Regulation and function of the proteasome in human platelets

Gründler, Katharina (2015): Regulation and function of the proteasome in human platelets. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: platelets, proteasome, NFkB, sepsis
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Mai 2015
• 1. Bericht­erstatter:in: Zahler, Stefan