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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 除了止血之外，已知血小板在免疫应答和炎症过程中发挥作用。与病原体的相互作用涉及病原体识别、颗粒内容物的释放和血小板中的细胞骨架重排。我们已经确定了血小板表面的Toll样受体（TLR）的表达，支持血小板在先天免疫反应中的作用。此外，TLR途径将炎症与动脉粥样硬化联系起来，因为TLR在动脉粥样硬化斑块中表达，并且鼠TLR 2刺激引起增强的动脉粥样硬化。细胞内因子XIIIA（FXIIIA）可在血小板活化时交联血小板细胞骨架蛋白。与免疫应答相关，FXIIIA是单核细胞吞噬过程中细胞骨架重塑所需的，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TLR 2和FXIIIA相关蛋白。在从血小板裂解物中免疫沉淀（IP）TLR 2和FXIIIA，然后进行凝胶分离后，切下蛋白条带，用胰蛋白酶消化并通过质谱法分析。应用蛋白质印迹分析来确认质谱结果。我们重复地直接用TLR 2特异性抗体免疫沉淀FXIIIA，而FXIIIA的IP拉下TLR 2的截短形式，如通过Western印迹鉴定的，其可能对应于TLR 2的可溶形式。通过质谱，我们确定了FXIIIA和FXIIIA相关蛋白：血小板反应蛋白，细丝蛋白和塔林。重要的是，talin以前没有被报道为FXIII底物或相关蛋白。血小板反应蛋白是一种已知的FXIII底物，储存在血小板颗粒中。FXIIIA-IP后的强凝血酶敏感蛋白带和TLR 2-IP后的弱带表明颗粒中部分存在FXIIIA和可溶性TLR 2。有趣的是，FXIIIA似乎与其在静息血小板中的底物相关。总之，我们的研究结果表明血小板中存在已知的和新的FXIIIA相关蛋白，并表明TLR 2和FXIIIA在血小板依赖性免疫应答中的作用。报告这些结果的手稿已被接受发表在血栓形成和止血。

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 .