, VII, IX and X by approximately 40%-70%. Accordingly, international normalized ratio and activated partial thromboplastin time were increased after IA by 59.1% and 32.7%, respectively. The d-dimer levels were generally very low with stable values after IA. Coagulation tests almost returned to baseline values within 24 h in patients without liver dysfunction. As a practical approach in patient care, the global coagulation tests should be monitored during IA therapies with single-use, multiple-pass protein A-adsorber, especially before invasive diagnostic or therapeutic procedures. KW - Immunadsorption KW - Protein A-Adsorber KW - Vitamin K-abhängige Gerinnungsfaktoren II, VII, IX, X Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-317075 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 - JOUR A1 - Leicht, Hans Benno A1 - Weinig, Elke A1 - Mayer, Beate A1 - Viebahn, Johannes A1 - Geier, Andreas A1 - Rau, Monika T1 - Ceftriaxone-induced hemolytic anemia with severe renal failure: a case report and review of literature JF - BMC Pharmacology and Toxicology N2 - Background: Drug induced immune hemolytic anemia (DIIHA) is a rare complication and often underdiagnosed. DIIHA is frequently associated with a bad outcome, including organ failure and even death. For the last decades, ceftriaxone has been one of the most common drugs causing DIIHA, and ceftriaxone-induced immune hemolytic anemia (IHA) has especially been reported to cause severe complications and fatal outcomes. Case Presentation: A 76-year-old male patient was treated with ceftriaxone for cholangitis. Short time after antibiotic exposure the patient was referred to intensive care unit due to cardiopulmonary instability. Hemolysis was observed on laboratory testing and the patient developed severe renal failure with a need for hemodialysis for 2 weeks. Medical history revealed that the patient had been previously exposed to ceftriaxone less than 3 weeks before with subsequent hemolytic reaction. Further causes for hemolytic anemia were excluded and drug-induced immune hemolytic (DIIHA) anemia to ceftriaxone could be confirmed. Conclusions: The case demonstrates the severity of ceftriaxone-induced immune hemolytic anemia, a rare, but immediately life-threatening condition of a frequently used antibiotic in clinical practice. Early and correct diagnosis of DIIHA is crucial, as immediate withdrawal of the causative drug is essential for the patient prognosis. Thus, awareness for this complication must be raised among treating physicians. KW - ceftriaxone KW - drug-induced immune hemolytic anemia KW - hemolysis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176637 VL - 19 IS - 67 ER - TY - JOUR A1 - Müller, P.A. A1 - Bröcker, E.B. A1 - Klinker, E. A1 - Stoevesandt, J. A1 - Benoit, S. T1 - Adjuvant treatment of recalcitrant Bullous pemphigoid with immunoadsorption JF - Dermatology N2 - Elimination of pathogenic autoantibodies by immunoadsorption (IA) has been described as an effective adjuvant treatment in severe bullous autoimmune diseases, especially in pemphigus. There is much less experience in the treatment of bullous pemphigoid (BP). BP was diagnosed in a 62-year-old Caucasian woman presenting a pruritic rash with multiple tense blisters. Standard treatments with topical and oral corticosteroids, steroid-sparing agents including dapsone, azathioprine, mycophenolate mofetil (MMF) and intravenous immunoglobulins were ineffective or had to be discontinued due to adverse events. An immediate clinical response could be achieved by two treatment cycles of adjuvant protein A immunoadsorption (PA-IA) in addition to continued treatment with MMF (2 g/day) and prednisolone (1 mg/kg/day). Tolerance was excellent. Clinical improvement remained stable after discontinuation of IA and went along with sustained reduction of circulating autoantibodies. Our data demonstrate that PA-IA might be a safe and effective adjuvant treatment in severe and recalcitrant BP. KW - Bullous pemphigoid KW - Immunoadsorption KW - Immunoapheresis Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-196620 SN - 1018-8665 SN - 1421-9832 N1 - This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. VL - 224 IS - 3 SP - 224 EP - 227 ER - TY - THES A1 - Hermann, Stephanie T1 - Adenosindiphosphat-vermittelte Funktion und Expression von purinergen Rezeptoren in gewaschenen humanen Thrombozyten T1 - Adenosindiphosphate-mediated function and expression of purinergic receptors in washed platelets N2 - Nach der Präparation von gewaschenen Thrombozyten, einem