Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.
Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden, the Netherlands.
J Thromb Haemost. 2019 Apr;17(4):627-634. doi: 10.1111/jth.14406. Epub 2019 Mar 1.
Essentials Prothrombotic extracellular vesicles (EV) carry agonist pathway-specific proteomes Agonists for protease activated receptor (PAR) 2 signaling have distinct effects on EV composition PAR2 signaling rapidly generates prothrombotic EV and slowly EV with inactive tissue factor (TF) FVIIa integrin ligation restricts TF incorporation into EV from endothelial cells SUMMARY: Background Cell injury signal-induced activation and release of tissue factor (TF) on extracellular vesicles (EVs) from immune and vessel wall cells propagate local and systemic coagulation initiation. TF trafficking and release on EVs occurs in concert with the release of cell adhesion receptors, including integrin β heterodimers, which control trafficking of the TF-activated factor VII (FVIIa) complex. Activation of the TF signaling partner, protease-activated receptor (PAR) 2, also triggers TF release on integrin β EVs from endothelial cells, but the physiological signals for PAR2-dependent EV generation at the vascular interface remain unknown. Objective To define relevant protease ligands of TF contributing to PAR2-dependent release on EVs from endothelial cells. Methods In endothelial cells with balanced expression of TF and PAR2, we evaluated TF release on EVs by using a combination of activity and antigen assays, immunocapture, and confocal imaging. Results and Conclusions PAR2 stimulation generated time-dependent release of distinct TF EVs with high coagulant activity (early) and high antigen levels (late). Whereas PAR2 agonist peptide and a stabilized TF-FVIIa-activated FX complex triggered TF EV release, stimulation with FVIIa alone promoted cellular retention of TF, despite comparable PAR2 activation. On endothelial cells, FVIIa uniquely induced formation of a complex of TF with integrin α β . Internalization of TF by FVIIa or anti-TF and activating antibodies against integrin β prevented PAR2 agonist-induced release of TF on EVs. These data demonstrate that intracellular trafficking controlled by FVIIa forcing interaction with integrin β regulates TF availability for release on procoagulant EVs.
基本凝血酶促细胞外囊泡（EV）携带激动剂途径特异性蛋白质组 蛋白酶激活受体（PAR）2 信号的激动剂对 EV 组成有不同的影响 PAR2 信号迅速产生促凝血酶 EV，而缓慢产生无活性组织因子（TF）的 EV FVIIa 整合素连接限制了 TF 从内皮细胞向 EV 的掺入 总结：背景 细胞损伤信号诱导免疫和血管壁细胞的细胞外囊泡（EV）上组织因子（TF）的激活和释放，从而促进局部和全身凝血起始。TF 在 EV 上的转运和释放与细胞粘附受体（包括整合素β异二聚体）的释放同时发生，后者控制 TF 激活的因子 VII（FVIIa）复合物的转运。TF 信号伴侣蛋白酶激活受体（PAR）2 的激活也触发内皮细胞 EV 上 TF 的释放，但血管界面 PAR2 依赖性 EV 生成的生理信号仍然未知。目的 确定与 PAR2 依赖性 EV 生成相关的 TF 相关蛋白酶配体。方法 在 TF 和 PAR2 表达平衡的内皮细胞中，我们通过活性和抗原测定、免疫捕获和共聚焦成像的组合来评估 EV 上 TF 的释放。 结果和结论 PAR2 刺激产生具有高凝血活性（早期）和高抗原水平（晚期）的时间依赖性释放的不同 TF EV。虽然 PAR2 激动肽和稳定的 TF-FVIIa 激活的 FX 复合物触发 TF EV 释放，但单独刺激 FVIIa 会促进 TF 在细胞内的保留，尽管 PAR2 激活相当。在内皮细胞上，FVIIa 独特地诱导 TF 与整合素αβ形成复合物。FVIIa 或抗 TF 对内皮细胞上 TF 的内化以及针对整合素β的激活抗体可防止 PAR2 激动剂诱导的 TF 在 EV 上的释放。这些数据表明，由 FVIIa 控制的细胞内转运通过强制与整合素β 相互作用调节 TF 用于促凝血酶 EV 释放的可用性。
