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.

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.

Atherosclerotic lesions are populated by cells of the innate and adaptive immune system, including CD8\(^+\) T cells. The CD8\(^+\) T cell infiltrate has recently been characterized in mouse and human atherosclerosis and revealed activated, cytotoxic, and possibly dysfunctional and exhausted cell phenotypes. In mouse models of atherosclerosis, antibody-mediated depletion of CD8\(^+\) T cells ameliorates atherosclerosis. CD8\(^+\) T cells control monopoiesis and macrophage accumulation in early atherosclerosis. In addition, CD8\(^+\) T cells exert cytotoxic functions in atherosclerotic plaques and contribute to macrophage cell death and necrotic core formation. CD8\(^+\) T cell activation may be antigen-specific, and epitopes of atherosclerosis-relevant antigens may be targets of CD8\(^+\) T cells and their cytotoxic activity. CD8\(^+\) T cell functions are tightly controlled by costimulatory and coinhibitory immune checkpoints. Subsets of regulatory CD25\(^+\)CD8\(^+\) T cells with immunosuppressive functions can inhibit atherosclerosis. Importantly, local cytotoxic CD8\(^+\) T cell responses may trigger endothelial damage and plaque erosion in acute coronary syndromes. Understanding the complex role of CD8\(^+\) T cells in atherosclerosis may pave the way for defining novel treatment approaches in atherosclerosis. In this review article, we discuss these aspects, highlighting the emerging and critical role of CD8\(^+\) T cells in atherosclerosis.

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.

Background Coronavirus disease 2019 (COVID-19) associated coagulopathy (CAC) leads to thromboembolic events in a high number of critically ill COVID-19 patients. However, specific diagnostic or therapeutic algorithms for CAC have not been established. In the current study, we analyzed coagulation abnormalities with point-of-care testing (POCT) and their relation to hemostatic complications in patients suffering from COVID-19 induced Acute Respiratory Distress Syndrome (ARDS). Our hypothesis was that specific diagnostic patterns can be identified in patients with COVID-19 induced ARDS at risk of thromboembolic complications utilizing POCT. Methods This is a single-center, retrospective observational study. Longitudinal data from 247 rotational thromboelastometries (Rotem®) and 165 impedance aggregometries (Multiplate®) were analysed in 18 patients consecutively admitted to the ICU with a COVID-19 induced ARDS between March 12th to June 30th, 2020. Results Median age was 61 years (IQR: 51–69). Median PaO2/FiO2 on admission was 122 mmHg (IQR: 87–189), indicating moderate to severe ARDS. Any form of hemostatic complication occurred in 78 % of the patients with deep vein/arm thrombosis in 39 %, pulmonary embolism in 22 %, and major bleeding in 17 %. In Rotem® elevated A10 and maximum clot firmness (MCF) indicated higher clot strength. The delta between EXTEM A10 minus FIBTEM A10 (ΔA10) > 30 mm, depicting the sole platelet-part of clot firmness, was associated with a higher risk of thromboembolic events (OD: 3.7; 95 %CI 1.3–10.3; p = 0.02). Multiplate® aggregometry showed hypoactive platelet function. There was no correlation between single Rotem® and Multiplate® parameters at intensive care unit (ICU) admission and thromboembolic or bleeding complications. Conclusions Rotem® and Multiplate® results indicate hypercoagulability and hypoactive platelet dysfunction in COVID-19 induced ARDS but were all in all poorly related to hemostatic complications..