Janus kinase inhibitors ruxolitinib and baricitinib impair glycoprotein-VI mediated platelet function

Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses <i>in vitro</i> in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin α_IIbβ₃ activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function <i>in vitro</i> and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.近年来已有多款贾纳斯激酶（Janus kinase, JAK）抑制剂（jakinibs）获批用于治疗炎症性、自身免疫性及血液系统疾病。尽管JAK与下游信号转导与转录激活因子（signal transducer and activator of transcription, STAT）蛋白在血小板中的功能已逐步被阐明，但JAK抑制剂是否会影响血小板功能仍未明确。 本研究针对5种临床相关的jakinibs，包括鲁索替尼（ruxolitinib）、乌帕替尼（upadacitinib）、奥克拉替尼（oclacitinib）、巴瑞替尼（baricitinib）与托法替布（tofacitinib），对血小板的体外（in vitro）生化与生理应答进行了系统性表征。 流式细胞术、显微镜成像及其他检测实验结果显示，强效JAK1/2抑制剂巴瑞替尼与鲁索替尼可降低血小板对胶原的黏附能力，同时可抑制糖蛋白VI（glycoprotein VI, GPVI）激动剂胶原相关肽（collagen-related peptide, CRP-XL）诱导的血小板聚集、颗粒分泌及整合素α_IIbβ₃活化。 蛋白质印迹法（Western blot）分析表明，jakinibs可降低GPVI活化后Akt的磷酸化水平与活化状态，其中鲁索替尼与巴瑞替尼还可阻断DAPP1的磷酸化。与之相反，jakinibs对凝血酶诱导的血小板应答无显著影响。 GPVI与JAK信号通路抑制剂同样可阻断CRP-XL刺激后血小板的STAT5磷酸化。后续药理学实验进一步证实，STAT5在血小板颗粒分泌、整合素活化及细胞骨架应答过程中发挥关键调控作用。 综上，本研究结果证实鲁索替尼与巴瑞替尼在体外（in vitro）可抑制血小板功能，并支持JAK/STAT5通路在GPVI/免疫受体酪氨酸激活基序（ITAM）介导的血小板功能调控中的核心作用。