TY - JOUR A1 - Rosa, Annabelle A1 - Butt, Elke A1 - Hopper, Christopher P. A1 - Loroch, Stefan A1 - Bender, Markus A1 - Schulze, Harald A1 - Sickmann, Albert A1 - Vorlova, Sandra A1 - Seizer, Peter A1 - Heinzmann, David A1 - Zernecke, Alma T1 - Cyclophilin a is not acetylated at lysine-82 and lysine-125 in resting and stimulated platelets JF - International Journal of Molecular Sciences N2 - Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed—neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets. KW - Cyclophilin A KW - acetylation KW - platelets KW - CD147 KW - EMMPRIN Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284011 SN - 1422-0067 VL - 23 IS - 3 ER - TY - THES A1 - Kurz, Hendrikje T1 - Regulation of ion conductance and cAMP/cGMP concentration in megakaryocytes by light T1 - Regulation der Ionenleitfähigkeit und cAMP/cGMP Konzentration in Megakaryozyten durch Licht N2 - Platelets play an essential role in haemostasis. Through granule secretion of second wave mediators and aggregation, they secure vascular integrity. Due to incorrect activation, platelet aggregation and subsequent thrombus formation can cause blood vessel occlusion, leading to ischemia. Patients with defects in platelet production have a low platelet count (thrombocytopenia), which can cause an increased bleeding risk. In vitro platelet generation is still in its development phase. So far, no convincing results have been obtained. For this reason, the health care system still depends on blood donors. Platelets are produced by bone marrow megakaryocytes (MKs), which extend long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Due to shear forces, platelets are then released into the bloodstream. The molecular mechanisms underlying platelet production are still not fully understood. However, a more detailed insight of this biological process is necessary to improve the in vitro generation of platelets and to optimise treatment regimens of patients. Optogenetics is defined as “light-modulation of cellular activity or of animal behaviour by gene transfer of photo-sensitive proteins”. Optogenetics has had a big impact on neuroscience over the last decade. The use of channelrhodopsin 2 (ChR2), a light-sensitive cation channel, made it possible to stimulate neurons precisely and minimally invasive for the first time. Recent developments in the field of optogenetics intend to address a broader scope of cellular and molecular biology. The aim of this thesis is to establish optogenetics in the field of MK research in order to precisely control and manipulate MK differentiation. An existing “optogenetic toolbox“ was used, which made it possible to light-modulate the cellular concentration of specific signalling molecules and ion conductance in MKs. Expression of the bacterial photoactivated adenylyl cyclase (bPAC) resulted in a significant increase in cAMP concentration after 5 minutes of illumination. Similarly, intracellular cGMP concentrations in MKs expressing photoactivated guanylyl cyclase (BeCyclop) were elevated. Furthermore, proplatelet formation of MKs expressing the light-sensitive ion channels ChR2 and anion channelrhodopsin (ACR) was altered in a light-dependent manner. These results show that MK physiology can be modified by optogenetic approaches. This might help shed new light on the underlying mechanisms of thrombopoiesis. N2 - Thrombozyten sind für die primäre Hämostase verantwortlich und unterstützen die Blutgerinnung. Durch ihre Aggregation und die Synthese bzw. Freisetzung von in Granula gespeicherten second wave Mediatoren, sichern sie die Integrität der Blutgefäße. Werden Thrombozyten fälschlicherweise aktiviert, kann es zu einem Gefäßverschluss durch Thrombusbildung mit daraus resultierender Ischämie kommen. Patienten mit einer defekten Thrombozytopoese weisen eine reduzierte Thrombozytenzahl (Thrombozytopenie) auf, die mit einer erhöhten Blutungsneigung assoziiert ist. Bisher gibt es keine überzeugenden Ansätze, die eine Thrombozytenproduktion in vitro ermöglichen. Aus diesem Grund ist das Gesundheitswesen, in der Versorgung der bedürftigen Patienten mit Thrombozytenkonzentraten, auf Blutspender angewiesen. Thrombozyten werden im Knochenmark von ihren Vorläuferzellen, den Megakaryozyten (MKs) produziert. Diese bilden