Regulation and function of the proteasome in human platelets

Even though platelets are the smallest cells in circulating blood, they play an integral role in blood clotting where they are activated, adhere to the vessel wall, and contribute to hemostasis. But over the years it was discovered that those anucleate cells have more extended functions. They organize their cellular vitality similar to nucleated cells and have an active protein metabolism performing protein de novo synthesis as well as protein degradation. One of the main degradation systems in cells is the proteasome. Besides protein quality control, the proteasome is involved in important cellular processes like cell survival, transcription, development, selective elimination of abnormal proteins and antigen processing. A dysregulation of this multicatalytic protein complex leads to various disease developments. Proteasome inhibitors, for instance, have been studied for treating cancer. Platelets like nucleated cells contain a proteasome. However, the impact of the proteasome on platelet functions remains poorly investigated until today. A better knowledge of signaling pathways in platelets aids in understanding how alterations in proteasome functions affect platelet-mediated processes and diseases. This study confirms the existence of a functional proteasome in human platelets and illustrates an important role in platelet biology, as well as sepsis. With this study the role of the proteasome in anucleate platelets is demonstrated in more detail and a signaling pathway regulating its activity was observed. Here, the proteasome in platelets is linked to platelet aggregation. First, proteasome inhibitors epoxomicin and bortezomib reduce ADP- and collagen-induced aggregation. Furthermore, the 26S chymotrypsin-like activity of the proteasome is enhanced when platelets are incubated with the platelet agonist collagen. Additionally, cytoskeletal proteins Filamin A and Talin-1, which are crucial for platelet activation, were identified as proteasome substrates and increased cleavage of these proteins occurs with proteasome activation. To investigate possible mechanisms of regulating the proteasome, the signaling pathway related to NFκB was analyzed under platelet agonist treatment. The NFκB pathway, that mediates aggregation, is initiated when platelets are treated with collagen and the inhibitory protein of NFκB, IκBα, is degraded in collagen-stimulated platelets. More interestingly, NFκB inhibitors prevent collagen-stimulated enhancement of the proteasome activity. In return the connection of the proteasome and the NFκB pathway is further demonstrated as NFκB inhibitors restrict cleavage of the proteasome substrate Talin-1. These results propose a novel pathway that involves the proteasome and that is in return connected with non-genomic functions of NFκB in regulating platelet aggregation. In a second part this work shows for the first time that mitochondrial membrane depolarization in platelets correlates with the disease course and disease severity in patients with sepsis. Additionally, during these studies increased proteasome activity was observed in sepsis patients compared to control patients and pathogenic bacteria intensified the 26S trypsin-like activity of human platelets. Therefore, molecular markers of platelet vitality may be valuable parameters to help evaluating the clinical outcome of sepsis patients. In summary, the study confirms the existence of a functional proteasome in human platelets, contributes to our understanding how the proteasome affects platelet functions such as aggregation and how this may be regulated on a molecular basis. Furthermore, it allows for new insights in the disease course of sepsis and identifies new molecular markers for assessing the disease severity and clinical outcome of sepsis patients

Platelet mitochondrial membrane depolarization reflects disease severity in patients with sepsis and correlates with clinical outcome

Introduction: Sepsis is still a leading cause of morbidity and mortality, even in modern times, and thrombocytopenia has been closely associated with unfavorable disease outcome. Decreases in mitochondrial membrane potential (depolarization) were found in different tissues during sepsis. Previous work suggests that mitochondrial dysfunction of platelets correlates with clinical disease activity in sepsis. However, platelet mitochondrial membrane potential (Mmp) has not been investigated in a clinical follow-up design and not with regard to disease outcome. Methods: In this study, platelet mitochondrial membrane depolarization was assessed by means of a fluorescent Mmp-Index with flow cytometry in 26 patients with sepsis compared with control patients. Platelet Mmp-Index on admission was correlated with the clinical disease scores Acute Physiology and Chronic Health Evaluation Score II (APACHE II), Sequential Organ Failure Score (SOFA), and Simplified Acute Physiology Score II (SAPS II). Finally, platelet Mmp-Index on admission and follow-up were compared in the group of sepsis survivors and nonsurvivors. Expression of the prosurvival protein Bcl-xL in platelets was quantified by immunoblotting. Results: Platelet mitochondrial membrane depolarization correlated significantly with the simultaneously assessed clinical disease severity by APACHE II (r = -0.867; P < 0.0001), SOFA (r = -0.857; P < 0.0001), and SAPS II score (r = -0.839; P < 0.0001). Patients with severe sepsis showed a significant reduction in platelet Mmp-Index compared with sepsis without organ failure (0.18 (0.12 to 0.25) versus 0.79 (0.49 to 0.85), P < 0.0006) or with the control group (0.18 (0.12 to 0.25) versus 0.89 (0.68 to 1.00), P < 0.0001). Platelet Mmp-Index remained persistently low in sepsis nonsurvivors (0.269 (0.230 to 0.305)), whereas we observed recovery of platelet Mmp-Index in the survivor group (0.9 (0.713 to 1.017)). Furthermore, the level of prosurvival protein Bcl-xL decreased in platelets during severe sepsis. Conclusion: In this study, we demonstrated that mitochondrial membrane depolarization in platelets correlates with clinical disease severity in patients with sepsis during the disease course and may be a valuable adjunct parameter to aid in the assessment of disease severity, risk stratification, and clinical outcome

