Michels Alison, Swystun Laura L, Mewburn Jeffrey, Albánez Silvia, Lillicrap David
Department of Pathology and Molecular Medicine, Queen's University.
Department of Medicine, Queen's University.
J Vis Exp. 2017 Aug 14(126):55917. doi: 10.3791/55917.
Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.
血管性血友病因子（VWF）是一种多聚体糖蛋白凝血因子，在内皮损伤部位介导血小板黏附和聚集，并在循环中携带凝血因子VIII。VWF由内皮细胞合成，要么持续释放到血浆中，要么储存在称为魏尔-帕拉德小体（WPB）的特殊细胞器中，以便在应对止血挑战时按需释放。促凝血和促炎刺激可迅速诱导WPB胞吐和VWF释放。内皮细胞释放的大部分VWF在血浆中循环；然而，一部分VWF锚定在内皮细胞表面。在生理剪切条件下，内皮锚定的VWF可与血小板结合，形成VWF-血小板链，这可能代表血栓形成的病灶。流动腔系统可用于直观观察VWF从内皮细胞的释放以及随后血小板的捕获，其方式具有可重复性且与VWF介导的血栓形成的病理生理学相关。使用这种方法，将内皮细胞培养在流动腔中，随后用促分泌剂刺激以诱导WPB胞吐。然后将洗涤过的血小板灌注到活化的内皮上。血小板被激活，随后在流体流动方向上与伸长的VWF链结合。使用细胞外组蛋白作为促凝血和促炎刺激物，我们观察到与未处理的内皮细胞相比，组蛋白处理的内皮细胞上VWF-血小板链的形成增加。本方案描述了在血栓形成和止血模型中对VWF-血小板相互作用激活的定量、可视化和实时评估。
