血管内皮细胞清除活化和凋亡的血小板调控血栓形成的作用与机制

Phosphatidylserine (PS) exposed on apoptotic or activated platelets not only provides a catalytic surface for the assembly of tenase and prothrombinase complexes but also critical “eat-me” signal for phagocytes. But the relationship between phagocytosis and coagulation is still not well understood. Endothelial cells (ECs) play an important role in balance of thrombosis and hematosis in vivo, however, the mechanisms of platelet clearance by non- professional ECs and effect on the thrombogenesis are still unclear. We first reported lactadherin and phagocytosis could cooperatively reduce procoagulant activity of APL (Blood 2012), and the ability of ECs to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells (Thromb Haemost 2013). The current project will explore the anti-thrombosis role of ECs in the clearance of PS exposed platelet. With PS-lactadherin as the center axis, the C2 domain of lactadherin (Lact-C2) is used to dynamically monitoring the distribution of PS in platelet membrane. We elucidate the mechanisms of platelet clearance by EC in vitro and vivo, and investigate the regulatory role of integrin-lactadherin-PS to thrombophilia by coagulation experiments on cell and overall level. Lactadherin functioned as an opsonin (bridge) between apoptotic cell and phagocyte, and an efficient anticoagulant for inhibition of coagulation, maybe more aggressive antithrombotic or cytoreductive strategies. This study aims to providing novel information to understand the balance of thrombosis and hematosis in vivo, establish theoretical and technical foundation for the treatment of thrombosis.

血小板凋亡或活化导致的磷脂酰丝氨酸（PS）外翻既是凝血过程中的重要调控分子又是重要的吞噬识别信号，然而，人们对血小板吞噬与凝血信号通路之间的关系所之甚少。血管内皮细胞在调节机体凝血平衡中起重要作用，然而，做为重要的兼职性吞噬细胞其吞噬血小板进而调控血栓与止血的作用尚不清楚。我们最近研究证明乳粘素介导内皮细胞吞噬APL细胞抑制其促凝活性（Blood 2012），吞噬凋亡的中性粒细胞调节炎症相关的凝血紊乱（Thromb Haemost 2013）。本项目将内皮细胞对血小板的清除及凝血调控贯穿思考，以PS-乳粘素-整合素为中心轴，利用Lact-C2小分子探针动态监测血小板膜PS的动态变化；通过体内、外实验证明内皮细胞清除血小板的机制；利用凝血相关实验从细胞和整体水平阐明调控吞噬信号途径对凝血通路的作用，将吞噬和抗凝有机整合，为体内出、凝血平衡的调控机制提供新的资料，为防治血栓性疾病提供新的思路。

血小板凋亡或活化导致的（phosphatidylserine，PS）外翻既是凝血过程中的重要调控分子又是重要的吞噬识别信号，目前，人们对血小板吞噬与凝血信号通路之间的关系所之甚少。血管内皮细胞在调节机体凝血平衡中起重要作用，然而，做为重要的兼职性吞噬细胞其吞噬血小板进而调控血栓与止血的作用尚不清楚。本研究选取两种易栓性疾病脓毒血症（sepsis）和原发性血小板增多症（ET）,通过流式细胞仪检测患者血小板PS外翻、微粒释放，利用内、外源FXa、凝血酶生成实验和凝血时间检测其促凝活性，阐明血小板PS外翻在此类疾病血栓形成中的作用。基于血小板PS外翻这一血栓形成中的关键环节和PS做为重要的吞噬识别信号这一理论，进一步探索血管内皮细胞和血小板之间的相互作用，利用流式细胞仪、激光共聚焦显微镜、扫描电镜和透射电镜从体外和动物体内实验证明内皮细胞能够吞噬活化的血小板（包括ET和sepsis患者血小板），通过促进和抑制吞噬信号通路证明内皮细胞通过整合素-乳粘素-磷脂酰丝氨酸途径（integrin-lactadherin-PS）吞噬活化的血小板，利用乳粘素调控这一吞噬途径及阻断凝血通路抑制血小板的数量和功能，进而抑制血栓形成。.本研究证明了血管内皮细胞的吞噬是活化和凋亡的血小板在外周血中清除的重要方式，帮助人们进一步认识内皮细胞与血小板之间的相互作用，补充血小板体内清除的机制，这一机制的阐明将进一步解释血小板低温保存后体内快速被清除的原因，为血小板保存及输注提出新的策略。重要的是证明了血管内皮细胞对血小板的吞噬机制及其在调控机体凝血平衡中的重要作用，利用乳粘素调控integrin-lactadherin-PS通路，促进血管内皮细胞清除表达PS的血小板和微粒，进而纠正血液高凝状态，可应用于血栓性疾病的防治。如骨髓增殖性肿瘤（ET、PV）、脓毒血症、心肌梗塞等病理情况下，外周血中大量暴露PS的血小板高度促凝，可考虑调控integrin-lactadherin-PS轴从降低细胞数和抗血栓形成两方面同时入手，为血栓性疾病的防治提供新的视角。