The Heart Research Institute, Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia.
Central Clinical School, The University of Sydney, Sydney, New South Wales, Australia.
Mol Cell Proteomics. 2024 Feb;23(2):100717. doi: 10.1016/j.mcpro.2024.100717. Epub 2024 Jan 16.
Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.
血小板激活会诱导促进血小板聚集和炎症的蛋白质分泌。然而，由于血小板在分离过程中容易预激活，以及缺乏用于低丰度释放物分析的敏感方案，因此详细分析释放的血小板蛋白质组受到了阻碍。在这里，我们详细介绍了迄今为止对血小板释放物蛋白质组最敏感的分析，检测到超过 1300 种蛋白质。在释放物蛋白中的翻译后修饰的无偏扫描突出了 O-糖基化作为主要成分。我们首次在以前未表征的位点上检测到 O-岩藻糖基化，包括多聚蛋白 1（MMRN1），它是一种主要的α颗粒蛋白，支持血小板与胶原的黏附，也是血小板因子 V 的载体。MMRN1 的弹性微纤维界面（EMI）结构域的 N 端，一个关键的蛋白-蛋白相互作用位点，在一个新的结构域环境中，在保守的苏氨酸上发生了 O-岩藻糖基化。我们的数据表明，蛋白质 O-岩藻糖基转移酶 1 或一种新的蛋白质 O-岩藻糖基转移酶可能负责这种修饰。在 EMI 结构域上突变这个 O-岩藻糖基化位点导致 MMRN1 分泌减少了 >50%，支持 EMI O-岩藻糖基化在 MMRN1 分泌中的关键作用。通过比较静止和凝血酶处理的血小板的释放物，发现 202 种蛋白质在高剂量凝血酶刺激后显著释放。血小板裂解物的补充定量鉴定出超过 3800 种蛋白质，通过相关性分析证实了释放物蛋白的血小板来源。低剂量凝血酶处理产生了较少的显著调节蛋白质，并且分泌途径酶也较少。这里提供的广泛的血小板蛋白质组资源（larancelab.com/platelet-proteome）允许鉴定出针对药物靶向的新型调节机制，以解决血小板功能障碍和血栓形成。
