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

Toll 样受体 2 的蛋白质组学

• 批准号: 8170899
• 负责人: JANE E Freedman
• 金额: $ 0.4万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170899
• 关键词: Alpha Granule; Antibodies; Arterial Fatty Streak; Atherosclerosis; Blood Platelets; Computer Retrieval of Information on Scientific Projects Database; Cytoplasmic Granules; Cytoskeletal Proteins; DNA Sequence Rearrangement; Funding; Gel; Grant; Hemostatic function; Immune; Immune response; Immunoprecipitation; Inflammation; Inflammatory; Institution; Link; Manuscripts; Mass Spectrum Analysis; Mediating; Mus; Pathway interactions; Phagocytosis; Platelet Activation; Play; Process; Proteins; Proteomics; Publishing; Reporting; Research; Research Personnel; Resources; Rest; Role; Source; Surface; TLR2 gene; Talin; Thrombosis; Toll-Like Receptor 2; Toll-Like Receptor Pathway; Toll-like receptors; Trypsin; United States National Institutes of Health; Western Blotting; crosslink; filamin; monocyte; novel; pathogen; protein protein interaction

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 was published in Thrombosis and Hemostasis (S Rex et al., Immune versus thrombotic stimulation of platelets differentially regulatessignalling pathways, intracellular protein-protein interactions, and alpha-granule release.Thromb Haemost. 2009, 102, 97-110 ).

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 除止血外，血小板还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识别，颗粒含量的释放以及血小板中的细胞骨架重排。我们已经确定了在血小板表面上的Toll样受体（TLR）的表达，从而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，而鼠TLR2刺激会导致动脉粥样硬化。细胞内因子XIIIA（FXIIIA）可以在血小板激活后交叉链接血小板细胞骨架蛋白。与免疫反应相关的是，单核细胞吞噬过程中的细胞骨架重塑需要FXIIIA，但其在血小板介导的免疫过程中的作用尚不清楚。我们采用了一种蛋白质组学方法来搜索静息血小板中与TLR2和FXIIA相关的蛋白。 TLR2和FXIIIA免疫沉淀（IP）后，从血小板裂解物中进行了凝胶分离后，切除蛋白质带，用胰蛋白酶消化，并通过质谱法分析。应用蛋白质印迹分析以确认质谱结果。我们反复用TLR2特异性抗体直接免疫沉淀的FXIIIA，而FXIIIA的IP降低了TLR2的截断形式，如Western blot所确定的，可能对应于TLR2的可溶形式。根据质谱，我们确定了FXIIIA和FXIIIA相关蛋白：血小板传播，丝蛋白和塔林。重要的是，塔林先前尚未被报道为FXIII-substrate或相关蛋白。血小板传播是一种已知的FXIII底物，存储在血小板颗粒中。在FXIIIA-IP之后，TLR2-IP后的弱带后，强烈的血小板传播表明FXIIIA和可溶性TLR2在颗粒中存在部分存在。有趣的是，FXIIIA似乎与其静止血小板中的底物有关。总之，我们的结果表明血小板中存在已知和新型FXIIIA相关蛋白，并提出了TLR2和FXIIIA在血小板依赖性免疫反应中的作用。报道这些结果的手稿在血栓形成和止血中发表（S REX等人，血小板的免疫与血小板刺激差异化调节途径，细胞内蛋白质 - 蛋白质相互作用以及α细晶粒球状释放。