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

除了它们在止血和血栓形成中的作用之外，有新的证据表明血小板参与免疫应答机制。例如，通常在吞噬细胞或树突细胞上表达的toll样受体（TLR）对于病原体模式的识别是重要的，并且在血小板上也是可检测的。据推测，血小板TLR通过结合细菌病原体或脂多糖（LPS）以及通过嗜中性粒细胞的连续活化参与免疫应答的起始。这样，它们可能支持炎症性疾病或输血相关的不良反应。以前已经证明TLR 2通过src/Syk/磷脂酶C-γ 2信号通路刺激血小板，而TLR 4的作用由促分裂原活化蛋白激酶介导。这两种受体都对核因子κ B调节途径有影响。然而，进一步探索TLR信号传导和与血小板活化的相互作用对于理解TLR在免疫或止血过程中的功能是必需的。由于作为无核细胞的血小板仅发挥有限的从头蛋白质合成，因此由蛋白酶体系统调节的蛋白质降解和周转可能对TLR功能和信号传导具有重要影响。血小板具有蛋白酶体系统的所有组分。最近的研究表明，蛋白酶体抑制与血小板减少症有关，例如在硼替佐米治疗的患者中，以及在小鼠模型中减少血栓形成。血小板活化过程中蛋白酶体活性增加，其作用通过钙蛋白酶和蛋白激酶C介导。TLR功能的调节蛋白酶体系统的上下文中与信号通路尚未系统地研究。因此，在这个项目中，蛋白酶体活性的改变和干扰激活和抑制血小板信号通路在不同水平上进行了研究后，TLR刺激血小板与TLR激动剂LPS或Pam 3CSK 4。通过测量信号传导途径的分子关键标志物和功能测试（如聚集测定法、流式细胞术和流动室实验）评估效果。通过额外使用阻断TLR受体抗体和信号传导途径的特异性抑制剂来确定结果的特异性。此外，通过使用蛋白酶体系统的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.