wichtigen Ausgangsmaterial für die experimentelle Forschung oder für die Transfusionsmedizin, tritt bekannterweise ein zunehmender Verlust der ADP-vermittelten Aggregationsfähigkeit ein. Die verminderte Funktionsfähigkeit von Thromboyzten nach dem Waschvorgang kann somit auch experimentelle Ergebnisse beeinflussten. Allerdings sind die dafür verantwortlichen molekularen Mechanismen bisher nicht aufgeklärt, sodass in dieser Dissertationsarbeit molekulare sowie auch funktionelle Vorgänge untersucht wurden, die zum bekannten Phänomen des raschen Verlustes der ADP-vermittelten Aggregationsfähigkeit gewaschener Thrombozyten führen. Die Wirkung von ADP wird über die drei purinergen Rezeptoren P2Y1, P2X1 und P2Y12 vermittelt wird. Daher wurde zunächst die ADP-induzierte Aggregationsfähigkeit alleine bzw. unter Kostimulation mit Epinephrin oder Serotonin - zwei Induktoren, deren Rezeptoren mit analogen Signalwegen wie die ADP-Rezeptoren P2Y1 bzw. P2Y12 gekoppelt sind - bestimmt. Um Hinweise zu erhalten, wie die Abnahme der ADP-vermittelten Reaktivität von gewaschenen Thrombozyten mit der purinergen Rezeptorexpression und -distribution sowie mit der nachgeschalteten Signalweiterleitung im Zusammenhang steht, wurde zudem die Expression purinerger Rezeptoren auf der Thrombozytenoberfläche bzw. die Konzentration von purinergen Rezeptoren im Zytosol gewaschener Thrombozyten mittels Durchflusszytometrie bzw. ELISA gemessen. Es zeigte sich, dass die Funktion der den purinergen Rezeptoren nachgeschalteten Signalwege während der Lagerungszeit zunehmend beeinträchtigt wird, aber zumindest teilweise erhalten bleibt, wie anhand von Effekten durch Kostimulation mit den Induktoren Epinephrin und Serotonin gezeigt werden konnte. Die Distribution der Rezeptoren zwischen der Thrombozytenoberfläche und den intrazellulären Kompartimenten unterliegt komplexen Prozessen, die induktorabhängig reguliert sind. Eine initiale Zunahme der Expression von ADP-Rezeptoren während der Lagerung von gewaschenen Thrombozyten geht dabei nicht einher mit der Aufrechterhaltung der ADP-induzierten Aggregation. In der Schlussfolgerung ist die fortschreitende Degeneration der ADP-vermittelten Aggregation - neben einem Rückgang der Rezeptorexpression nach mehr als einer Stunde Lagerungszeit - vor allem auf einen funktionellen Verlust der purinergen Rezeptoren zurückzuführen. N2 - Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research, or transfusion medicine. As a well-known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets. Therefore, washing of platelets may affect experimental results. The underlying molecular mechanisms of the rapid loss of ADP-mediated aggregation of washed platelets have not been thoroughly studied. Aim of this dissertation was to elucidate the molecular and functional processes of this phenomenon. ADP mediates its action via three purinergic receptors P2Y1, P2X1 and P2Y12. At first the ADP-induced aggregation of washed platelets was determined by using ADP alone or ADP combined with the stimulators epinephrine or serotonin. Both mediate their effects via the same signaling pathways as ADP but use different receptors. To get information if the reduced responsiveness to ADP in washed platelets is linked with the receptor expression and distribution, the expression and concentration of purinergic receptors on the platelet surface and in the cytosol of washed platelets was measured by using flow cytometry and ELISA. It was shown that the function of the signaling pathways downstream of the purinergic receptors was increasingly impaired during storage of washed platelets. However, it could be at least partially retained by co-stimulation with epinephrine or serotonin. The distribution of receptors between the platelet surface and the intracellular compartments is based on complex processes which are regulated depending on the different stimulators. An initial increase in the expression of ADP receptors during storage of washed platelets was not associated with the maintenance of ADP-induced aggregation. In conclusion, the progressive decrease of ADP-mediated aggregation is - next to a decrease of expression - mainly caused by functional loss of the purinergic receptors. KW - ADP KW - Aggregation KW - G-Protein gekoppelte Rezeptor KW - Purinozeptor KW - Thrombozyt KW - adenosine diphosphate KW - aggregation KW - purinergic receptors KW - g-protein-coupled receptor KW - washed platelets KW - P2Y1 KW - P2Y12 KW - P2X1 KW - ELISA KW - flow cytometry Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-185201 ER -