受体相互作用蛋白3在调控血小板参与止血与血栓形成过程中的作用及其机制研究

Receptor-interacting protein kinase 3 (RIP3) is vital for regulating necroptosis, but its role in thrombosis and hemostasis remains unclear. In our previous studies, using RIP3-deficient (RIP3-/-) mice, we found RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild-type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. Wild-type mice repopulated with RIP3-/- bone marrow-derived cells had longer occlusion times than RIP3-/- mice repopulated with wild-type bone marrow-derived cells, suggesting a role of RIP3-deficient platelet in thrombosis. In consistent with these findings, we found that RIP3 was expressed in both human and mice platelets. ATP release and aggregation in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619, were attenuated in the platelets lacking RIP3, and addition of exogenous ADP rescued the reduced aggregation. Finally, we found RIP3 was associated with Gα13 in both resting and activated platelets. RIP3 deficiency impaired U46619- or thrombin-induced integrin activation. Moreover, RGDS peptides abolished agonist-induced ATP secretion, and platelet spreading on fibrinogen in the presence of thrombin was markedly reduced in the deficiency of RIP3. In addition, we found RIP3 involves in regulating the function of glycoprotein (GP) Ib-IX. These data demonstrate a novel role for RIP3 in amplifying platelet activation by mediating ADP secretion and integrin signaling in vitro, and in promoting thrombus formation and hemostasis in vivo. However, although the roles of RIP3 in platelet activation and in vivo thrombosis and hemostasis have been confirmed, the mechanisms remain unclear. In the current study, we will further explore the mechanisms of RIP3 in regulating platelet secretion, the GP Ib-IX signaling, and integrin activation. The findings will verify that RIP3 plays an important role in platelet activation which should be physiologically or pathophysiologically significant. RIP3 may represent a novel signaling model leading to platelet activation and a potential new target for antithrombotic strategy.

蛋白激酶受体相互作用蛋白3（Receptor-interacting protein 3，RIP3）在坏死性凋亡中的关键作用已被证实，但其在止血与血栓形成中是否有作用尚未见报道。申请人使用RIP3基因敲除小鼠研究发现，RIP3在血栓形成和止血过程中起着关键作用，初步研究证实，RIP3通过调节血小板ADP的释放，调控凝血酶和血栓素A2受体介导的血小板功能，但其机制尚未知；研究还提示，RIP3可能参与血小板整合素和膜糖蛋白Ib-IX功能的调控。本研究，将揭示RIP3调节ADP释放的机制；探明RIP3调控整合素和膜糖蛋白Ib-IX受体功能及其机制。因此，本研究将揭示RIP3在血小板参与止血与血栓形成过程中的作用和机制，从而提出一种新的血小板功能调控模式。不仅对血小板相关基础研究，而且，对于揭示血栓与出血性疾病发病机制，建立新的诊断、预防方法具有重要价值，还将为研制新的抗血栓药物提供新的靶点。

蛋白激酶受体相互作用蛋白3（Receptor-interacting protein 3，RIP3）在坏死性凋亡中的关键作用已被证实，但其在止血与血栓形成中是否有作用尚未见报道。本项目中申请人研究发现，RIP3基因敲除（RIP3-/-）小鼠尾出血时间较野生型（WT）小鼠显著延长，FeCl3诱导的RIP3-/-小鼠微动脉阻塞时间较WT小鼠延迟，而且小鼠骨髓移植实验显示，RIP3-/-→RIP3-/-（供体移植给受体）及RIP3-/-→WT小鼠的的FeCl3诱导肠系膜微动脉血管阻塞时间比WT→WT及WT→RIP3-/-小鼠长，证明RIP3在血栓形成和止血过程中起着关键作用。进一步研究发现，凝血酶或血栓素A2类似物U46619刺激RIP3-/-小鼠血小板出现聚集抑制、致密颗粒分泌的减弱、血小板铺展和血块回缩均受抑制的现象，研究结果显示：1）RIP3缺失导致U46619诱导的、依赖于PI3K/Akt信号通路的血小板二相ADP释放缺陷；2）凝血酶或U46619刺激的RIP3-/-血小板中，Akt的磷酸化程度减弱；3）激活RIP3-/-血小板中的Akt信号通路可以纠正血小板聚集反应的缺陷；4）受Akt所调控的GSK3β的磷酸化程度在RIP3-/-血小板中也有所减弱；5）PI3K抑制剂在WT及RIP3-/-血小板均可抑制低浓度凝血酶或U46619诱导的聚集，证明RIP3是通过PI3K/Akt信号通路调节血小板ADP的释放，进而调控凝血酶和血栓素A2受体介导的血小板功能，而且RIP3还可以通过Gα13蛋白参与调控血小板整合素由外向内的信号转导。本研究揭示了RIP3在血小板参与止血与血栓形成过程中的作用和机制，不仅对血小板相关基础研究，而且对于揭示血栓与出血性疾病发病机制，建立新的诊断、预防方法具有重要价值，为研制新的抗血栓药物提供新的靶点。