调节血栓形成的新途径：整合素失活及血小板聚集体解离机制的研究

Growing evidence suggests that activation of the major platelet integrin, αIIbβ3, and subsequent platelet aggregation are intrinsically dynamic and reversible processes. When aggregation is not robust enough to activate feed-forward autocrine amplification pathways, platelet integrins revert to their inactivated state and fibrinogen dissociates, resulting in platelet disaggregation. Defective platelet disaggregation has been known for many years to correlate with occlusive arterial diseases and diabetes mellitus. However the mechanisms by which integrins become inactivated leading to dissociation of fibrinogen is not well understood. It has been shown that both talin and kindlin-3 binding to integrin β3 is the prerequisite of integrin activation in platelets stimulated by agonists. And phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling is required to keep integrins in an activated state to form stable platelet aggregates. Recently, we found that in platelets stimulated with ADP or collagen-related peptide (CRP), PI3K/Akt signaling is negatively regulated in an aggregation-dependent manner. And, both talin and kindlin-3 translocate to αIIbβ3 upon ADP stimulation, but dissociate from it during the process of platelet disaggregation. Blocking ADP-induced platelet aggregation with integrin antagonist inhibited dissociation of talin and kindlin-3 from the integrin. Furthermore, platelets missing molecules, which play a positive role downstream of αIIbβ3-mediated outside-in signaling, are less prone to disaggregate following low-dose agonist stimulation than their wild-type counterparts. Based on these observations, we hypothesize that platelet aggregation and integrin αIIbβ3-mediated outside-in signaling not only initiates signals to amplify integrin activation and platelet aggregation, but also guards against inadvertent activation by shifting activated integrin αIIbβ3 back to its resting state, leading to dissociation of platelet aggregates. Therefore, in the next four year grant period, co-immunoprecipitation analysis, subcelluar fractionation assays, immunostaining assays and confocal laser scanning microscopy will be used to 1) Examine the dynamic interaction of intergrin with talin and kindlin3 during the process of platelet aggregation-disaggregation in response to various agonists stimulation, 2) Examine the effect of platelet aggregation on the interaction of talin with phosphatidylinositol 4,5-bisphosphate (PIP2) and Ras-related protein 1 (Rap1), 3)the role of PP2A and PTEN in the inhibitory effect of platelet aggregation on PI3K/Akt signaling,4)the role of platelet disaggregation in thrombosis. Together, these studies will provide important new information about the platelet biology that is platelet aggregation can function as a negative feedback of integrin activation, and shed a new light on the development of novel strategy to prevent and treat platelet related clinical disorders.

血小板是人体血液循环中的有形成分之一，不但具有可聚集性，而且还具有内在的解聚功能。血小板聚集功能的异常升高和解聚功能的降低是导致血栓形成和血栓栓塞性心脑血管疾病的主要原因。血小板表面活化的整合素αIIbβ3同其主要的配体纤维蛋白原的结合是血小板聚集的主要介质。 血小板聚集和整合素αIIbβ3介导的"由外向内(outside-in)"的信号传导不仅启动了正反馈机制，增强血小板的激活和聚集，也同时启动了内源性的负调控机制,中断了PI3K/Akt介导的信号传导途径,导致整合素αIIbβ3的失活和血小板聚集体的解离，防止了正常循环中异常血栓的形成。通过对血小板聚集机制的研究开发出了许多在临床上行之有效的治疗和预防血栓栓塞性心脑血管性疾病的药物。本课题旨在通过对整合素αIIbβ3的失活和血小板解聚机制的研究，为开发新的预防和治疗血栓栓塞性疾病的药物打下坚实的理论基础并提供新的靶点。

整合素αIIbβ3介导的“outside-in（outside-in）”的信号转导被广泛的认为是扩大血小板激活反应的，然而越来越多的证据表明“outside-in”的信号转导在某些情况下可以抑制血小板的激活。为了弄明白整合素αIIbβ3介导的“outside-in”信号通路对血小板激活的确切作用，我们通过血小板聚集仪，流式细胞仪，免疫印迹及免疫共沉淀等方法发现（1）整合素αIIbβ3的抑制剂RUC2和RGD-peptide对低剂量CRP和TRAP引起的血小板ATP释放起促进作用。说明整合素αIIbβ3介导的“outside-in”信号通路抑制血小板ATP释放。所以整合素αIIbβ3介导的“outside-in”信号通路具有负调控血小板聚集的作用。（2）在ADP为刺激剂的情况下，血小板聚集时Talin和Kindlin-3结合到整合素β3亚基上，当血小板解聚时，Talin和Kindlin-3从整合素β3上脱落下来。加入RGD-peptide抑制Talin和Kindlin-3从整合素αIIbβ3上脱落。说明是整合素αIIbβ3介导的“outside-in”信号通路参与调节Talin和Kindlin-3从整合素β3上脱落。（3）当刺激剂诱导整合素αIIbβ3激活时，Akt发生磷酸化；而激活的整合素和配体纤维蛋白原结合，血小板发生聚集后，Akt发生去磷酸化，PI3K信号通路失活。加入整合素抑制剂RGD-peptide后，Akt的去磷酸化作用被抑制。说明Akt失活是由整合素αIIbβ3 “outside-in”信号通路介导的。（4）我们加入SHIP-1抑制剂3AC后， Akt去磷酸化作用被抑制。说明SHIP-1参与调节Akt的去磷酸化作用。（5）SHIP-1的抑制剂3AC抑制血小板解聚，同时恢复ADP诱导的血小板ATP释放，而且能消除整合素介导的从外到内信号通路对血小板ATP释放的抑制效应。说明整合素介导的从外到内的信号通路通过激活SHIP-1，进而抑制血小板ATP的释放。这些结果暗示整合素αIIbβ3介导的“outside-in”的信号转导可能启动了一个负反馈通路通过促进Talin和Kindlin-3从整合素上脱落使得整合素失活和激活SHIP-1导致P13K/Akt信号通路失活这两个方面抑制整合素的激活。本文我们研究血小板解聚的机制，通过研究血小板的解聚机制为开发出不引起出血