lange zytoplasmatische Fortsätze aus, die Proplättchen genannt werden. Durch die Scherkräfte des Blutstroms in den sinuosoidalen Blutgefäßen, schnüren sich Thrombozyten von den Proplättchen ab. Bisher sind die molekularen Prozesse der Thrombozytenproduktion noch weitgehend unverstanden. Ein besseres Verständnis des Vorgangs ist die Voraussetzung für eine Weiterentwicklung der in vitro Thrombozytengenerierung und einer optimierten Patientenbehandlung. Unter Optogenetik versteht man die Übertragung lichtempfindlicher Proteine in zuvor nicht lichtempfindliche Zellen. Dadurch wird eine nicht-invasive Beeinflussung von Zellvorgängen oder des Verhaltens von Tieren durch Licht ermöglicht. Das Feld der Optogenetik, besonders der lichtempfindliche Kanal Channelrhodopsin 2 (ChR2), hatte einen großen Einfluss auf die neuronale Forschung. Durch ihn war es möglich, Neuronen gezielt nicht-invasiv zu aktivieren und Kreisläufe zu untersuchen. Mittlerweile wurde das Spektrum auf eine Vielzahl von Forschungsgebieten und Zelltypen ausgeweitet. Das Ziel dieser Arbeit ist es, die Methoden der Optogenetik in MKs zu etablieren. Dadurch soll ein Weg gefunden werden, die Megakaryozytenreifung gezielt zu kontrollieren bzw. zu manipulieren. Die bereits vorhandene „optogenetische Toolbox“ wurde verwendet, um die intrazellulären Konzentrationen bestimmter Signalmoleküle und Ionen in MKs zu verändern. Durch die Expression der bakteriellen fotoaktivierbaren Adenylatzyklase (bPAC), wurde die cAMP Konzentration nach 5 min Lichtgabe signifikant erhöht. Ebenfalls ist es durch die Expression der fotoaktivierbaren Guanylatzyklase (BeCyclop) gelungen, die intrazelluläre cGMP Konzentration in MKs durch Belichtung zu erhöhen. Darüber hinaus konnte der Vorgang der Proplättchenformierung in MKs, welche die lichtempfindlichen Ionenkanäle ChR2 und Anion Channelrhodopsin (ACR) exprimierten, durch Licht beeinflusst werden. Die Ergebnisse zeigen, dass eine Beeinflussung der Megakaryozytenphysiologie durch Optogenetik möglich ist. Die Erkenntnisse können dazu beitragen, die Vorgänge der Thrombozytopoese in Zukunft besser zu verstehen. KW - optogenetics KW - megakaryocytes KW - Optogenetik KW - Megakaryozyt KW - proplatelets KW - second messenger Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216947 ER - TY - JOUR A1 - Perrella, Gina A1 - Montague, Samantha J. A1 - Brown, Helena C. A1 - Garcia Quintanilla, Lourdes A1 - Slater, Alexandre A1 - Stegner, David A1 - Thomas, Mark A1 - Heemskerk, Johan W. M. A1 - Watson, Steve P. T1 - Role of tyrosine kinase Syk in thrombus stabilisation at high shear JF - International Journal of Molecular Sciences N2 - Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s\(^{−1}\)). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A\(_2\) (TxA\(_2\)), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen. KW - disaggregation KW - platelet KW - Syk KW - thrombus KW - tyrosine kinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284243 SN - 1422-0067 VL - 23 IS - 1 ER - TY - JOUR A1 - Schanbacher, Constanze A1 - Bieber, Michael A1 - Reinders, Yvonne A1 - Cherpokova, Deya A1 - Teichert, Christina A1 - Nieswandt, Bernhard A1 - Sickmann, Albert A1 - Kleinschnitz, Christoph A1 - Langhauser, Friederike A1 - Lorenz, Kristina T1 - ERK1/2 activity is critical for the outcome of ischemic stroke JF - International Journal of Molecular Sciences N2 - Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke. KW - ERK1/2 KW - tMCAO KW - ischemic stroke KW - RKIP Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-283991 SN - 1422-0067 VL - 23 IS - 2 ER - TY - JOUR A1 - Gupta, Shishir K. A1 - Osmanoglu, Özge A1 - Minocha, Rashmi A1 - Bandi, Sourish Reddy A1 - Bencurova, Elena A1 - Srivastava, Mugdha A1 - Dandekar, Thomas T1 - Genome-wide scan for potential CD4+ T-cell vaccine candidates in Candida auris by exploiting reverse vaccinology and evolutionary information JF - Frontiers in Medicine N2 - Candida auris is a globally emerging fungal pathogen responsible for causing nosocomial outbreaks in healthcare associated settings. It is known to cause infection in all age groups and exhibits multi-drug resistance with high potential for horizontal transmission. Because of this reason combined with limited therapeutic choices available, C. auris