整合素激活因子点燃素-3在深静脉血栓形成中作用的研究

Deep vein thrombosis (DVT) is a major cause of morbidity and mortality worldwide. Where arterial thrombosis is normally triggered by direct platelet accumulation on a denuded vessel wall, DVT is often initiated by innate immune cells (mainly neutrophils) in flow-restricted deep veins. These cells are recruited and then release neutrophil extracellular traps (NETs) and present tissue factor to induce blood coagulation, together with local platelet activation and aggregation, eventually leading to DVT formation. Therefore, DVT formation involves both the innate immune cells and platelets. However, the molecular basis underlying these involved cells remains largely unknown. Kindlin-3 is a newly identified integrin β-subunit binding partner and an essential integrin activator in hematopoietic cells. The functional importance of kindlin-3 in both innate immune cells and platelets suggests that kindlin-3 may play a critical role in DVT formation. This proposal focuses on understanding the role of integrin-kindlin-3 signaling in hematopoietic cells in DVT formation. The hypothesis to be tested is that kindlin-3 in neutrophils and platelets contributes to DVT formation at different stages by supporting both integrin-mediated cell adhesion/aggregation and NET release. First, we will determine the respective contribution of kindlin-3 in neutrophils and platelets to DVT formation. The role of kindlin-3 signaling in DVT formation will be measured by performing an established inferior vena cava (IVC) ligature procedure to induce DVT in mice with kindlin-3 knockouts specific for neutrophils and platelets. Next, we will evaluate the role of integrin-dependent kindlin-3 signaling in neutrophils and platelets in DVT formation by employing a strain of kindlin-3 knock-in mice that carry an integrin-interaction defective mutation that allows us to specify the integrin-kindlin-3 signaling. Finally, we will focus on understanding how kindlin-3 signaling supports NET release. We will perform both in vitro and in vivo NET formation assays on isolated neutrophils and in ligated IVC to determine whether NET release is supported by the integrin-kindlin-3 signaling. All these studies are essential for further understanding of the molecular basis of DVT formation, and will generate new information that may lead to more targeted and effective treatments for DVT.

深静脉血栓症是目前世界上致死和致残的一个重要病因，其形成的诱因一般是局部深静脉血流凝滞，进而促使中性粒细胞的粘附和血小板的聚集，并诱发凝血反应，最终导致血栓的形成。目前深静脉血栓形成的分子基础尚不清楚。本课题将对在中性粒细胞和血小板中调控多种整合素激活的关键因子点燃素-3进行系统的研究，并重点分析点燃素-3信号通路在不同血细胞中对深静脉血栓形成的影响。为此，我们制作了点燃素-3基因条件性剔除小鼠，然后与组织特异性Cre小鼠杂交分别获得了在中性粒细胞和血小板中点燃素-3表达缺失的小鼠。另外，我们还制作了点燃素-3基因点突变敲入小鼠，阻断点燃素-3在血细胞中与整合素的识别。同时，我们通过腹腔静脉结扎手术建立了小鼠体内深静脉血栓形成的病理模型。由此，我们可利用这些点燃素-3基因模式小鼠分析整合素信号通路在不同血细胞中对深静脉血栓形成的作用，进而阐明深静脉血栓发生的分子机理。

本项目主要内容是评价血小板和中性粒细胞中Kindlin-3分子对深静脉血栓形成的影响和相应的调控机制。 Kindlin-3分子表达在血细胞中，是重要的胞内整合素激活因子。首先，利用Kindlin-3基因突变敲入小鼠（K3KI）模型来阻断其与整合素的结合，我们证明了K3KI小鼠相对于野生型小鼠深静脉血栓的形成明显降低，说明血细胞中整合素信号通路是影响深静脉血栓形成的关键因素。因为血液细胞中血小板和中性粒细胞对深静脉血栓形成都有重要的促进作用，所以我们制作了Kindlin-3的条件性基因剔除小鼠（Kindlin-3fl/fl）并获得了血小板中Kindlin-3特异敲除的Kindlin-3fl/fPF4-Cre小鼠和髓系粒细胞中Kindlin-3特异敲除的Kindlin-3fl/fLysM-Cre小鼠。通过功能验证，我们发现血小板中Kindlin-3分子缺失可显著抑制深静脉血栓的形成；但是我们意外地发现Kindlin-3分子在粒细胞中表达缺失对深静脉血栓的形成反而有明显的促进作用。进一步机理研究发现中性粒细胞中Kindlin-3的表达缺失可促进其细胞核物质(NETs)的释放，从而容易诱发深静脉血栓的形成，但是该过程是受到血小板Kindlin-3信号的正向调控。这些发现不仅进一步揭示了不同血细胞中Kindlin-3信号对深静脉血栓形成的影响和调控机理，而且也为将来开发更安全高效的抗深静脉血栓形成药物提供了新的靶点和理论基础。