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Real time imaging analysis of platelets aggregation and thrombus formation under shear stress

剪切应力下血小板聚集和血栓形成的实时成像分析

基本信息

批准号：
18590204
负责人：
URANO Tetsumei
金额：
$ 2.44万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590204/
关键词：
thrombus formation intra vital confocal microscopy vo Willebrand Factor ADAMTS13 Thrombotic Thrombocytopenic Purpura platelet vascular endothelinl cell von Willebrand因子

项目摘要

[Background] After secretion from Weibel-Palade body in vascular endothelial cells (VECs), von Willebrand factor (vWF) circulates as ultra-large multimer forms (ULM-vWF) and adheres to subendothelial collagen, to which platelets subsequently adheres to form platelet thrombi. Lacking of vWF leads to bleeding tendency (vWF disease) and excess amounts of ULM-vWF leads to thrombotic disease (thrombotic thrombocytopenic purpura). [Aim] Employing gene deficiency mouse of vWF cleaving enzyme (ADAMTS13) hybridized with green fluorescent protein (GFP) expressing transgenic mouse (KO mouse), we analyzed the dynamics of vWF exocytosis as well as platelet adhesion and its aggregation under shear stress by intra-vital confocal microscopy. [Results] (1) DDAVP, known to stimulate vWF exocytosis, increased ULM-vWF which was visualized as long strings using fluorescent labeled anti-vWF antibody. (2) The strings were longest at 5th minute, and longer in KO mice (5.28±4.28 vs 2.89±2.08 μm). (3) GFP expressing platelets adhered to being secreted ULM-vWF. (4) After topical infusion of 2.5% FeC12, longer (26.34±8.28 vs 10.73±3.11 μm) and more stable (2.35±1.23 vs 0.63±0.21 s) strings were visualized in KO mice. [Conclusion] (1) Platelets adhesion and aggregation were successfully monitored by intra-vital confocal microscopy. (2) ADAMTS13 appeared to cleave ULM-vWF under shear stress when it is being secreted. (3) The enhanced secretion itself, however, did not develop microthrombus even in KO mice. (4) Longer and more stable ULM-vWF adhered to injured VECs by ferric chloride treatment in KO mice. (5) Larger thrombus was formed in KO-mice at the initial phase after laser-irradiated injury of vascular wall, whereas there was no difference in the size of stabilized thrombus. (6) ADAMTS13 was shown to be responsible for the cleavage of ULM-vWF under shear stress, and lack of its activity appeared to be thrombogenic.

[背景]血管性假血友病因子(von Willebrand factor，vWF)经血管内皮细胞(VECs)分泌后，以超大多聚体形式(ULM-vWF)循环，并与内皮下胶原蛋白黏附，随后与血小板黏附形成血小板血栓。缺乏vWF会导致出血倾向(vWF病)，过多的ULM-vWF会导致血栓性疾病(血栓性血小板减少性紫癜)。[目的]利用vWF裂解酶基因缺陷小鼠(ADAMTS13)与表达绿色荧光蛋白(GFP)的转基因小鼠(KO鼠)杂交，利用活体共聚焦显微镜观察切应力作用下vWF胞吐及血小板黏附和聚集的动态变化。[结果](1)DDAVP可刺激vWF胞吐，荧光标记的抗vWF抗体可使ULM-vWF增多。(2)5分钟时最长，KO组最长(5.2 8±4.2 8比2.89±2.0 8μm)。(3)表达GFP的血小板黏附于分泌的ULM-vWF。(4)局部滴注2.5%FeC_(12)后，KO小鼠可见更长(26.34±8.28vs 10.73±3.11μm)和更稳定(2.35±1.23vs 0.63±0.21 S)串。[结论](1)活体共聚焦显微镜成功监测了血小板的黏附和聚集。(2)在ULM-vWF的分泌过程中，ADAMTS13在剪切力作用下表现出明显的切割作用。(3)即使在KO小鼠体内，增强的分泌物本身也不会形成微血栓。(4)KO小鼠损伤血管内皮细胞经三氯化铁处理后，ULM-vWF黏附在损伤的血管内皮细胞上更长、更稳定。(5)KO-小鼠在激光照射血管壁损伤后的初期形成较大的血栓，而稳定的血栓大小无明显差异。(6)在剪切力作用下，ADAMTS13参与了ULM-vWF的切割，其活性缺失可能与血栓形成有关。