infection has been acknowledged as a potential risk for causing a future pandemic, and thus seeking a promising strategy for its treatment is imperative. Here, we combined evolutionary information with reverse vaccinology approach to identify novel epitopes for vaccine design that could elicit CD4+ T-cell responses against C. auris. To this end, we extensively scanned the family of proteins encoded by C. auris genome. In addition, a pathogen may acquire substitutions in epitopes over a period of time which could cause its escape from the immune response thus rendering the vaccine ineffective. To lower this possibility in our design, we eliminated all rapidly evolving genes of C. auris with positive selection. We further employed highly conserved regions of multiple C. auris strains and identified two immunogenic and antigenic T-cell epitopes that could generate the most effective immune response against C. auris. The antigenicity scores of our predicted vaccine candidates were calculated as 0.85 and 1.88 where 0.5 is the threshold for prediction of fungal antigenic sequences. Based on our results, we conclude that our vaccine candidates have the potential to be successfully employed for the treatment of C. auris infection. However, in vivo experiments are imperative to further demonstrate the efficacy of our design. KW - T-cell epitope KW - epitope prediction KW - positive selection KW - evolution KW - immune-informatics Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293953 SN - 2296-858X VL - 9 ER - TY - JOUR A1 - Koo, Chek Ziu A1 - Matthews, Alexandra L. A1 - Harrison, Neale A1 - Szyroka, Justyna A1 - Nieswandt, Bernhard A1 - Gardiner, Elizabeth E. A1 - Poulter, Natalie S. A1 - Tomlinson, Michael G. T1 - The platelet collagen receptor GPVI is cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 molecular scissors JF - International Journal of Molecular Sciences N2 - The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein. KW - ADAM10 KW - GPVI KW - tetraspanin KW - platelet KW - shedding KW - TspanC8 KW - metalloproteinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284468 SN - 1422-0067 VL - 23 IS - 5 ER - TY - JOUR A1 - Bieber, Michael A1 - Schuhmann, Michael K. A1 - Bellut, Maximilian A1 - Stegner, David A1 - Heinze, Katrin G. A1 - Pham, Mirko A1 - Nieswandt, Bernhard A1 - Stoll, Guido T1 - Blockade of platelet glycoprotein Ibα augments neuroprotection in Orai2-deficient mice during middle cerebral artery occlusion JF - International Journal of Molecular Sciences N2 - During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte–platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2\(^{-/-}\)) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα–von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2\(^{-/-}\) mice. During ischemia–reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2\(^{-/-}\) mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke. KW - ischemic penumbra KW - Orai2 KW - glycoprotein receptor Ibα KW - ischemic stroke KW - thrombo-inflammation KW - middle cerebral artery occlusion Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286038 SN - 1422-0067 VL - 23 IS - 16 ER - TY - JOUR A1 - Navarro, Stefano A1 - Starke, Andreas A1 - Heemskerk, Johan W. M. A1 - Kuijpers, Marijke J. E. A1 - Stegner, David A1 - Nieswandt, Bernhard T1 - Targeting of a conserved epitope in mouse and human GPVI differently affects receptor function JF - International Journal of Molecular Sciences N2 - Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6\(^{tg/tg\)). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets. KW - glycoprotein VI KW - JAQ1 KW - platelet receptors KW - platelet activation KW - platelet inhibition Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286227 SN - 1422-0067 VL - 23 IS - 15 ER - TY - JOUR A1 - Butt, Elke A1 - Howard, Cory M. A1 - Raman, Dayanidhi T1 - LASP1 in cellular signaling and gene expression: more than just a cytoskeletal regulator JF - Cells N2 - LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included. KW - LASP1 KW - AKT KW - CXCR4 KW - structure KW - cytoskeleton KW - phosphorylation KW - transcriptional regulation KW - epigenetics KW - nucleus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297447 SN - 2073-4409 VL - 11 IS - 23 ER -