Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36.

特异性氧化磷脂（oxPCCD36）可通过清道夫受体CD36（scavenger receptor CD36）促进高脂血症状态下的血小板高反应性与血栓形成，但oxPCCD36在血小板中诱导的信号通路尚未得到明确阐释。本研究采用基于质谱的酪氨酸、丝氨酸及苏氨酸磷酸化蛋白质组学技术，对oxPCCD36与强效生理激动剂凝血酶诱导的血小板信号通路开展无偏分析。oxPCCD36与凝血酶分别诱导了115种蛋白（162个磷酸化位点）与181种蛋白（334个磷酸化位点）的差异磷酸化修饰。两种激动剂诱导的多数磷酸化蛋白质组变化此前均未在血小板中被报道，因此可为血小板信号通路研究提供候选靶点。研究人员通过对蛋白质磷酸化依赖应答的生物信息学分析，对（去）磷酸化的偏好基序进行分类、预测信号通路与激酶活性，并构建了调控整合素激活的磷酸化蛋白质组网络。本研究在oxPCCD36激活的血小板中鉴定出一条涉及Src家族激酶、SYK与PLCγ2的潜在信号通路。后续在人源血小板中开展的离体实验证实，该通路位于清道夫受体CD36的下游，且对oxPCCD36介导的血小板激活至关重要。本研究结果为血小板激活机制，尤其是oxPCCD36对血小板的调控机制提供了多项新见解。