Proteasome-mediated regulation of toll-like receptor function in human platelets

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In addition to their role in hemostasis and thrombus formation, there is emerging evidence that platelets are involved in immune response mechanisms. For example, toll-like receptors (TLRs), usually expressed on phagocytes or dendritic cells, are important for the recognition of pathogen patterns and also detectable on platelets. Presumably, platelet TLRs participate in the initiation of immune responses by binding bacterial pathogens or lipopolysaccharides (LPS) and by consecutive activation of neutrophils. In that way, they may support inflammatory diseases or transfusion related adverse reactions. It has been previously demonstrated that TLR 2 stimulates platelets via the src/Syk/phospholipase C-gamma2 signaling pathway, whereas TLR 4 effects are mediated by the mitogen-activated protein kinase. Both receptors have influence on nuclear factor kappa-B regulated pathways. However, further exploration of TLR signaling and interaction with platelet activation is mandatory for the understanding of TLR function in immunological or hemostatic processes. Since platelets as anucleated cells exert only limited de-novo protein synthesis, protein degradation and turnover regulated by the proteasome system may have essential effects on TLR function and signaling. Platelets possess all components of the proteasome system. And in recent studies, it was shown that proteasome inhibition is associated with thrombocytopenia, e.g. in patients under bortezomib treatment, and reduced thrombus formation in mouse models. Proteasome activity increases during platelet activation and effects are mediated via Calpain and proteinkinase C. The regulation of TLR function by the proteasome system in context with signaling pathways has not been systematically investigated. Therefore, in this project, alterations of the proteasome activity and the interference with activating and inhibitory platelet signaling pathways on different levels are investigated after TLR stimulation of platelets with the TLR agonists LPS or Pam3CSK4. The effects are assessed by the measurement of molecular key markers for signaling pathways and functional tests like aggregometry, flow cytometry and flow chamber experiments. The specificity of the results is ascertained by additional use of blocking TLR receptor antibodies and specific inhibitors of signaling pathways. Furthermore, the significance of the proteasome activity for TLR stimulation is proved by using inhibitors of the proteasome system. Novel insights into immune responses triggered by platelets will help to understand mechanisms contributing to inflammatory processes or transfusion related adverse reactions.