PROTEOMICS OF TOLL-LIKE RECEPTOR 2 & INTRACELLULAR FACTOR XIIIA IN PLATELETS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 除了止血，我们还知道血小板在免疫反应和炎症过程中发挥作用。与病原体的相互作用涉及病原体的识别、颗粒内容物的释放和血小板中的细胞骨架重排。我们已经鉴定了血小板表面Toll样受体(TLR)的表达，支持血小板在先天性免疫反应中发挥作用。此外，TLR途径将炎症与动脉粥样硬化联系起来，因为TLRs在动脉粥样硬化斑块中表达，而小鼠TLR2刺激导致动脉粥样硬化加剧。细胞内因子XIIIA(FXIIIA)可在血小板活化时使血小板细胞骨架蛋白发生交联。与免疫反应相关，FXIIIA是单核细胞吞噬过程中细胞骨架重塑所必需的，但其在血小板介导的免疫过程中的作用尚不清楚。我们应用蛋白质组学方法在静息血小板中寻找TLR2和FXIIIA相关蛋白。从血小板裂解液中免疫沉淀TLR2和FXIIIA后，凝胶分离，切下蛋白条带，胰酶消化后进行质谱分析。用Western印迹分析对质谱学结果进行验证。我们反复用TLR2特异性抗体直接免疫沉淀FXIIIA，而FXIIIA的IP经Western印迹鉴定，下调了TLR2的截短形式，可能与TLR2的可溶性形式相对应。通过质谱分析，我们鉴定了FXIIIA和FXIIIA相关蛋白：凝血酶原蛋白、细丝蛋白和Talin。重要的是，Talin之前还没有作为FXIII底物或相关蛋白的报道。凝血酶敏感蛋白是一种已知的FXIII底物，储存在血小板颗粒中。FXIIIA-IP后有一条较强的凝血酶原蛋白条带，TLR2-IP后有一条较弱的条带，提示FXIIIA和可溶性TLR2部分存在于颗粒中。有趣的是，FXIIIA似乎与其在静息血小板中的底物有关。总之，我们的结果表明在血小板中存在已知的和新的FXIIIA相关蛋白，并提示TLR2和FXIIIA在血小板依赖的免疫反应中发挥作用。报道这些结果的手稿已被接受发表在《血栓与止血》杂志上。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Beside hemostasis, platelets are known to play a role in immune responses and inflammatory processes. Interaction with pathogens involves pathogen recognition, release of granule-content, and cytoskeletal rearrangements in the platelet. We have identified the expression of toll-like receptors (TLR) on the platelet surface, supporting a role for the platelet in innate immune responses. In addition, the TLR pathway links inflammation to atherosclerosis, as TLRs are expressed in atherosclerotic plaques and murine TLR2 stimulation causes enhanced atherosclerosis. Intracellular Factor XIIIA (FXIIIA) can crosslink platelet cytoskeletal proteins upon platelet activation. Relevant to the immune response, FXIIIA is required for cytoskeletal remodeling during monocyte phagocytosis, but its role in platelet-mediated immune processes is unknown. We have applied a proteomics approach to search for TLR2- and FXIIIA-associated proteins in resting platelets. After immunoprecipitation (IP) of TLR2 and FXIIIA from platelet lysates followed by gel separation, protein bands were excised, digested with trypsin and analysed by mass spectrometry. Western blot analysis was applied to confirm the mass spectrometric results. We repeatedly immunoprecipitated FXIIIA directly with TLR2-specific antibodies, whereas the IP of FXIIIA pulled down a truncated form of TLR2, as identified by Western blot, that may correspond to the soluble form of TLR2. By mass spectrometry, we identified FXIIIA and FXIIIA-associated proteins: thrombospondin, filamin and talin. Importantly, talin has not been previously reported as FXIII-substrate or associated protein. Thrombospondin, a known FXIII substrate, is stored in platelet granules. A strong thrombospondin-band after FXIIIA-IP and a weak band after TLR2-IP suggests the partial presence of FXIIIA and soluble TLR2 in granules. Interestingly, FXIIIA appears to be associated with its substrates in the resting platelet. In conclusion, our results indicate the presence of known and novel FXIIIA-associated proteins in platelets and suggests a role for TLR2 and FXIIIA in platelet-dependent immune responses. A manuscript reporting these results has been accepted for publication in Thrombosis and Hemostasis .