PECAM-1及其亲和功能调控在弥散性血管内凝血中的作用及机制研究

Endothelial damage and excessive inflammation are pivotal pathological changes to develop disseminated intravascular coagulation (DIC), if a key molecule can be found and targeted in the pathological progress, the treatment of DIC will make effective improvement and breakthrough progress. Studies show that the role of platelet endothelial cell adhesion molecule-1(PECAM-1) on maintaining endothelial integrity and regulating inflammation is particularly critical，because its hemophilic and heterophilic affinity underlies the interactions between endothelial cells with endothelial cells and inflammatory cells. In our previous studies，we confirmed that the PECAM-1 KO mice was more inclined to occur DIC, and we firstly revealed that the affinity of PECAM-1 can be regulated by its structural domain Ig D6; furthermore, its hemophilic affinity can be enhanced by PECAM-1 IgD6 antibody through conformational changes. Therefore, we innovatively proposed that targeted regulation of the affinity of PECAM-1 on the cell surface may restore the imbalance of endothelium and inflammation, thus effectively interfering with the DIC pathological process. In this project, the aim is to elucidate the relationship between PECAM-1 and DIC through the chimeric animal models；and to explore the effects of antibody-driven affinity modulation of PECAM-1 on endothelial barrier restoration and anti-inflammatory response by the technologies of ECIS, nanodisc, etc. All the above-mentioned is to reveal the function of PECAM-1 and its affinity regulation in DIC, providing novel potential therapeutically approaches for DIC.

内皮损伤和过度炎症是弥散性血管内凝血(DIC)的重要病理环节，寻找其中关键分子并实施靶向干预，有望突破DIC治疗瓶颈。研究证实血小板内皮细胞粘附分子-1（PECAM-1）介导的同/异嗜性亲和是内皮-内皮、内皮-炎症细胞间识别粘附的基础，在维持内皮完整及调节炎症方面至关重要；我们前期研究发现PECAM-1KO小鼠更易发生DIC；首次确定PECAM-1结构域D6是其亲和功能调节区，针对D6区抗体可能通过构象改变增强PECAM-1亲和力；因此，创新性提出靶向调控细胞膜PECAM-1亲和功能以纠正内皮与炎症失衡，从而实现对DIC的有效干预。.本项目将通过嵌合体模式动物明确不同细胞来源PECAM-1与DIC的关系；应用ECIS、纳米盘等技术探讨IgD6抗体驱动PECAM-1亲和力改变对DIC内皮屏障修复、抗炎效应的影响及机制，从而揭示PECAM-1及其亲和功能调控在DIC中的作用，为治疗提供新思路。

内皮损伤和过度炎症是弥散性血管内凝血(DIC)的重要病理环节，寻找其中关键分子并实施靶向干预，有望突破DIC治疗瓶颈。研究证实血小板内皮细胞粘附分子-1（PECAM-1）介导的同/异嗜性亲和是内皮-内皮、内皮-炎症细胞间识别粘附的基础，在维持内皮完整及调节炎症方面至关重要。本研究在细胞及动物水平探索PECAM-1在脓毒症DIC中的作用及机制；深入探索PECAM-1同嗜/异嗜性识别与其构象改变对其功能的影响，明确PECAM-1及其同嗜/异嗜性调节在脓毒症DIC中的精确作用机制，为靶向干预脓毒症DIC病理奠定理论基础。前期，我们利用临床数据分析发现血浆中可溶性PECAM-1与脓毒症DIC的发生及预后密切相关，推测PECAM-1参与脓毒症DIC的病理机制。利用CRISPR基因编辑技术构建并繁殖了大量PECAM-1 基因敲除（KO）小鼠。利用脂多糖及盲肠结扎穿孔术在野生型和KO型小鼠中模拟脓毒症DIC模型，发现PECAM-1可抑制脓毒症DIC的发生；通过嵌合体模式动物，发现内皮细胞来源和造血细胞来源的PECAM-1均参与该过程。PECAM-1可通过平衡促炎/抗炎因子平衡、抑制巨噬细胞焦亡、修复血管屏障等多种机制抑制DIC。另外，通过Pymol结构分析软件新解析的PECAM-1同嗜性结合区域的晶体结构，预测可能参与到PECAM-1同嗜性结合功能的氨基酸残基。进一步验证发现PECAM-1 IgD1/IgD1界面中的D33R，IgD1/IgD2界面中的D52R，R152D/I突变，可完全破坏PECAM－1的同嗜性作用功能。随后进一步探索PECAM-1同嗜性功能的缺失对内皮细胞功能的影响。研究表明，PECAM-1结构上的变化，不仅可以影响其同嗜性识别功能，且能进一步影响内皮细胞的血管新生和屏障修复功能，为靶向干预脓毒症DIC提供了新的思路。