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。

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.