血小板整合素αIIbβ3激活的分子机理和功能重要性研究

Integrin αIIbβ3 plays a major role in platelet aggregation, and thus is an attractive pharmaceutical target for treating thrombosis-triggered cardiovascular disease. Integrin αIIbβ3 can be activated by various agonists at the site of damaged blood vessel, which enable to associate their receptors on platelet surface and induce inside signals to initiate extracellular ligand binding to integrin (termed inside-out signaling). The terminal effectors of the inside-out signaling pathway are integrin αIIbβ3 cytoplasmic tail (CT) binding partners, which can dynamically interact with integrin CT and control the conformational changes of integrin extracellular domain. Kindlin family members are recently identified integrin β CT binding proteins, in which kindlin3 is specifically expressed in platelets. However, how kindlin3 regulates integrin αIIbβ3 activation is mechanistically unknown. Here, we generated two strains of kindlin3 gene manipulated mice, one with floxed kindlin3 which can be used to breed with Pf4-Cre mice to make platelet specific kindlin3-/- mice (conditional knockout), and the other with a built-in mutation in kindlin3 to disable its binding to integrin β3 CT (knock-in mice). These kindlin3 mice will be used to systematically map the functional contribution of kindlin3 and β3 CT interaction to integrin activation and platelet aggregation. In addition, by employing multiple biochemical approaches, we will be making efforts to identify kindlin3 upstream signaling binding partners, and reconstitute the kindlin3 signaling pathway. In summary, our studies will insight into the molecular mechanism of integrin αIIbβ3 activation and shed light on developing novel therapeutics for heart disease and stroke.

血小板整合素αIIbβ3是介导血小板凝聚的主要粘附分子，也是治疗由血栓导致的急性心脑血管病变的重要药物靶分子。整合素αIIbβ3配体结合活性是受细胞内激活信号严格调控的。激活信号诱导胞内的激活因子和整合素胞内序列结合，进而调控整合素胞外区域的构象变化。前期研究证明Kindlin3在整合素αIIbβ3的激活中起着重要作用，但作用机理尚不清楚。本申请项目将用Kindlin3的条件性基因敲除小鼠和定点突变敲入小鼠来系统的评价Kindlin3与αIIbβ3的识别对αIIbβ3激活和血小板凝聚功能的影响。同时，利用免疫共沉淀和质谱分析等生化手段，并结合整合素αIIbβ3激活的细胞模型来进一步鉴定Kindlin3的上游结合信号分子，从而构建整合素αIIbβ3激活的信号通路网络。本项研究成果将有助于揭示血小板整合素αIIbβ3激活的分子机制，为开发新一代抗栓药物和防治心脑血管疾病提供理论基础。

本项目主要内容是评价血小板中整合素激活因子Kindlin-3对整合素的激活作用以及在血栓形成中的重要功能，并在此基础上进一步阐述Kindlin-3参与激活整合素的分子机理。首先，利用Kindlin-3基因敲入小鼠模型来阻断其与整合素的结合并通过系列体外和体内的分析，我们证明了Kindlin-3与整合素αIIbβ3的直接结合对整合素αIIbβ3所介导的血小板聚集，凝血块的收缩以及动脉血栓的形成有重要的促进作用。其次，通过酵母双杂交筛选和免疫共沉淀等生化分析手段，我们鉴定了多种Kindlin-3的结合蛋白因子，并且我们证明了其中一些信号接头蛋白因子对整合素的激活有着明显的促进作用，说明这些蛋白因子可能通过结合Kindlin-3调控整合素αIIbβ3的激活。这些发现不仅进一步揭示了整合素αIIbβ3激活的分子机理，而且对开发更安全高效的抗栓药物提供了新的靶点。