Centre for Microvascular Research, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
MRC Laboratory of Molecular Cell Biology, University College London, London, UK.
J Thromb Haemost. 2018 Sep;16(9):1873-1886. doi: 10.1111/jth.14218. Epub 2018 Aug 12.
Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo.
Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.
基本原理 内皮细胞激活启动多种过程，包括止血和炎症。促成这些过程的分子共同储存在分泌颗粒中。细胞如何控制颗粒内容物的释放以允许分化反应？选定的激动剂募集一个与胞吐相关的肌动蛋白环来促进亚群货物的释放。
背景 内皮细胞拥有专门的储存细胞器，Weibel-Palade 体（WPB）。WPB 内容物向血管腔中的胞吐作用启动了由 von Willebrand 因子（VWF）介导的原发性止血，以及由包括 P-选择素在内的多种蛋白介导的炎症。在完全融合期间，这种大型止血蛋白和较小的促炎蛋白的分泌被认为是不可分割地联系在一起的。
目的 确定 WPB 货物的分泌依赖性差异释放是否发生，以及这是否由胞吐过程中肌动球蛋白环的形成介导。
方法 我们使用 VWF 字符串分析、白细胞滚动测定、ELISA、旋转盘共聚焦显微镜、高通量共聚焦显微镜以及抑制剂和 siRNA 处理来证明存在允许 WPB 货物蛋白差异释放的细胞机制。
结果 肌动球蛋白环的抑制以不同的方式影响 WPB 胞吐调节的两个过程；它扰乱了 VWF 字符串的形成，但对白细胞滚动没有影响。环募集的效率与 VWF 释放相关；当环募集被抑制时，VWF 与小货物的释放比例降低。肌动球蛋白环的募集是时间依赖性的（刺激后直接发生的融合事件比稍后的事件更不可能引发止血），并且被蛋白激酶 C（PKC）同工型激活。
结论 不同的分泌剂募集肌动球蛋白环，从而证明了内皮细胞激活的促血栓形成效应可以被调节的一种机制。这可能会限制血栓形成，同时允许正常的炎症反应。这些结果对内皮细胞 WPB 融合、货物内容物释放的评估以及 von Willebrand 病患者的治疗具有重要意义。
