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

除了在止血和血栓形成中的作用外，越来越多的证据表明血小板还参与免疫反应机制。例如，toll样受体（TLRs）通常在吞噬细胞或树突状细胞上表达，对识别病原体模式很重要，也可以在血小板上检测到。据推测，血小板tlr通过结合细菌病原体或脂多糖（LPS）和连续激活中性粒细胞参与免疫应答的启动。这样，它们可能支持炎症性疾病或与输血相关的不良反应。先前已经证明，tlr2通过src/Syk/磷脂酶C-gamma2信号通路刺激血小板，而tlr4的作用是通过丝裂原活化蛋白激酶介导的。这两种受体都对核因子κ b调控的途径有影响。然而，进一步探索TLR信号及其与血小板活化的相互作用对于了解TLR在免疫或止血过程中的功能是必要的。由于血小板作为无核细胞仅发挥有限的de-novo蛋白合成，由蛋白酶体系统调节的蛋白降解和转换可能对TLR功能和信号传导有重要影响。血小板具有蛋白酶体系统的所有组成部分。最近的研究表明，蛋白酶体抑制与血小板减少有关，例如在接受硼替佐米治疗的患者中，并在小鼠模型中减少血栓形成。在血小板活化过程中，蛋白酶体活性增加，其作用是通过钙蛋白酶和蛋白激酶c介导的。蛋白酶体系统在信号通路背景下对TLR功能的调节尚未得到系统的研究。因此，本项目将研究TLR激动剂LPS或Pam3CSK4刺激血小板后，蛋白酶体活性的变化以及对活化和抑制血小板信号通路的不同程度的干扰。通过测量信号通路的分子关键标记物和功能测试（如聚集学、流式细胞术和流室实验）来评估其效果。结果的特异性是通过额外使用阻断TLR受体抗体和信号通路的特异性抑制剂来确定的。此外，通过使用蛋白酶体系统抑制剂证明了蛋白酶体活性对TLR刺激的重要性。对血小板引发的免疫反应的新见解将有助于理解炎症过程或输血相关不良反应的机制。

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.