血小板RIP2的功能及吉非替尼抗血小板作用机制的研究

Predominantly expressed in macrophages, RIP2 (receptor-interacting protein kinase 2) plays a pivotal role in innate immunity. We recently found that platelets express robust RIP2 with function unknown (Circulation 2015)..Gefitinib (Iressa) is an EGFR tyrosine kinase inhibitor clinically used for NSCLC (non-small cell lung cancer) bearing activating mutation of EGFR. As a target-directed anticancer drug (non-cytotoxic agent), gefitinib is generally well-tolerated. Gefitinib-related hemorrhage was reported with unknown mechanism. At nanomolar concentrations, gefitinib was found to inhibit RIP2 tyrosine phosphorylation in nucleated cells (Genes Dev 2010). We hypothesize that gefitinib may inhibit platelet activation as a RIP2 tyrosine kinase inhibitor and thus contribute to the bleeding events clinically. Our preliminary studies shows that 1) at 10 - 100 nM, gefitinib concentration-dependently inhibited human platelet aggregation induced by multiple agonists; 2) at 100 nM, gefitinib abolished thrombin-induced RIP2 tyrosine phosphorylation in platelets; 3) gefitinib orally given to mice prolonged bleeding time. We therefore propose that 1) platelet RIP2 plays an essential role in platelet activation, thrombosis, and hemostasis; 2) gefintib-induced bleeding results from platelet inhibition by gefitinib as a RIP2 tyrosine kinase inhibitor. We will explore the role of RIP2 in platelet activation, thrombosis, and hemostasis using RIP2 knockout mice. We will also investigate the role of gefitinib on platelet activation, thrombosis, and hemostasis using platelets from healthy donors (in vitro) and patients on gefitinib treatment (ex vivo). Mice (wild type and RIP2 knockout) dosed with and without gefitinib will also be used to study the role of gefitinib. RIP2 phosphorylation (Ser176 and Tyr) and downstream signal pathways (NFκB, MAPK, TXA2 et al) will be explored to elicit the mechanism of RIP2 in platelet activation and the antiplatelet effects of gefitinib..These studies will clarify the role of platelet RIP2 and the mechanism of gefitinib-induced bleeding. It will also shed novel insight into the prevention of gefitinib-related bleeding events as well as the use of gefitinib as a potential antiplatelet agent targeting RIP2.

受体相互作用蛋白激酶2 (RIP2) 主要在白细胞表达，在先天免疫中发挥重要作用。我们发现血小板表达高水平的RIP2，可能参与血小板活化(Circulation 2015)。EGFR酪氨酸激酶抑制剂吉非替尼临床上有出血副作用，原因不明。吉非替尼有RIP2酪氨酸激酶抑制剂活性。我们发现明显低于临床有效血药浓度的吉非替尼抑制血小板聚集和血小板RIP2酪氨酸磷酸化，提示吉非替尼可能通过抑制血小板RIP2发挥抗血小板作用。拟主要用RIP2 -/-小鼠研究：1) RIP2在血小板激活、血栓形成中的作用及机制；2) 吉非替尼抗血小板作用与RIP2的关系、机制; 3) 吉非替尼的抗血栓作用及机制。本研究将在RIP2功能、血小板激活、血栓形成的机制、吉非替尼的作用(明确其出血副作用的原因)方面提供许多非常重要的新知识, 并为吉非替尼出血副作用的防治、作为抗血小板药的临床应用提供重要的理论和实验依据。

受体相互作用蛋白2（RIP2）激酶作为包括NOD2在内的模式识别受体下游调控分子，在炎症与先天免疫中发挥重要作用。我们发现血小板表达高水平的RIP2，但其在血小板活化、血栓和心梗中作用仍不清楚。我们的研究发现血小板缺失RIP2后，增强了GPIb和GPVI受体活化所引起的致密颗粒释放，加速了血小板在动脉剪切力下的黏附。相应地，RIP2抑制剂WEHI-345抑制RIP2磷酸化，促进了凝血酶和胶原引起的血小板致密颗粒释放。RIP2缺失所引起的血小板功能增强，进一步导致了小鼠断尾出血时间缩短与三氯化铁诱导的肠系膜动脉血栓形成加快。值得注意的是，我们在冠心病病人与动脉粥样硬化的模型小鼠中均发现血小板RIP2的表达下降，同时RIP2缺失加重了小鼠缺血再灌注模型的严重程度。机制上，我们发现血小板表达细胞分化蛋白8（DOCK8），而磷酸化的RIP2能够通过抑制DOC8调控的Cdc42活化，进而抑制血小板致密颗粒释放。我们的研究阐明了血小板RIP2在血小板过度活化、血栓和心梗中扮演着保护性分子的角色。同时我们揭示了磷酸化RIP2-DOCK8-Cdc42这一全新通路，并发现其能负向调控GPIb和GPVI受体激活引起的血小板活化。这些结果提示血小板RIP2可能是动脉血栓性疾病，包括早期动脉粥样硬化发生发展，动脉血栓以及心梗所共同的潜在治疗靶点。该部分的研究结果已经投稿Blood, 外审中（BLD-2020-010364, under review）。