Regulation and function of the proteasome in human platelets

Even though platelets are the smallest cells in circulating blood, they play an integral role in blood clotting where they are activated, adhere to the vessel wall, and contribute to hemostasis. But over the years it was discovered that those anucleate cells have more extended functions. They organize their cellular vitality similar to nucleated cells and have an active protein metabolism performing protein de novo synthesis as well as protein degradation. One of the main degradation systems in cells is the proteasome. Besides protein quality control, the proteasome is involved in important cellular processes like cell survival, transcription, development, selective elimination of abnormal proteins and antigen processing. A dysregulation of this multicatalytic protein complex leads to various disease developments. Proteasome inhibitors, for instance, have been studied for treating cancer. Platelets like nucleated cells contain a proteasome. However, the impact of the proteasome on platelet functions remains poorly investigated until today. A better knowledge of signaling pathways in platelets aids in understanding how alterations in proteasome functions affect platelet-mediated processes and diseases. This study confirms the existence of a functional proteasome in human platelets and illustrates an important role in platelet biology, as well as sepsis. With this study the role of the proteasome in anucleate platelets is demonstrated in more detail and a signaling pathway regulating its activity was observed. Here, the proteasome in platelets is linked to platelet aggregation. First, proteasome inhibitors epoxomicin and bortezomib reduce ADP- and collagen-induced aggregation. Furthermore, the 26S chymotrypsin-like activity of the proteasome is enhanced when platelets are incubated with the platelet agonist collagen. Additionally, cytoskeletal proteins Filamin A and Talin-1, which are crucial for platelet activation, were identified as proteasome substrates and increased cleavage of these proteins occurs with proteasome activation. To investigate possible mechanisms of regulating the proteasome, the signaling pathway related to NFκB was analyzed under platelet agonist treatment. The NFκB pathway, that mediates aggregation, is initiated when platelets are treated with collagen and the inhibitory protein of NFκB, IκBα, is degraded in collagen-stimulated platelets. More interestingly, NFκB inhibitors prevent collagen-stimulated enhancement of the proteasome activity. In return the connection of the proteasome and the NFκB pathway is further demonstrated as NFκB inhibitors restrict cleavage of the proteasome substrate Talin-1. These results propose a novel pathway that involves the proteasome and that is in return connected with non-genomic functions of NFκB in regulating platelet aggregation. In a second part this work shows for the first time that mitochondrial membrane depolarization in platelets correlates with the disease course and disease severity in patients with sepsis. Additionally, during these studies increased proteasome activity was observed in sepsis patients compared to control patients and pathogenic bacteria intensified the 26S trypsin-like activity of human platelets. Therefore, molecular markers of platelet vitality may be valuable parameters to help evaluating the clinical outcome of sepsis patients. In summary, the study confirms the existence of a functional proteasome in human platelets, contributes to our understanding how the proteasome affects platelet functions such as aggregation and how this may be regulated on a molecular basis. Furthermore, it allows for new insights in the disease course of sepsis and identifies new molecular markers for assessing the disease severity and clinical outcome of sepsis patients

Zeitsprünge. Forschungen zur Frühen Neuzeit

Studies in Early Modern History, Culture and Science: Since its foundation by Klaus Reichert in 1997, Zeitsprünge has offered a forum for interdisciplinary early modern research in Europe. The editors welcome contributions concerning the history of ideas, knowledge, science, literature, art, and culture that are historically systematic while pursuing questions that transcend their respective disciplinary boundaries. We welcome contributions that shed new light on their respective field of research by both daring to take 'tiger leaps into the past' (Walter Benjamin) and critically glancing into the present and the future. Publication languages are German, English, French, Italian and Spanish). Articles in other languages may be considered after consultation with the editors. Zeitsprünge publishes open issues and thematic volumes as well as, less commonly, editions and monographs. Starting in 2022, subscriptions will be available in print and online (campus license). Zeitsprünge uses double-blind peer review process for all submitted manuscripts